1^'
Copyright HO. Berchman Austin Vaz 1993
ACKNOWLEDGEMENTS I would like to express my appreciation to Dr. David Hentges, Dr. Earl Ritzi, Dr. Doris Lefkowitz, Dr. Abdul Hamood and Dr. Stanley Lefkowitz for all of their strong support throughout the course of these studies. I am extremely grateful to all of them for their helpful suggesticnis during committee meetings and for the time they spent in reviewing this manuscript. Sincere thanks are due to the faculty, office staff and graduate students of the Department of Microbiology and Immunology at Texas Tech University Health Sciences Center, for their friendship and help during the past four years. I am indebted for the open door policy of the faculty members and especially my mentor. Dr. Lefkowitz, who was approachable at all times to discuss my research woric I would like to extend my sincerest appreciation for the friendships of George and Marie Riddell, and Margaret Fuller during the time I spent at TTUHSC. Heartfelt thanks are due to my best fiiend and fiancee, Cherylyn, for her support and encouragement from across the miles. I could not have achieved my goals without her patience, understanding and love. I would also like to thank my parents and family members for always expecting only the best from me and sacrificing a lot for my education. Most importantly, I would like to extend sincere thanks to my advisor. Dr. Stanley Lefkowitz for his patience andftiendshipduring the time I woriced as his student It has been a tremendously positive experience learning a lot about life and science from him. I hope to continue my association with him for many years in the future as a friend and collaborator.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ABSTRACT LIST OF TABLES LIST OF HGURES LIST OF ABBREVL\TIONS I. INTRODUCTION Macrophages Macrophage functions Phagocytosis Chemiluminescence Macrophage activation: Macrophage-mediated cytotoxicity Mechanisms: Reactive nitrogen intermediates Tumor necrosis factor Macrophage receptors/ surface proteins Cocaine Brief history of cocaine Routes of administration Metabolism of cocaine Effects of cocaine Pharmacological effects Other adverse effects of cocaine Previous studies on the effects of cocaine on the immune system The objectives of the currentresearcheffort iii
ii vi viii ix xii 1 1 3 3 4 6 7 7 8 11 11 12 13 14 14 15 16 20
n. MATERIALS AND METHODS Experimental animals Cells and tissue culture media Dnigs Collection ofresidentperitoneal macrophagesfix)mC57BL/6 mice Collection ofresidentmurine alveolar macrophages Measurement ofrespiratoryburst by chemiluminescence Materials Zymosan solution Luminol solution Assay procedure Phagocytosis assay (in vitro) using opsonized zymosan Phagocytosis assay (in vivo) using sheep erythrocytes Macrophage-mediated cytotoxicity assay Measurement ofreactivenitrogen intermediates (RNI) Assay for tumor necrosis factor Direct immunofluorescence analysis by flow cytometry Antibodies and FACS reagents
22 22 22 23 23 23 24 24 24 25 25 26 26 29 30 31 32 32
Macrophage labelling for direct immunofluorescence analysis by flow cytometry Flow cytometry and direct immunofluorescence analysis Statistical analysis of experimental m. RESULTS Effects of cocaine on phagocytosis Effects of cocaine on therespiratoryburst Effects of cocaine on phagocytosis, in vitro iv results 33 34 35 36 36 36 52
Effects of cocaine on phagocytosis, in vivo Effects of cocaine on macrophage activation Effects of cocaine on macrophage-mediated cytotoxicity Effects of cocaine on TNF secretion Effects of cocaine on production of reactive nitrogen intermediates Effects of cocaine metabolites on production ofreactivenitrogen intermediates (RNI) Effects of cocaine on macrophage receptors/ surface proteins IV. DISCUSSION Effects of cocaine on phagocytosis Effects of cocaine on macrophage activation (macrophage-mediated cytotoxicity) Effects of cocaine on macrophage receptors/ surface proteins Future studies V. CONCLUSIONS LITERATURE CITED
52 55 55 61 61
66 72 85 86 92 95 100 102 104
ABSTRACT
One of the most "fashionable" as well as dangerous drugs abused in this country is cocaine. Very little is known about the effects of cocaine on the immune system. Since macrophages play a central role in both the cellular and the humoral arms of the immune system, this study focused on the effects of cocaine on murine peritoneal macrophage functions, viz., phagocytosis and activation to the cytotoxic state. Phagocytosis is one of the most important components of the host defenses against invading microorganisms once the outer epithelial surface of the body has been breached. In the present study, cocaine injected intraperitoneally was found to increase phagocytic activity of isolated macrophages in vitro; but decreased it when measured by an in vivo assay. Therespiratoryburst, which is usually correlated with phagocytosis, was enhanced by cocaine. Macrophage activation was studied by measuring macrophage-mediated cytotoxicity (MMC), i.e., the acquisition of competence to destroy neoplastic cells in the absence of antibody. Macrophages from cocaine-injected mice demonstrated a reduced capacity for killing neoplastic cells; as well as a reduction in the secretion of reactive nitrogen intermediates. The latter represent a major product used by macrophages to kill ceUs. Macrophage receptors/ surface proteins involved in phagocytosis and activation were studied using flow cytometry. A number of these surface receptors were dramatically altered by exposure to cocaine. The presumed toxic metabolites of cocaine, benzoylecgonine and norcocaine, were found to have similar effects on macrophage functions. The present studies clearly demonstrate that cocaine affeas phagocytosis and activation to the cytotoxic state. This translates into impairment of certain non-specific immuneresponses.Theseresultstaken in their entirety suggest that the ability of cocaine to
VI
alter macrophage functions could modify specific immuneresponsesresultingin a compromised immune system.
Vll
UST OF TABLES
1. Corticosterone levels of mice exposed to cocaine 2. Effects of cocaine on phagocytosis of opsonized zymosan in vitro by peritoneal macrophages 3. Effects of cocaine on phagocytosis of sheep erythrocytes in vivo by peritoneal macrophages 4. Effects of cocaine on macrophage-mediated cytotoxicity 5. Effects of cocaine inhalation on induction of tumor necrosis factor 6. Effects of cocaine on production ofreactivenitrogen intermediates 7. Effeas of cocaine on macrophage-mediated cytotoxicity (MMC) and production ofreactivenitrogen intermediates 8. Effects of cocaine on F4/80 surface marker of peritoneal macrophages 9. Effects of cocaine on Mac-1 receptor 3 hours after exposure 10. Effects of cocaine on Mac-1 receptor 24 hours after exposure 11. Effects of cocaine on la expression 3 hours after exposure 12. Effects of cocaine on la expression 24 hours after exposure
38 53 54
60 63 69 70 75 77 78 83 84
vm
LIST OF HGURES 1. Scheme describing studies done in dissertation 2. Photomicrograph of macrophages with ingested opsonized zymosan (in vitro phagocytosis assay) 3. Photomicrograph of macrophages with ingested sheep erythrocytes (in vivo phagocytosis assay) 4. Detection of cocaine metabolites (benzoylecgonine) in sera of mice injected intravenously 10 min earlier with different amounts of cocaine 5. Enhancement of therespiratoryburst of peritoneal macrophages 60 min after (A) a single 2.5 mg/kg i.p. injection of cocaine or (B) a single 2.5 mg/kg i.p. injection of cocaine 21 27 29 37
40
6. Enhancement of therespiratoryburst of peritoneal macrophages 24 hr after (A) a single 2.5 and 5 mg/kg i.p. injection of cocaine or (B) a single 2.5 mg/kg i.p. injection of cocaine 41 7. Enhancement of therespiratoryburst of peritoneal macrophages 48 hr after (A) a single 2.5 and 5 mg/kg i.p. injection of cocaine or (B) a single 2.5 mg/kg i.p. injection of cocaine 42 8. Enhancement of therespiratoryburst of peritoneal macrophages 24 hr after 4 consecutive daily 5 mg/kg i.p. injections of cocaine 9. Enhancement of therespiratoryburst of peritoneal macrophages 24 hr after (A) a single i.v. injection of cocaine, (B) a single i.m. injection of cocaine, (C) cocaine inhalation (168 mg/m^ for 1 hr) 10. Enhancement of therespiratoryburst of alveolar macrophages (A) 60 min and (B) 24 hr after a single 10 mg/kg i.p. injection of cocaine 11. Effects of in vitro cocaine on therespiratoryburst of peritoneal macrophages 60 minutes after exposure 12. Effects of (A) a 5mg/kg injection of either cocaine or ecgonine methyl ester HCl (B) a 5 mg/kg injection of either cocaine or ecgonine HQ on the respiratory burst of peritoneal macrophages 60 min after exposure 13. Effects of a 5mg/kg injection of either cocaine or its metabolite, ecgonine methyl ester HCl, on the respiratory burst of peritoneal macrophages, 1 and 24 hr after exposure 14. Effects of a 5mg/kg i.p. injection of either (A) cocaine or norcocaine and (B) ecgonine HCl, ecgonine methyl ester HQ, or benzoylecgonine, 3 hr after exposure, on the respiratory burst of peritoneal macrophages ix 43
45 47 48
49
50
51
15. Effects of a single ip injection of cocaine on MMC measured at different intervals after exposure. Thisfigureis a composite of 4 experiments. Groups of 3 mice per experiment were injected with 10 mg/kg cocaine or saline. Peritoneal macrophages wereremovedat various intervals after injection and cultured and evaluated for cytotoxicity. 16. Effects of a single ip injection of cocaine on MMC measured at different intervals after exposure. Thisfigureis a composite of 3 experiments. Groups of 3 mice per experiment were injected with 25 mg/kg cocaine or saline. Peritoneal macrophages wereremovedat various intervals after injection and cultured and evaluated for cytotoxicity. 17. Effects of a single ip injection of cocaine on MMC measured 24 hr after exposure. Thisfigureis a composite of 3 experiments. Groups of 3 mice per experiment were injected with 5,10, or 25 mg/kg cocaine. Matched controls were injected with saline. Peritoneal macrophages were removed 24 hr after injection, cultured and evaluated for cytotoxicity. 18. Effects of a one injection of cocaine/day for 5 days on MMC measured 24 hr after the last injection. Thisfigureis a composite of two experiments. Matched controls were injected with saline. Peritoneal macrophages were removed 24 hr after the last injection, cultured and evaluated for cytotoxicity. 19. Effects of a single i.p injection of cocaine on MMC (using WEHI 164 as target cells) measured 24 hr after injection. Thisfigureis a composite of two experiments. Groups of three mice were injected with 25 mg/kg cocaine. Matched controls were injeaed with saline. Peritoneal macrophages were removed 3 and 24 hr after the last injection, cultured and evaluatal for cytotoxicity. 20. Effects of a single 5, 10, or 25 mg/kg i.p. injection of cocaine on the production of RNI (nitrites) by murine peritoneal macrophages. Macrophages were removed (A) 3 hr after exposure and (B) 24 hr after exposure 21. Effects of a single 5, 10 or 25 mg/kg injection of cocaine on the production of reactive nitrogen intermediatesrepresentedas a percentage of control. Macrophage wereremoved3 and 12 hours after exposure. 22. Effects of a single 5 mg/kg injection of cocaine on the production of nitrites by murine peritoneal macrophages. Macrophage wereremoved3,12 and 24 hours after exposure. 23. Effects of a single 10 mg/kg injection of cocaine, 3, 12 and 24 hours after exposure on the production of (A) nitrites and (B) nitrates by murine peritoneal macrophages 24. Effects of a single 5 mg/kg ip injection of cocaine or its metabolites on the production of RNI (nitrites) by murine peritoneal macrophages. Macrophages wereremoved3 hr after exposure 25. Effects of cocaine on F4/80 surface marker of peritoneal macrophages, 3 and 24 hr after exposure x
56
57
58
59
62
64
65
67
68
71 74
26. Effects of cocaine on Mac-1 receptor of peritoneal macrophages, 3 and 24 hr after exposure 27. Effects of cocaine on IgG2a receptor of peritoneal macrophages, 3 and 24 hr after exposure 28. Effects of cocaine on IgG2b receptor of peritoneal macrophages, 3 and 24 hr after exposure 29. Effects of cocaine on mannose-fucose receptor of peritoneal macrophages, 3 and 24 hr after exposure 30. Effects of cocaine on la expression of peritoneal macrophages, 3 and 24 hr after exposure
76 79 80 81 82
XI
LIST OF ABBREVIATIONS
ACTH, adrenocorticotropic hormone AIDS, autoimmune deficiency syndrome ATCC, American Type Culture Collection BSA, bovine serum albumin C, complement CL, chemiluminescence CO2, carbon dioxide dl, deciliter DMEM, Dulbecco's Modified Eagle's medium FACS,fluorescentactivated cell sorting FBS, fetal bovine serum FcR, Fc receptor fig, figure FTTC,fluoresceinisothiocyanate FL, fluorescence HCl, hydrochloride HEPES, N-2-hydroxyethyl-piperazine-N'-2-ethane-sulfonic acid HTV, human immunodeficiency virus H2O, water H2O2, hydrogen peroxide hr, hour(s) IFN, Interferon IL-l,interleukin-l IL-2, interleukin-2 xii
i.m., intramuscular i.p., intraperitoneal i.v., intravenous kg, kilogram LPS, lipopolysaccharide MMC, macrophage-mediated cytotoxicity M0, macrophage(s) MFR, mannose-fucose receptor m, meter M, moles/molar mg, milUgram min, minutes |i.g, microgram \il, microliter ^iM, micromolar NaN3, sodium azide ng, nanogram NK, natural killer NMA, N-monomethyl-L-arginine NO, nitric oxide NO2, nitrites N03, nitrates NRD, neutral red dye O2, oxygen 02', superoxide OH", hydroxy] xiu
PBS, phosphate buffered saline PE, phycoerythrin PI, propidium iodide PMT, photomultiplier PMA, phorbol myristate acetate RB,respiratoryburst RNI, reactive nitrogen intermediates ROI,reactiveopxygen intermediates rpm,revolutionsper minute S.D., standard deviation SEM, standard error of the mean SSC, side scatter TGF, transforming growth factor TNF, tumor necrosis factor uv, ultraviolet
XIV
CHAPTER I INTRODUCTION
One of the very important problems facing contemporary society is substance abuse. This problem has caused increased crime as well as destruction of the health of a significant portion of society. At the present time, one of the most commonly abused as well as dangerous drugs in this country is cocaine. This substance is most commonly administered via "snorting" or by smoking thefireebaseform known as "crack." The net effects of these drugsresultin significant alterations in bodily functions. Cocaine addiction with its attendant medical complications (including transmission of AIDS) has become a major problem, particularly among lower socioeconomic class drug abusers. Many of the fi"equently abused drugs, under specific conditions, are capable of interfering with immune responses. The effects of cocaine on immune function have not been adequately determined. Because of the multifunctional capability of macrophages (M0) in both humoral and cellular responses, this study focuses on the effects of cocaine on these cells. Cocaine was given to experimental mice as the model system for these investigations. The purpose of this research was to ascertain the effects of cocaine and its metabolites on M0 functions, viz., phagocytosis and activation to the cytotoxic state.
Macrophages The mononuclear phagocyterepresentsa cell which is widely distributed throughout most tissues. The term "macrc^hage" was first used nx)re than 100 years ago by Elie Metchnikoff in Messina, to describe the large mononuclear phagocytic cells he observed in tissues (Kamovsky, 1981). The M0 is the major differentiated cell of the mononuclear phagocyte system. This is a system of "professional" phagocytes that have a common
1
origin, similar morphology and identifying features (van Furth, 1980). The progenitor cell of the M0 (the promonocyte) is located in the bone manx)w. In humans, monocytes leave the bone marrow approximately 2 1/2 days after they develop. Macrophages are mature phagocytic leukocytes which have exited the blood stream and have establishedresidencein various tissues throughout the body, in contrast to monocytes which remain in the general circulation (Rook, 1989). M0 have been identified as to the tissue in which they have taken upresidence.Examples include alveolar M0 of the lungs, Kupffer cells of the liver, microglial ceUs of the brain, Langherhans' cells of the skin, osteoclasts of the bone, and peritoneal M0 of the peritoneal serous cavity (Lanier, 1991; Auger and Ross, 1992). The life span of the mature M0 is a few months (Stites et al., 1984). In the mouse, the life-span is 20-40 days for peritoneal M0, 60 days for Kupffer cells in the liver, and 50 days for alveolar M0. The total monocyte production in the mouse is approximately 1.5 X 10^ cells per 24 hours, and all of these cells eventually leave the bone marrow and become tissue M0 (van Furth, 1989) Macrophages play an importantrolein the immune system They are intimately involved in both cellular and humoral immunity. One of the earliest recognized functions of these cells is endocytosis. The M0 has numerous surface receptors that allow it to interact with hormones, exogenous and endogenous proteins, polysaccharides, and lipids. Interactions of these receptors with their complementary ligands mayresultin the engulfment of these substances as well as various particles which exhibit these ligands. Following binding and/or internalization, these cells synthesize and express bioactive molecules which modulate theresponseof these and other cells to the environment. In addition to the process of phagocytosis, M0 secrete more than 100 different substances, each of which have significant immunobiologic actions. Secreted molecules include substances such as prostaglandins, leukotrienes, complement proteins, enzymes, growthpromoting substances and cytokines such as interleukin 1 (IL-1), tumor necrosis factor
(TNF) and interferon (IFN). These cells also producereactiveoxygen intermediates (ROI) which are strongly correlated with phagocytic activity aiKireactivenitrogen intermediates (RNI) which are involved with macrophage-mediated cytotoxicity (MMC). Other immunologic functions of M0 are antigen processing and presenting,regulationof lymphocyte reactivity via theirreleaseof positive and negativeregulatorymolecules, and as effector cells in cellular immunity and delayed hypersensitivity. Macrophages participate in inflammation and healing of tissues following injury by production of certain of the factors discussed. These cells also play a key role in infections because they rapidly mobilize to the infected sites, participate as accessory cells for activation of T cells, and rapidly release mediators whichresultin the acute phaseresponsefollowing infection. In addition, they also are believed to function as one of the major guardians eliminating aberrant cells (malignant) which arise spontaneously. Because of their extensive involvement in many facets of the immune response, numerous studies have been done to assess M0 function as a measure of immunosuppression by exogenously added substances. Among the various capacities and functions measured are phagocytosis, spreading, activation to the cytotoxic state, and production of specific cytokines.
Macrophage functions Phagocytosis One of the nK)st prominent functional properties of M0 is their ability to recognize and phagocytize foreign material and damaged cells. In addition to eliminating aberrant cells, M0 also serve to eliminate bacteria and other foreign invaders. Phagocytosis is the ability of M0 to bind and ingest various particles. Phagocytosis is characterized by the ingestion and eventual digestion of particles greater than 4 nm in diameter (Fauve, 1978). During phagocytosis, particles are bound to either specific or non-specific receptors. The particle is
then surrounded by the plasma membrane. During phagocytosis, "zippering" of the membrane occurs. The membrane surrounds the particle so that it is located within a vacuole called a phagosome. This vacuole fuses with another lysosome to form the phagolysosome. Once this fusion vacuole is formed, killing and digestion can occur. Phagocytosis is frequently used as a measure of immune function. Phagocytosis can be studied using both in vivo and in vitro assays (Lanzavecchia, 1990; Lanier 1991).
Chemiluminescence The production and intracellular release of reactive oxygen intermediate species is a major microbicidal mechanism employed by monocytes and macrophages. Thus, the interaction of certain substances with their appropriate receptors on the plasma membrane of a M0 or monocyte results in a coordinated series of biochemical events known as the respiratory burst (RB). This results in a dramatic increase in the consumption of oxygen and the activation of a membrane associated oxidase. This enzyme reduces molecular oxygen to superoxide anion. The latter dismutates spontaneously to H2O2. Superoxide and H2O2 can interact to give rise to hydroxy] radicals and singlet oxygen which are highly cytotoxic. Because of the sharp increase in the oxygen uptake, this series of changes has come to be known as the "respiratory burst," although its purpose is to generate cytotoxic agents rather than to produce energy. Phagocytosis is usually coupled to the RB. Phagocytosis by M0 is therefore usually accompanied by an increase in the production of 02" which spontaneously dismutates to O2 and H2O2. Myeloperoxidase is present in a subpopulation of cytoplasmic granules (azurophilic or primary granules) and is discharged into the phagosome by the degranulation process following particle ingestion. Myelq>eroxidase reacts with a halide and H2O2, generated by the respiratory burst, to form the cytotoxic triad or microbicidal system. Although H2O2 can react with chloride, bromide, iodide and the pseudohalide
thiocyanate, the physiologic halide is chloride, which is oxidized to hypochlorous acid/ hypochlorite (HOCIAXH')- Hypochlorous acid, at acid pH, can react with excess chloride to form chlorine. These are powerful oxidants which are highly toxic to microorganisms. Hypochlorous acid is also known toreactwith certainreactivenitrogen intermediates, such as nitric oxide, to form monochloramines and dichloramines withretentionof some oxidizing activity and toxicity (Hibbs, 1991). Macrophages, as weU as other phagocytic cells, emit light while ingesting organisms or particles (Allen and Loose, 1976). The exact light emitting specie(s) of chemiluminescence (CL) has/have not yet been identified. However, both superoxide and H2O2 have beenreportedtoreactpreferentially with luminol (Wilhehn and Vladmir, 1986). The H2O2 formed oxidizes luminol (5-amino-2,3 dihydro-l,4-phthalazinedione) which amplifies the light emitted Thus an increase in the phagocytic activity of M0, inresponseto the addition of a stimulant, can be measured by determining the increase in chemiluminescence in the presence of luminol. Engagement of Fc receptors, complement receptors, receptors for mannose terminal glycoproteins, or exposure to the phorbol ester, PMA, can stimulate the oxidative burst (Johnston, 1981). Activation of the oxidase by intact cells is complex and incompletely understood; requirements differ to some degree with the nature of the stimulus. Stimulation is generally, but not always, initiated by the binding of the stimulant to specific highaffinity receptors on the surface of the cell. Examples include receptors for the Fcregionof immunoglobulins and for complement products (C3b, C3bi) which recognize ligands on opsonized particles, f-met-leu-phe receptor, etc. The secretion of the reactive oxygen intermediates play an extremely importantrolein numerous cytotoxic reactions of M0. The processes that control phagocytosis and the RB may be closelyrelatedand interdependent (Grebner ct al., 1977: Bryant and Hill, 1981; Easmon et al., 1980). In addition, the importance of the oxidative burst is exemplified byreportsof depressed CL by M0 fix)m
patients with chronic granulomatous disease (Stjemholm et al., 1973; Cumutte et al., 1974).
Macrophage activation: Macrophage-mediated cvtotoxicitv (MMC^ The study of M0 activation began with the dissection of acquired cellularresistanceto facultative or obligate intracellular parasites by George Mackaness and his colleagues (Adams and Hamilton, 1984). The concept of macrophage activation was subsequendy broadened when a number of workers, including Hibbs and Remington, and Evans and Alexander, observed that M0 activated for non-specific hostresistanceto parasites quite effectively and selectively destroyed neoplastic cells in culture (Adams and Hamilton, 1992). Macrophage activation is a complex phenomenonresultingin the ability of these cells to destroy aberrant or cancer cells. Acquisition of the competence to destroy neoplastic cells in the absence of antibody is known as macrophage-mediated cytotoxicity (MMC). It is believed that induction of such a function occurs through a series of steps, each one of which raises the M0 to a higher level of function (Somers, Johnson and Adams, 1986) Only M0 raised to the highest level are capable of killing the appropriate target cells. Inflammation may be the first signal which starts the process and produces the "inflammatory" or "responsive" M0. This is frequentiy followed by stimulation with a cytokine which raises the M0 to a higher level, the "primed" M0, which may be subsequendy triggered by exposure to trace amounts of endotoxin. The ability of the M0 to remain at the highest level of activation, the "activated" M0, lasts for hours. Nanogram levels of lipopolysaccharide can enhance tumor cell killing (Balkwill and Burke, 1989). If any interruption in the activation sequence occurs these cells will be unable to kill. Macrophages are capable of killing target cells by a number of mechanisms after their activation by cytokines, including interferon y or following exposure to high levels of
lipopolysacharride (LPS). Among the products of M0 involved in killing are reactive oxygen intermediates (ROI),reactivenitrogen intermediates (RNI), cytolytic protease, and tumor necrosis factor (TNF)(Adams and Marino, 1984; Weaver and Unanue, 1991). Because of the complex series of steps associated with activation, this phenomenon represents a logical target to study effects of cocaine on M0 function.
Mechanisms: Reactive nitrogen intermediates One of the major methods of killing by activated M0 is through the production of reactive nitrogen intermediates (RNI) (Hibbs, Tainter, and Vavrin, 1987; Keller and Keist, 1989). Activation of M0resultsin the conversion of arginine to RNI. The most active of these is nitric oxide which is highly toxic to bacteria, fiingi, and mammalian cells (Hibbs et al., 1987). Nitric oxide is subsequendy rapidly converted to NO2" and NO3". Because of the importance of these recentiy recognized mediators in M0 activation and target cell killing, studies were initiated to determine the presence of NO2" following exposure of mice to cocaine. Nitric oxide (NO), one of the nK)st important of the RNI, is currentiy thought to be a major M0 product involved in cell killing. In addition, NO plays other important roles in the body as a neurotransmitter and as a vasodilator and physiologic mediator of blood vesselrelaxation(Snyder, 1992).
Tumor necrosis factor TuiiK)r necrosis factor is a cytokine secreted by M0. At low concentrations, TNF acts locally as a paracrine and autocrineregulatorof leukocytes and endothelial cells. At higher concentrations, TNF is an endogenous pyrogen involved in systemic reactions, where it can act as an endocrine hormone affecting numerous physiologic functions. Activated M0 secrete tumor necrosis factor-alpha inresponseto an appropriate stimulus, such as lipopolysaccharide, phorbol myristate acetate, muramyl dipeptide, bacillus Calmette
8 Guerin, or a variety of peroxidative enzymes (Lefkowitz et al., 1988; Jue et al., 1990; Oppenheim et al., 1991; Lefkowitz et al., 1992). Tumor necrosis factor may be bound to the plasma membrane of the activated M0 and/or secreted into the extracellular environment (Decker et al., 1987). This factor is involved in the destruction of tumor cells as well as immunomodulation of the immune system (Asher et al., 1987; Beutier and Cerami, 1989; Kelso, 1989). These range from co-mitogenic influences on T lymphocytes to enhance major histocompatibility complex antigen and interleukin-2 receptor expression, to enhancing neutrophil chemotaxis and activation of the neutrophilrespiratoryburst and degranulation. Direct tumor cell cytotoxicity is also observed with this cytokine (Wilson et al., 1989). Tumor necrosis factor is an endogenous pyrogen (i.e., cachectin) which induces a febrileresponseupon dissemination to aid immunologicresponsesto invasive agents, and is known to enhance lipolysis and mediate endotoxic shock (Cerami and Beutier, 1988).
Macrophage receptors/ surface proteins Intercellular and intracellular communication is vital for development and function of multicellular organisms. An understanding of various cell surface receptors is essential for the determination of mechanisms involved in inflammation and immuneresponses.The presence and density of these receptors mayreflecteither normality or an altered pathological state. A number of different receptors have been defined on cells of the mononuclear phagocyte system. These receptors include those involved in signal transduction, cell adhesion, and cell differentiation. The receptor-ligand interactions of these molecules have been implicated in altering chemotaxis and phagocytosis. Cell-ceU adhesion receptors, such as Mac-1, also function as important transducers of information to the interior of the cell, influencing cell differentiation and proliferation. Among the receptors involved in signal transduction are the cytokine receptors. These glycoproteins
interact with TNF, IFN, IL-1, etc. After receptor-ligand interaction and internalization of the complex, signals are transduced which alter the behavior of therespondingcell (i.e., macrophage spreading, phagocytosis, secretion of cytokines). F4I80: F4/80 is a surface marker used to identify mature macrophages. The F4/80 antigen is a single chain glycoprotein which displays microheterogeneity on Western blot analysis of lysates prepared from various tissues and cells. Its function is not known at the present time. Expression of F4/80 by peritoneal macrophages is down-regulated by inflammatory stimuli, which induce the recruitment of immature cells, by short term adhesion in ceU culture and by exposure to lymphokines, especially interferon-y (Gordon et aL, 1986). Mac-1: Mac-1 (CDl lb/CD 18) is a receptor, found on all nx)use M0, which binds a multitude of ligands (Larson and Springer, 1990). The binding of Mac-1 to one of its ligands, iC3b, suggests that Mac-1 is involved in both attachment and phagocytosis of iC3b coated particles (Ding et al.,1987). It has been observed that antibody to Mac-1 can affect M0 by: (1) inhibiting IgG coated particle phagocytosis, (2) inhibiting Fc receptor mediated phagocytosis, (3) increasing expression of la and (4) mimicking the effect of certain cytokines by causing M0 activation. However, it should be noted that quantitative differences in these receptors may not be sufficient for a particular cellular event to be initiated (Kishimoto et al., 1989). It is clear that the integrins such as Mac-1 play a major role in various host defenses. Since Mac-1 is involved with phagocytosis, the possibility exists that this integrin may be altered following cocaine administration. Fc Receptors: Fc receptors on M0 participate in the selective binding and ingestion of bacteria, viruses, and foreign cells. In addition to theirrolein phagocytosis, Fc receptors are important in antibody-dependent cell-mediated cytotoxicity (ADCC). Engagement of the receptors induces secretion of superoxide anion and various enzymes which play arolein die host defense system. Murine FcRl (IgG2a), FcR2 (IgGi and IgG2b) and FcR3 (IgG3)
10 have been identified. Drug-induced suppression of these receptors couldresultin an aberrant immune response. Mannose-fucose Receptor (MFR): Accumulating evidence indicates that various humoral and cellular immunereactionsareregulatedby specific cellular interactions involving the carbohydrate portion of surface glycoconjugates on immunologically competent cells (Gabius, 1987). It has been proposed that a primitive immune recognition system based on carbohydrate-specific interactions may operate in mammals and possibly lower vertebrates (Stahl, 1990). The mannose receptor (MFR) is important in botii molecular scavenging and host defenses (Stahl, 1990). This receptor mediates the recognition and presumed killing of microorganisms coated with mannose, glucose, and similar sugars (Ezekowitz, 1989). Theroleof the mannose receptor is inferred in a number of immunological activities including opsonization, monocyte cytotoxicity, antigen-induced T-lymphocyte proliferation and adhesive interactions involved in lymphocyte recirculation (Kuhlman et al., 1989; Stutman et al.,1980; Monsigney et al., 1983). In addition, it has been suggested that binding to the MFR results not only in thereleaseof numerous cytokines but mayresultin activation of M0 to the cytotoxic state (Lefkowitz et al., 1989, 1991). Further justification to study the MFR comes from earlierreportsthat inhalation of certain anesthetic agentsresultsin impaired lymphocyte reactivity, granulocyte function and bone marrow depression under both in vivo and in vitro conditions (Stevenson et al., 1986; Nunn et al., 1986). Recent studies by Bardosi et al. (1990) suggest that this depression may be caused by reduction of the MFR. The "snorting" of cocaine could parallel this exposure to anesthetic agents. Finally, uptake of Pneumocystis carinii, which causes the fatal pneumonia associated with AIDS, may be mediated by the MFR (Ezekowitz et al., 1991). la Expression: The role of M0 as antigen presenting cells is well-documented. After processing the antigen, the M0 presents an antigen-la complex to a T helper cell. Increased
11 la expression has been correlated with enhanced M0 function and/or activation. An alteration in the number of cells expressing la or the amount of la expressed on individual cells could alter antigen presentation to the T helper which could ultimately affect the immune response.
Cocaine Brief history of cocaine Cocaine has been praised as a cure-all, as well as cursed as a scourge to humanity at various times in history. Cocaine has been documented to have been used as early as 500 A.D. A grave in Peru was found to contain cocaine, as well as other things, for the occupant to take into the "afterlife." During the 16th century, the Spanish conquistadors in South America noted the use of coca leaves by the Peruvians working at high altitudes. The plantreachedthe European continent around 1750. Cocaine was first isolated and purified from coca leaves in 1859 by Albert Niemann, a German chemist. During the 19th century, cocaine was being tried as a panacea for a host of illnesses. It was recommended for toothaches, digestive disorders, nervous exhaustion, syphilis, asthma, hayfever, and postnasal catarrh (Musto, 1991; Kleber, 1988). Cocaine became available to the general public through John Styth Pemberton's formulation in 1885. He combined coca with kola nut, aromatized it, added syrup and therewith gave birth to Coca-Cola, "the brain tonic and intellectual soda-fountain beverage." Cocaine was also tried as aremedyfor opiate and alcohol addiction at this time (Benowitz, 1993). Cocaine was first used as a local anesthetic during surgery by Karl Koller in 1884. It was soon used for surgery of the eye, ear, nose and throat as well as a nerve block for hemia operations and amputations. It was not until the late 1800s that negative experiences with cocaine use, began beingreported.As aresultof excessive use and negative outcome, legislation was initiated to control cocaine. The Pure Food and Drug Act of 1906 attempted
12 to limit the use of cocaine for medical purposes alone. The Harrison Narcotic Act of 1906 labeled cocaine as a narcotic. Finally, the Contolled Substances Act of 1970 was the first act in which the illegal possession of cocaine was made a Federal crime. Cocaine abuseremainedat a low level until the 1960s and the early 1970s when it reemerged among American youth. At first, the users were affluent (entertainers, sports figures and business people), and cocaine was viewed as a costiy party drug, promoting conviviality and good spirits, and considered to be safe. Cocaine was taken at this time primarily by nasal sniffing of the hydrochloride salt Presumably because ofrelativelylow doses and/or slow rate of absorption via the nasal route, serious toxicity was uncommon. In the mid-1980s cocaine became widely available in another form, namely chunks of alkaloidal cocaine called crack (for the cracking noise made when the chunks are heated). In the United States at present, crack cocaine is plentiful,relativelyinexpensive, and is commonly smoked, often in combination with injecting heroin and/or drinking alcohol. At the present time, cocaine abuse appears to be declining in the U.S., at least among less frequent users (i.e., middle class).
Routes of administration Cocaine is an alkaloid derived from the leaves of Erythroxyion coca. It is readily absorbed through mucous membranes of the lungs and of the gastrointestinal tract Cocaine is most commonly administered via "sncMting" or by smoking thefreebaseform in "rocks" known as crack. Variousroutesof administration have been tried by the cocaine user, and each produces its own characteristic absorption pattern and associated problems. Oral (chewing leaves). The natives of South America chew coca leaves by mixing them with alkali (ashes). Low concentrations of cocaine are obtained in the blood, which arc only half that obtained by snorting.
13 Nasal inhalation (snorting). Cocaine in the hydrochloride salt form is readily absorbed from the nasal mucosa. Ischemic nasal damage is common in cocaine snorters. Intravenous administration. The "high" appears 15 to 20 seconds after i.v. injection and lasts about 20 minutes. This is not a popular method of administration because of problems associated witii needles such as hepatitis and AIDS. Intraperitoneal. This is the preferredrouteof use in experimental animals, especially in rodents, as evidenced by a survey of the literature. Smoking (freebasing). Alkaloidal cocaine, but not the salt, is volatile and so can be smoked. Because of the intense high and ease of self-administration, methods have been devised to free the cocaine from its hydrochloride salt. "Crack" is a form of freebase cocaine processed from the salt using baking soda (sodium bicarbonate).
Metabolism of cocaine Cocaine is rapidly and extensively metabolized with less than 10% excreted unchanged in the urine. The majority of cocaine is hydrolyzed by plasma pseudocholinesterases (PCh) or liver esterases into ecgonine, ecgonine methyl ester and benzoylecgonine. In humans, the distribution of cocaine metabolites after 12 hr is benzoylecgonine, 46%; and ecgonine methyl ester, 41%. These metabolites arerelativelyinactive and are excreted in the urine where they can be readily detected (Stewart et al., 1979). Regardless of therouteof admiiustration, within 24 hours after a single dose, cocaine is undetectable in the blood (Ambre, 1985). A minor pathway of cocaine metabolism mayresultin the production of a number of highlyreactivetoxic intermediates. This pathway involves cytochrome P-450 metabolism in the liver andresultsin the production of highly reactive con^xmnds including norcocaine, N-hydroxynorcaine, and norcocaine nitroxide. A hepatotoxin is formed from norcocaine either through A/-hydroxylation to N-hyroxynorcocaine or through oxidation to
14 norcocaine nitroxide (Thompson, Shuster and Shaw, 1990). These reactive products result in extensive hepatic damage in rodents (Roth et al., 1992; Holsapple, 1992). Norcocaine and benzoylecgonine have also been shown to have neurotoxic effects (Williams, 1992). Norcocaine has properties of a local anesthetic, inhibits norepinephrine uptake, and alters behavior in humans. The following diagram explains the two metabolic pathways of cocaine. Minor Pathway cyt ^^ . norcocaine "^ cocame P450 Major pathway PCh i^v.11 ecgonine ^ * ecgonine methyl ester benzoylecgonine ^
The plasma clearance of cocaine averages about 20-30 ml'l.min"l.kg"l, and the halflife (after intravenous dosing) averages 60-90 min (Jeffcoat et al., 1989; Ambre et al., 1988). Benzoylecgonine and ecgonine methyl ester, the major metabolites of cocaine have average half-lives of 7.5 and 3.6 hr,respectively(Ambre, 1985). Cocaine and norcocaine are highly lipophilic, accounting for a rapid crossing of the blood-brain barrier, benzoylecgonine and ecgonine are highly polar and do not significantly cross this barrier. These two latter metabolites, therefore, are excreted in the urine (in amounts equivalent to one-quarter to one-half of the original dose of cocaine) within 24 to 36 hours. Depending on urine acidity (greater excretion with lower pH), from 9.5 to 20 percent of cocaine is excreted unchanged in the urine. Cocaine can also be detected in the bile.
Effects of cocaine Pharmacological effects Cocaine has two major pharmacological effects. It is (1) a local anesthetic and (2) an indirect acting sympathomimetic which potentiy enhances the effect of neural transmission. Cocaine prevents conduction of sensory impulses by interfering with the neuron membrane to block ion channels. As aresultof this block, the ion exchange which is normally
15 responsible for the electric signals cannot be propagated along the axon and therefore sensory impulses are not received in the central nervous system. The second major pharmacological effect of cocaine is its ability to potentiate neurotransmission in neurons that use one of the the monoamines, norepinephrine (NE), dopamine (DA), or serotonin (5-HT), as a neurotransmitter. In the periphery this occurs mainly at the noradrenergic terminals of the sympathetic component of the autonomic nervous system, and in the brain at monoaminergic terminals (i.e., those that use NE, DA, or 5-HT), for which there are manyfibernetworks. Neurons normally transmit or modulate electrical signals across the synaptic gap byreleasinga chemical which induces a change in ion permeability in the receivingtissue.This allows further conduction to take place. Normally, monoamine neurons terminate the transmission process and then recycle the transmitter by actively taking the chemical back up into the terminal from which it was released. Wlien this process is blocked by cocaine, the effect of nervous system activity is greatiy enhanced In the peripheral nervous system, one of the effects of sympathetic nervous system activation is vasoconstriction. The effects of cocaine on the nervous system are dosedependent. At low doses, cocaine increases arousal and motor activity. At moderate doses, heart rate increases. Hypertensionresultsfrom the increased peripheralresistance(due to vasoconstriction) in addition to the increase in the heart rate. Body temperature is increased and dilatation of the pupils may occur as well. At high doses, cocaine can induce convulsions. Cardiac arrest is not uncommon due to a direct action of the drug on the heart muscle.
Otiier adverse effects of cocaine Cocaine causes an intense "high" which is the reason for its abuse. There is an intense rush of feelings of energy, power and competency. It soon subsides and is replaced by a
16 phase of restiess irritability. During the phase of direct cocaine action, there is autonomic and central activation such as tachycardia. Cardiac arrhythmias are not uncommon at high doses. On prolonged use of cocaine, there is considerable weight loss due to an anorectic action. Cocaine "bugs" or "formication" can occur in which the user feels like there are insects crawling under the skin. Long term use can also produce cocaine psychosis. This manifests in the form of paranoia. Manic behavior is expressed as hyperactivity and distractibility. Delirium which also may develop, includes disorientation and confusion. Liver damage has been found in cocaine addicts. Cocaine is metabolized via the cytochrome P450 to the hepatotoxic intermediate norcocaine. A hepatotoxin is formed from norcocaine either through A^-hydroxylation to N-hydroxynorcocaine or through oxidation to norcocaine nitroxide (Thompson, Shuster and Shaw, 1990). Pulmonary damage may result through ischemic injury. Cocaine is also known to be teratogenic. Cocaine use can cause a high incidence of miscarriage and behavioral deficits in those infants normally delivered. Infants bom to mothers who admitted using cocaine showed a reduced ability to interact with environmental stimuli (Chasnoff et al., 1985), Infants bom to cocaine abusing mothers are at considerably higherriskfor growthretardation,lower birth weight, neurobehavioral impairment and various congenital malformations (VanDette and Comish, 1989).
Previous studies on the effects of cocaine on the immune system The effects of cocaine on immunity has beenreviewedrecentiy (Watson, 1990). For the most part, many of the frequentiy abused drugs, under specific conditions, are capable of interfering with immuneresponses.Certain of these drugs affect the immune system through a stress-induced endocrine alteration. Regardless of the stress, the common pathways are: stimulation of adrenocortical secretion, with consequent increases in serum
17 glucocorticoids and activation of the sympathetic nervous system, followed by areleaseof catecholamines. The interactions of the immune system with various neurotransmitters and hormones has only recentiy been recognized The effects of cocaine and its metabolites on various neurotransmitters are also known. These studies have not lead to any consistent conclusionsregardingthe effects of cocaine on the immune system. Under certain conditions, cocaine has been immunosuppressive; and under other conditions, immunoenhancing. The effects of cocaine on lymphoid cells have been investigated. Studies by Watson et al. (1983) indicated that cocaine was immunosuppressive when administered to mice. Using levels of 15-60 mg/kg, inhibition of both plaque-forming cells and delayed hypersensitivity using DNFB was apparent Exposure of mice to 30 or 60 mg/kg cocaine (Faith and Valentine, 1983) decreased both spleen and thymus weight and suppressed both theresponsivenessof B cells to LPS and the antibody response to sheep erythrocytes. In this same study, cocaine also increased responsiveness to mitogens and delayed hypersensitivity. Havas et al. (1987) showed that high doses of cocaine (50 mg/kg) administered 3timesdaily had no effect on antibodies to pneumococcal polysaccharide but resulted in a 2-fold increase of anti-DNP plaque-fOTming cells in mice. When 5 mg/kg cocaine were administered by i.m. injection 24 hr prior to assay, phagocytic activity of peritoneal M0 was decreased about 75% (Ou et al., 1989). This was also accompanied by a decrease in both the number of thymocytes and the number of leukocytes. These investigators showed that cocaine caused a reduction in the number of plaque-forming cells. Using Fisher rats injected witii 1.25-2.5 mg/kg, Bagasra and Forman (1989) reported an increase in the ability of animals to mountresponsesto both T dependent and T independent antigens. At higher concentrations (5 mg/kg) these reactions were depressed These investigators noted a significant increase in B cell numbers in cocaine treated animals. Cocaine augments proliferation of human T-lymphocytes when the cells were
18 activated through the T-cell receptor complex by increasing Ca^* mobilization and subsequent IL-2 production (Matsui, Friedman and Klein, 1992). Elevated serum levels of IgG is one of the many immunological abnormalities exhibited in intravenous drug abusers. However, a recent report has shown that cocaine does not affect B cellfiinctionin vitro, suggesting that cocaine in vivo may exert its immunomodulatory effects via indirect mechanisms (Martinez and Watson, 1990). Chronic administration of cocaine to laboratory miceresultsin a reduction of immunocompetence and increased lethality to Friend leukemia vims (Starec et al., 1991). Very few studies have been done to investigate the effects of cocaine on phagocytic cells, such as neutrophils and macrophages. Ou et al. (1989) reported that 5 mg/kg cocaine administered by i.m. injection, 24 hr prior to assay, decreased phagocytic activity of murine peritoneal M0 by about 75% as noted earlier. Recent studies have shown that transforming growth factor-B (TGF-6), a cytokine which inhibits various cell functions, was induced in human monocyte cultures exposed to cocaine (Chao et al., 1991). In these studies TGF-B also interfered with superoxide production which may be involved in cell killing. Of interest is the fact that this suppression was naloxone sensitive. Recent studies by Peterson et al. (1991) suggested that cocaine may increase human immunodeficiency vims (HIV) replication by human peripheral blood mononuclear cells in vitro through the prxxiuction of transforming growth factor B (TGF-B). Cocaine and its derivatives have beenreportedto inhibit neutrophil functions including superoxide induction and cell surface receptor expression (Haines et al., 1990). Studies by Peterson et al. (1987) indicated that both morphine and B-endorphin inhibit the respiratory burst of human peripheral mononuclear cells stimulated with opsonized zymosan and phorbol myristate. Isolated cellsfrompatients on methadone also had an impaired capacity for generation of superoxide (Peterson et al., 1989). Watson et al. (1993)
19 havereportedthat cocaine, in vitro, inhibited production of tumor necrosis factor by M0 from uninfected andretrovirallyinfected mice. In vitro studies also do not give a clear picture of the effect of cocaine on immune cells. Studies of leukocyte migration in vitro, underscore the enonnous complexity associated with determinations using cultured cells (Mason and Van Epps, 1989; Van Epps et al., 1991). Significant but reversible inhibition of human peripheral blood mononuclear and NK cell activity occurred only after very high exposures to cocaine (Welch, 1983). Another study utilizing NK ceU activity also showed that plasma levels following cocaine use had no effect in vitro (Van Dyke et al., 1986). In contrast 100 M-g/ml of cocaine inhibited blastogenesis of lymphocytes to the T cell mitogen concanavalin A by 26% and 200 |ig/ml inhibited it by 73% (Klein et al., 1988). Human lymphocyte subsets exposed to high concentrations of cocaine were not affected (Bagasra and Forman, 1989). In vitro cytotoxicity mediated by natural killer and cytotoxic T-lymphocytes was not affected by cocaine levels similar to those observed in vivo (Lu and Ou, 1989). Differences reported by the various investigators could be explained by differences in dosages, routes, and schedules of administration. Studies of the effects of cocaine on immuneresponsesin man are limited. Plasma levels of cocaine in man rangefix)m0.1-1 ^.g/ml. These concentrations decline rapidly because cocaine has a one hour biological half-life. Van Dyke et al. (1986) reported that a single intravenous injection of 0.6 mg/kg resulted in an increase in NK cell activity. They attributed this activity to potentiation of endogenous catecholamines. Another study by Donahoe et al. (1986) demonstrated that cocainereversedthe E-rosette depression by heroin. Cocaine abuse is also associated with an increase the level of neopterin (Guynn et al., 1986). Knowledge of the effects of cocaine on the immune responses of man are virtually unknown.
20 A review of the literature shows that cocaine can be immunosuppressive as well as immunoenhancing under different circumstances. The studies described above and others, taken in their entirety, suggest that low levels of cocaine could be stimulatory to the immune system. Higher levels are probably inhibitory to the immune system. Furthermore most of these studies utilized exposure schedules of 2 weeks or less. There are no studies utilizing chronic exposure to cocaine in order to measure immunosuppressive effects. Continuous exposure may be required to demonstrate definite immunomodulatory effects of this substance. Most of the studies have been done using different species of mice or with human subjects which might explain conflictingresultsobtained. Also, very littie is known about the effects of cocaine on macrophages and their various functions.
The objectives of the current research effort The major hypothesis of this study was that cocaine administration would result in marked effects on immuneresponsiveness,especially macrophage (M0) functions. Cocaine is metabolized in the body to toxic and non-toxic metabolites via two distinct pathways. It was important to understand whether the effects of cocaine on M0 functions occur through the production of certain metabolites. Oxaine or its metabolites were administered to C57BL/6 mice via the intraperitonealrouteand its effect was studied on macrophage phagocytosis and activation to the cytotoxic state. Phagocytosis was studied using in vivo and in vitro assays. Since therespiratoryburst usually correlates closely with phagocytic activity; it was investigated to understand the mechanisms by which cocaine might produce changes in phagocytosis. Macrophage activation was studied by measuring their ability to kill specific target cells (macrophagemediated cytotoxicity). Some of the known mechanisms through which macrophagemediated cytotoxicity (MMC) occurs are via the production of reactive oxygen (ROI) and reactive nitrogen intermediates (RNI). These were studied to obtain a better understanding
21 of pathways involved in alteration of the MMC. Surface receptors and proteins involved in phagocytosis and macrophage activation such as Mac-1, Fc receptor, mannose-fucose receptor, and la were also studied using flow cytometry. Figure 1representsa scheme which summarizes the studies described in this dissertation. It was hoped that these studies would delineate some of the effects of cocaine on macrophage functions and elucidate the mechanisms and pathways involved.
Cocaine
Cocaine Metabolites
MACROPHAGE Phagocytosis
Respiratory Burst Reactive Oxygen Receptors: Fc, MFR, Mac-1
Cytotoxicity
Reactive Intermediates Nitrogen / Oxygen Receptors: la, Mac-1
Figure 1. Scheme describing studies done in dissertation.
rssi
CHAPTER n MATERIALS AND METHODS
Experimental animals Male or female, 12-16-week-old C57BL/6 mice were purchased from Jackson Laboratories, Bar Harbor, ME. All mice were housed in the Texas Tech University Health Sciences Center (TTUHSC) vivarium or in the facilities at the Biological Sciences Building of the Texas Tech University. The care and use of these animals conformed to the policies andregulationsof the TTUHSC Institutional Animal Care and Use Committee as approved under Protocol No. 90016-03.
Cells andtissueculture media Media used for the studies were Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO, Long Island, NY), Auto-Pow EMEM without phenol (Flow Lab Inc., McLean, VA) or RPMI1640 (GIBCO, Long Island, NY). DMEM and RPMI were supplemented with 10% fetal bovine serum (FBS) (Intergen, l^fY), 25 mM N-2-hydroxyetiiyl-piperazineN'-2-etiiane-sulfonic acid (HEPES) (Research Organics, Cleveland, OH) and 50 mg/1 gentamicin sulfate (U.S. Biochemicals, Cleveland OH). These were designated as complete media. Macrophages were cultured for most of tiie studies in complete DMEM at 37^0 under 5% C02. For studying chemiluminescence and the production of reactive nitrogen intermediates, Auto-Pow EMEM witiiout phenol was used The tumor necrosis factor resistant, P815 mastocytoma cell line and the tunwr necrosis factor sensitive, WEHI 164 fibrosarcoma cells were obtainedfix>mAmerican Type Culture Collection (ATCC), Rockville, Maryland The P815 cells were cultured in complete DMEM and the WEHI 164 cells were grown using complete RPMI.
22
23
Dmgs Cocaine hydrochloride, norcocaine, benzoylecgonine, ecgonine methyl ester hydrochloride and ecgonine hydrochloride were obtained from National Institute of Dmg Abuse (NIDA). All the above were dissolved in phosphate buffered saline (PBS), at pH 7.2, and injected intraperitoneally utilizing various schedules. Otherroutes,including intravenous, intramuscular and inhalation were used for a limited number of studies.
Collection ofresidentperitoneal macrophages from C57BL/6 mice Methods used to collect peritoneal M0 have beenreportedpreviously by Lefkowitz (1987) and are described briefly as follows: Mice were sacrificed by cervical dislocation. With the ventral side up, the skin was swabbed with 70% alcohol, cut along the midline and removed. Each animal was injected intraperitoneally with 8 ml of cold PBS at pH 7.2. The abdomen was gentiy massaged for 1 min, and the fluid slowly withdrawn. Cells were centrifiiged at 150 X g for 10 min at 5C, counted using a hemocytometer, and resuspended at the required concentration in the appropriate medium. Efforts were made to remove the peritoneal cells with a minimum of erythrocyte contamination because of interference with both macrophage activity and chemiluminescence. If contamination occurred, erythrocytes were lysed with 0.83% ammonium sulfate. Cells were kept on ice until utilized
Collection ofresidentmurine alveolar macrophages The alveolar M0 were collected from 12-16 week old C57BL/6 mice using methods described previously by Holt in 1989. Mice were sacrificed by a lethal 0.5 ml ip injection of 130 mg/ ml phenobarbital (EUdns-Sinn Inc., Cherry Hill, NJ). A lavage tube (O.D. < 1 mm) was carefully inserted through a small incision in the trachea. Two 10 ml syringes
24 were inter-connected through a three-way stopcock. The lungs were filled witii 0.5 ml PBS containing 40 mg/ml Xylocaine (Astra, Westborough, MA) and the chest was massaged gentiy and fluid withdrawn into the other syringe by changing the direction of the stopcock. This process wasrepeateduntil the lungs were lavaged with 10 ml of media. Approximately, 2 X 105 cells were obtained per animal. The cells were kept on ice until utilized.
Measurement ofrespiratoryburst by chemiluminescence Materials Luminol (5-amino-2,3-dihydro-l,4-phthalazinedione), BSA essentially globulin free, and zymosan were obtained from Sigma, St Louis, MO. A Turner luminometer Model 20e (Tumer Designs, Mountainview, CA) with a temperature controlled counting chamber was utilized.
Zymosan solution The following procedure was cairied out under aseptic conditions. Ten mg/ml of zymosan were suspended in water and boiled for 30 to 45 min. After cooling, the suspension was centrifuged at 250 x g for 10 min. The pellet wasresuspendedin equal amounts of PBS and guinea pig complement (GIBCO, Long Island, NY). After incubation at room temperature for 30 min, the suspension was centrifuged and the pellet resuspended in equal amounts of complement and PBS as before. The zymosan suspension was incubated at room temperature for an additional 30 min, then centrifuged. The supematant was discarded and 10 ml PBS was added to die pellet. The solution was vortexed aiui centrifuged. This procedure wasrepeatedonce. After the last washing, the pellet was resuspended in PBS at a concentration of 1 mg/ml. Aliquots of the zymosan solution were
25 kept frozen at - 7 0 ^ . At this temperature, the opsonized zymosan was stable for at least one year.
Luminol solution One mg each of luminol and crytallized globulin-free BSA per ml of PBS was mixed in a beaker. The mixture was stirred for 10 min, and then filtered using a 0.2 ^imfilter.The filtrate was aliquoted and stored at 4^ in a covered container to prevent exposure to light. This solution was stable for at least four months.
Assay procedure The chemiluminescence (CL) medium was prepared using Auto-Pow EMEM without phenol (Flow Lab Inc., McLean, VA) containing bovine semm albumin (essentially globulin-free) 1.0 g/dl (Sigma, St. Louis, MO); HEPES 0.6 g/dl (U.S. Biochemicals, Cleveland, OH); and sodium bicarbonate 0.2 g/dl (Sigma). M0 were suspended in this medium at 1 X lO^/ml for the peritoneal cells and 2.5 X 105/ml for the alveolar cells. Exactiy 100 |il of cell suspension were added to each 8 x 50 mm tube (Evergreen Scientific, Los Angeles, CA). After incubation for one hr at 31^C under 5% CO2, the cell monolayers were washed and 100 |il of fi^sh media were added. After incubation for another 30 minutes, the media were discarded and the monolayers washed 3 times with 1(X) |il of media. Exactiy, 100 p.] of zymosan (Sigma) opsonized with guinea pig complement (GIBCO, Long Island, NY) and 30 ^il of luminol (Eastman Kodak, Rochester, NY) were added to the tubes and the amount of light measured using the luminometer at 3 7 ^ . The luminometer was programmed for 5readingsof 2 min each. Theresultswere recorded and plotted as time versus light emission.
26 Phagocytosis assay (in vitro) u.sing opsonized zymosan The method used was described by Greenberg, in 1991. Resident peritoneal M0 from cocaine treated mice or saline-injected controls, were obtained from C57BL/6 mice and adjusted to a concentration of 1 x lO^/ml. Cells were resuspended in DMEM (GIBCO) witii 10% fetal bovine semm (FBS) (Intergen, NY) and 100 ^1 of cells were added to each well of a 16-well Lab-Tek chamber slide (Nunc Inc., Naperville, IL). After a 2 hr incubation at 37^0 under 5% CO2, the cells were washed twice witii warm PBS. Exactiy 10 p.1 of opsonized zymosan and 90 ^1 of media were added to each well. After 30 minutes incubation, the slide was washed and fixed with 10% formalin. This was followed by staining for 30 minutes with 1 |ig/ml acridine orange (Sigma) in PBS at room temperature. The slide was then washed, blotted dry and mounted using glycerol and viewed at lOOOX using a microscope with UV light source. The ingested zymosan appeared as fluorescent particles surrounded by "black holes" within a blue staining cytoplasm. One hundred M0 were countedfromeach well and the percentage of M0 ingesting zymosan was recorded. Three wells were counted for each control and cocaine-treated animal. Figure 2 is a photomicrograph showing macrophages with ingested opsonized zymosan.
Phagocytosis assay (in vivo) using sheep erythrocytes The method was similar to thatreportedby Ou et al., 1989 and is briefly described as follows: Mice were injected i.p. with 5 mg/kg cocaine or saline. On the day of the assay, approximately 0.5 ml of 1% v/v sheep erythrocyte suspension (Micropure Medical Inc., Stillwater, MN) were injected i.p. into the mice. After thirty minutes, the mice were sacrificed by cervical dislocation and 2 ml of PBS were used to harvest the cells. Cells were washed with saline and cytospinned for 2 min, onto a sUde. Each slide was then stained using Wright's stain (Harleco, Gibbstown, NJ). A total of 300 M0 were counted per mouse. Percentage of phagocytosis was calculated as die percentage of M0 ingesting
27
m
l-iuuiv 2. PhoioniKToizraph of inacTi>ph;ii:cs wiih inticsicd t>ps(>iii/al /yniosan {in
28 erythrocytes. Figure 3 is a photomicrograph showing macrophages witii ingested shep erythrocytes.
Macrophage-mediated cytotoxicity assay Methods used were those of Russell, Pace and Varesio (1986), and are described as follows: Exactiy 4 hr prior to tiie assay, tiie TlVF-resistant P815 mastocytoma ortiieTNFsensitive WEHI 164fibrosarcomatarget cells, were centrifuged at 200g X 5 min and resuspended at a concentration of 10^ cells/0.7 ml DMEM. Exactiy 0.3 ml containing 300 ^iCi of 51Cr was added to the cells which was then incubated for 1 hr in a ?n^C waterbath. Uptake of 51 Cr by the target cells was terminated by diluting the cells to 50 ml with DMEM. Cells were gentiy mixed, without vortexing, and centrifuged at 200g X 10 min to getridof the excess 51Cr. The pellet wasresuspendedin 10 ml media and retumed to the waterbath at 3 7 ^ for another 1 hr. The cells were centrifuged, supematant was discarded and the resuspended cells adjusted to a concentration of 2 x 105/ ml. Resident peritoneal M0 were collectedfix)meither cocaine-exposed mice or saline injected controls. Cells were counted and adjusted to a concentration of 1 x 10^ / ml in Dulbecco's Minimal Essential Medium (DMEM) with 10% fetal bovine semm (FBS). Exactiy 200 |xl containing 2 X 10^ M0 were added per well in 96 well culture dishes (Costar) and allowed to adhere for 2 hours at 37^0 under 5% CO2. The cells were washed three times with DMEM and then 2.5 units/ ml of interferon y (IFNy) (obtained through courtesy of Dr. Sam Barron, Department of Microbiology, University of Texas Medical Branch, Galveston) combined with 10 ng/ml lipopolysaccharide (LPS) (Sigma, St Louis, MO) were added. Appropriate control wells of M0 without IFNy and LPS were included to measure spontaneous lysis of P815 or WEHI 164 target cells. After a 6 hr incubation, 100 ^il containing 2 X 10^ P815 target cells, which had been incubated previously witii 3(X) ^iCi 5^Cr (Dupont, Wilmington, DE) were added to each well.
29
^
Figure 3. Photomicrograph of macrophages with ingested sheep erytiirocytes (in vivo phagocytosis assay).
30 The plate containing the activated M0 and the target cells was incubated for 18 hr. Lysis of the target cells by activated M0resultedin the release of ^^Cr which was measured using a Packard gamma counter. Experimental release was calculated as the amount of 51Cr released from the target cells which were added to the IFNy and LPS treated M0. Spontaneousreleasewas obtained from the release of 51Cr from target cells added to the untreated M0. Total incorporation was obtainedfrom51cr incorporated target cells which were incubated with an equal amount of distilled water andfrozenat - 2 0 ^ overnight The cells were then altemately thawed in ethanol and frozen with dry ice for total release of ^^Cr from tiie target cells. The % specific release was calculated using the formula: % lysis = Experimental Release - Spontaneous Release Total Incorporation (freeze-thaw) - Spontaneous Release X 100.
Measurement of reactive nitrogen intermediates (RND Methods used for quantitating the amount of nitrites (N02') produced by macrophages were those described by Ding, Nathan and Stuehr (1988). Peritoneal M0fromcocaine treated or saline control mice were harvested using Auto-Pow MEM (Flow Lab Inc., McLean, VA) containing 10% fetal bovine semm (FBS). The cells were cultured at 2 X 10^ cells/ well in 96 well microtiter plates (Costar) at 37^0 under 5% CO2 to facilitate adherence. After 2 hr, cells were washed twice witii Auto-Pow MEM containing 10% FBS. Cells were then exposed to 2.5 units/ ml of interferon y combined witii 10 ng/ ml lipopolysaccharide (LPS) (Sigma, St. Louis, MO). Cells were maintained in tiie incubator overnight. After 22 hr, plates were centrifuged at 150g for 10 min. Exactiy 50 |il of culture supematants were harvested and mixed with an equal volume of freshly prepared Griess reagent (1% sulfanilamide/0.1% naphthyl etiiylene diamine dihydrochloride/ 2.5% phosphoric acid). The color change was read at 550 nm using a Bio-Tek El-312 microplate reader after 10 minutes. N-monomethyl-L-arginine (NMA) (Sigma), a competitive inhibitor
31 of arginine, was used to block thereactionthereby determining specificity. A dose dependent inhibition was obtained witii NMA intiierange of 15 ^iM to 1000 ^iM. Sodium nitrite (Sigma) was diluted serially and used as a standard Totalreactivenitrogen intermediate (RNI) production assay was done by Dr. Matthew Grisham, Department of Physiology, Louisiana State University Medical Center, Shreveport, LA. Total RNI assay included production of nitrites as well as nitrates by M0. Nitrates were converted to nitrites with the use of E. coli reductase to obtain total RNI; and nitrate production was calculated as the nitrite production subtracted from the total RNI.
Assay for tumor necrosis factor The method of Morgan et al. (1991) was used to demonstrate TNF-mediated cytotoxicity and calculate TNFtiters.Briefly, PRIMARIA 96-well flat-bottomed microtiter plates (Baxter Scientific, Grand Prairie, TX) were seeded with 2x10^ WEHI 164fibrosarcomacells in 1(X) jxl of RPMI supplemented with 10% FBS. The cells were incubated 4-6 hr at 37C under 5% CO2 to facilitate cell adherence. M0 supematants were diluted two-fold across the plate by the serial transfer of 100 |il of well mixed sample. Control wells received fresh RPMV 10% FBS or a TNF standard of known titer. Actinomycin D was added to all wells at a concentration of 1-2 |ig/ml in 100 |il to inhibit target cellreplicationand to ensure maximum assay sensitivity. The plates were then incubated for an additional 18.5 hr at 370C under 5% CO2. Neutral red dye (NRD) was prepared at a concentration of 0.033 g% in 6.7 mM phosphate buffer, pH 7.4, containing 100 mM sodium chloride, as described by Ruff and Giffoid (1981). The solution was gentiy swirled to enhance crystal dissolution and filtered by gravity flow through a double thickness of Whatman filter paper No.l. Seventy-five |il oftiiefilteredNRD solution were added to all wells,resultingin a final well volume of 275 ^il. The plates were then incubated for 1.5 hr at 3 7 ^ under 5% CO2, for a total incubation
32 time of 20 hr. The well contents were aspirated without disturbing the target cells and the monolayers were gentiy washed twice by the addition of 2(X) ^1 warm PBS, pH 7.4, followed by gentie aspiration. NRD taken up by surviving target cells was extracted by the addition of 100 |il of 50% absolute ethanol in 100 mM dibasic sodium phosphate followed by forceful repetitive pipetting. The optical absorbance at 550 nm of each well was measured with a BioTek El-312 microtiter plate reader. The absorbance values were then used to calculate tiie percentage of cytotoxicity attributed to TNF using the following formula: % cytotoxicity = I-IA550 pf Target ceUs exposed to TNF1 x 100. [A550 of Control cells] TNFtiterswere then calculated using two simultaneous equations of the forms y = ax + h , where y-% cytotoxicity above and below the theoretical 50% point, and ;c = the
reciprocd of the corresponding equations.
Direct immunofluorescence analysis by flow cytometry Antibodies and FACS reagents F4/80 rat monoclonal antibody to mouse macrophages: Fluorescein isothiocyanate conjugated (FTTC) was purchasedfromSerotec, Harlan Bioproducts for Science, Indianapolis, IN. Anti-Mac-1 mousefluoresceinconjugate, Anti la mouse phycoerythrin conjugate, Anti-Mac-1, and Anti la were obtainedfromBoehringer Mannheim Co, Indianapolis, IN. Mouse IgG2b FTTC, mouse IgG2b unlabelled, mouse IgG2a RPE and mouse IgG2a unlabelled were obtained from Southem Biotechnology Associates, Inc., Birmingham, AL. a-D-Mannosylated-FTTC-albumin (MBSA-FTTC) was obtained from Sigma, St. Louis, MO. Chrompure rat IgG Fc fragment (2.2 mg/ml), used as blocking antibody, was obtained from Jackson ImmunoResearch Lab Inc., Indianapolis, IN. Propidium iodide (PI), used for determining cell viability, was purchased from Sigma.
33
Macrophage labelling for direct immunofluorescence analysis by flow cvtometry Peritoneal M0 cells were collected fix)m C57B1V6 mice and suspended in PBS containing 1% BSA and 0.02% sodium azide (PBS/ 1% BSA/0.02% NaN3). Two milliliters of cell suspension at a concentration of 1 X 10^ cells/ml were aliquoted into polymetiiylpentene (PMP) jars (Nalgene, Rochester, NY). The jars were placed at 37^0 under 5% CO2 to facilitate cell adherence. After 20 minutes the cells were washed vigorously 3 to 4 times with PBS/ 1% BSA/ 0.02% NaN3 to getridof contaminating nonadherent cells. The jars were then kept on ice for another 20 minutes and thereafter vortexed to remove attached M0. The cells were resuspended in PBS/ 1% BSA/ 0.02% NaN3 at a concentration of 2.5-5 x 105 cells/ml. The cell suspensions were dispensed into sterile polypropylene microtubes (Biorad Laboratories) at a volume of 1 ml/tube. The microtubes were then centrifuged at 13(X) rpm for 6 min at 4^0 to pack the macrophages. The suspension medium was aspiratedfromall microtubes without disturbing the cell pellets. Ten |jJ of Chrompure rat IgG Fcfragment(2.2 mg/ml) was added prior to direct staining with Anti-Mac-1-fluorescein conjugate and Anti-la, mouse Phycoerythrin conjugate. Direct staining was then performed with addition of 100 [i\ dilutions of 1:100 F4/80fluoresceinconjugate; 1:200 Anti-Mac-1-fluorescein conjugate and Anti-la, mouse Phycoerythrin conjugate; 3:100 mouse IgG2b-FrrC and mouse IgG2a-RPE; and 5:100 aD-Mannosylated-FTTC-albumin (MBSA-FTTC) totiieirrespective"dry" cell pellets. The cell pellets wereresuspendedby mild vortexing and incubated on ice for 30 to 60 minutes. The tubes were mildly vortexed every 15 minutes to maintain the cells in suspension. Each microtube received 1.3 ml of PBS/ 1% BSA/0.02% NaN3. The microtubes were centrifuged at 1300 rpm for 1 minutes at 40C. The suspension medium was aspirated without disturbing the cell pellets. The washing procedure was repeated twice and final antibody labelled cell pellets were resuspended in 1 ml of PBS/ 1% BSA/ 0.02% NaN3.
34 Propidium Iodide (PI) was added to a single control microtube at a volume of 10 ^il from a 50 M^g/ml stock solution in PBS. The final cell suspensions were then transferred to 10 x 75 mm polystyrene sample tubes (Falcon) and maintained on ice until flow cytometric analysis. Appropriate controls were included prior to mnning series of experiments. These included: (1) macrophages only (no labelled antibody) to measure autofluorescence; (2) macrophages labelled with rat naonoclonal antibody to mouse macrophages (F4/80); (3) macrophages with isotype-matched primary antibody (affinity purified rat myeloma IgG2b); (4) macrophages with unlabelled blocking antibody and labelled antibody. Optimum concentrations or dilution of labelled antibody were determined in preliminary titration studies.
Flow cytometry and direct immunoflurescence analysis Macrophage cell monolayers preparedfipomperitoneal exudate cells were analyzed by flow cytometry. A FACStar Plus flow cytometer (Beckton Dickinson Immunocytometry Systems, Mountainview, CA) was used for data acquisition. A single argon laser was employed for FTTC and PI excitation at a wavelength of 480 nm. Instrument photomultiplier tube (PMT) detector voltage settings were as follows for cell suspensions: Side Scatter (SSC), 300 volts; Fluorescence 1 (FLl), 750 volts; and Fluorescence 2 (FL2), 700 volts. Compensation was not employed since thefluorochromeconjugate was FTTC or phycoerythrin for single staining detection and analysis. Typically, ten thousand cells were passed through the flow cytometer for each cell suspension. Oil viability was determined by PI uptake. Data acquisition for the macrophage suspension typically required 1 min under these conditions and instrument settings. Data analysis was convicted using FACStar Plus Research Software Version 1.0 Menu 2.4.2. Dot plots and/or histogram representations were employed to visualize positively stained macrophagesfix)mall sample
35 suspensions. Data acquisition and and analysis were performed by specialized personnel at the Flow Cytometry Laboratory, Department of Cell Biology and Anatomy, TTUHSC.
Statistical analysis of experimental results The arithmetic average (mean), the standard deviation (S.D.), and the standard error of the mean (S.E.M.) were calculated as descriptive statistics for each set ofreplicativedata. Where appropriate, comparisons between the experimental means were made with student's t test for paired and unpaired observations; significant differences were denoted at p < 0.05.
CHAPTER m RESULTS
It was important to determine the minimum levels of cocaine metabolites that could be detected in mice after exposure to cocaine. Mice were injected intravenously with 0.5, 1, and 2.5 mg/kg cocaine. After 10 minutes these animals were bled and their sera assayed for benzoylecgonine, a major metabolite of cocaine. The assays were done by Dr. J. Wilkins, UCLA School of Medicine, Los Angeles. It can be seen that as little as 50 ng/ml benzoylecgonine can be detected in the semm of the mouse exposed to 0.5 ml/kg cocaine (Figure 4). Amounts detected in sera paralleled the dose of cocaine injected Cocaine has been postulated to produce some of its effects on the immune system by modulating the neuroendocrine pathways. This system involves the hypothalamus, pituitary and adrenal glands. Corticosterone is one of the major endocrine hormones involved in this system. To investigate whether cocaine injections could affect corticosterone levels in the experimental animals, 3 mice were injected ip with 5 mg/kg and 3 were injected with PBS. Two hours later, the mice were bled and their sera saved. Semm corticosterone levels were measured by Dr. Bums, Department of Biological Sciences, Texas Tech University, Lubbock, Texas. Table 1 indicates that animals exposed to cocaine have higher levels of corticosterone than comparable controls.
Effects of cocaine on phagocytosis Effects of cocaine on therespiratoryburst Therespiratoryburst (RB) closely correlates witii phagocytic activity of macrophages. Initial studies were done to determine the effects of cocaine on production of reactive oxygen intermediates (ROI) using chemiluminescence. The RB was measured as
36
37
400 T
300 -
o
CA
200
C
100 -
0.5
1
2.5
Cocaine mg/Kg
Figure 4. Detection of cocaine metabolites (benzoylecgonine) in sera of mice injected intravenously 10 min earlier with different amounts of cocaine.
38 Table 1. Corticosterone Levels^ of Mice Exposed to Cocaine^ Control 316.3 + 95.85C ^ ng/ml semm, ^ Three mice were injected with 5 mg/kg cocaine i.p. and bled 2 hours later. Corticosterone was measured using a commercial kit (I.C.N. Biomedicals). ^ standard deviation, significance < 0.05% Cocaine (5mg/kg i.p.) 499.3+ 50.2
39 chemiluminescence or emission of light per unit time. The Y-axis records the relative emission of light as counts obtained directiy from the luminometer. Time was recorded as two minute intervals which arerepresentedon the X-axis. At each interval, M0 cultures were assayed in triplicate and results expressed as tiie mean S.E.M. Results indicated that administration of cocaine in vivoresultedin a major increase in the RB of isolated peritoneal M0. Figures 5 to 10 and 12 to 14 will illustrate the experiments comparing the RB of M0 from control mice with M0 from cocaine or cocaine-metabolite injected mice. After various intervals, the respiratory burst of peritoneal macrophages was measured using doses from 1.25 to 25 mg/kg cocaine. The responses were found to be dose and time dependent. The effects of cocaine on the RB of murine peritoneal M0 after exposure in vivo was measured after 60 minutes (Figure 5), 24 hr (Figure 6) and 48 hr (Figure 7). Mice were injected i.p. with 2.5 or 5 mg/kg cocaine in PBS. Control animals were injected with the PBS vehicle alone. The peritoneal M0 were collected after the above intervals and cultured in tubes as described in "Materials and Methods." It was observed that cocaine caused an enhancement of the RB as early as 1 hr after exposure to either 2.5 or 5 mg/kg cocaine and remained elevated even when M0 were harvested at 24 and 48 hr after injection. Control values were approximately 300 counts, whereas the RB of M0 from cocaine-treated animals were 20 times higher following a 1 hr exposure to cocaine in vivo. WTien M0 were cultured 24 hr after exposure they were 10-fold higher than controls. This enhancement persisted when M0 were isolated 48 hr after exposure and were essentially at control values by 72 hr (data not shown). Multiple injections of cocaine were also employed. In Figure 8, mice were injected i.p. with 5 mg/kg cocaine per day for 4 days. The RB was measured on day 5, and was found to be higher in M0fromcocaine-treated mice than the saline-injected controls. Other experiments were done using M0fixMncocaine treated and control mice to show that the RB was not stimulated when opsonized zymosan was omittedfromtiiereactionmixture (data not shown).
40 A)
(0
3
o u
4 6 8 TIME (MINS)
B)
8000 O 6000 control 1 cocaine 1 control 2 cocaine 2 4000-
o
o
2000 -
4
6 8 TIME (MINS)
Figure 5. Enhancement of the respiratory burst of peritoneal macrophages 60 min after (A) a single 2.5 mg/kg ip injection of cocaine and (B) a single 5 mg/kg ip injection of cocaine. Readings were obtained in triplicate for each control or cocaine-treated animal.
41 A)
300O control cocaines mg/kg cocaine 2.5 mg/kg
C O
2000-
O
o
1000-
I
I
I
I
12 TIME (MINS)
B)
3000 -
0)
2000 -
3
control 1 cocaine 1 control 2 cocaine 2 control 3 cocaine 3
O
O
1000 -
4 6 8 TIME (MINS)
12
Figure 6. Enhancement of therespiratoryburst of peritoneal macrophages 24 hours after (A) a single 2.5 and 5 mg/kg i.p. injection of cocaine and (B) single 5 mg/kg i.p. injection of cocaine. Readings were obtained in triplicate for each controlOTcocaine-treated animal.
42
A)
2000
(0 3 1000-
o
o
4 6 8 TIME (MINS)
B)
1000800en o o 60040O200control 1 cocaine 1 control 2 cocaine 2
6
8
10
12
TIME (MINS)
Figure 7. Enhancement of the respiratory burst 48 hrs after (A) a single 2.5 mg/kg ip injection of cocaine and (B) a single 5 mg/kg ip injection of cocaine. Readings were obtained in triplicate for each control or cocaine-treated animal.
43
3000-
control 1 cocaine 1 control 2 cocaine 2
(0
2000-
o o
1000-
TIME
(MINS)
Figure 8. Enhancement oftiierespiratoryburst of peritoneal macrophages 24 hours, after 4 consecutive daily 5 mg/kg i.p. injections of cocaine. Readings were obtained in triplicate for each control or cocaine-treated animal.
44 Different routes of exposure to cocaine were also employed to ascertain if they would affect generation of the RB. Cocaine was administered via the intravenous, intramuscular and inhalationroutesin mice and its effect on the RB compared. Figures 9A and 9B show an enhancement of the RB was obtained with both i.v. and i.m. route of administration. Mice injected i.v. or i.m with 2.5 or 5 mg/kg cocaine showed an increase intiierespiratory burst of peritoneal M0 isolated 24 hours after injection. Mice were also exposed to aerosolized cocaine (particle size 1.7 nm) at a concentration of 167 mg/m3 or to air without cocaine, for 1 hr using special chambers. The animals were sacrificed 24 hr later and the peritoneal M0removedand the RB measured M0frommice exposed to cocaine via inhalation showed an enhancement of therespiratoryburst (Figure 9C). The respiratory burst of alveolar M0 was similarly examined for theirresponseto cocaine. M0 were collected from the lungs of mice at various intervals after i.p. injections of cocaine and cultured in tubes. A 2 to 4-fold increase in the RB of isolated alveolar M0 was noted at 60 minutes (Figure lOA) and at 24 hours (Figure lOB) after a single i.p. injection of 10 mg/kg cocaine. The chemiluminescence was generally lower than that obtained using peritoneal M0. Peritoneal M0 were exposed to cocaine, in vitro, and its effect on the RB studied. No changes were detected when cocaine (25-400 |ig/ml) was incubated directiy witii M0 for 15 min to 2 hr. Figure 11 shows that there was no effect on the RB 60 min after exposure to cocaine in vitro. This finding suggests that cocaine may either act through its "active" metabolites which are formed in vivo,OTit noay act secondarily through the induction of other pharmacologically active compounds. Various metabolites of cocaine were examined for effects on the RB (Figures 12 to 14). Intervals of 1, 3, and 24 hr after exposure to treatment were employed When 5 mg/kg of ecgonine methyl ester HClOTecgonine HCl (Figures 12 and 13) were injected i.p., and the peritoneal M0 removed after 1 or 24 hour, the RB was not affected Figure 14
45 A)
3000" control cocaine 5 mg/kg cocaine 2.5 mg/kg (0 2000-
o o
1000-
6 8 TIME (MINS)
10
12
B)
3000control cocaine 5 mg/kg cocaine 2.5 mg/kg
0) O
2000-
o
1000-
4
6 8 TIME (MIN)
12
Figure 9. Enhancement of therespiratoryburst of peritoneal macrophages 24 hours after (A) a single i.v. injection of cocaine, (B) a single i.m. injection of cocaine. Readings were obtained in triplicate for each control OT cocaine-treated animal.
46
C)
3000
2000(0 Z
o o
1000-
TiME
(MINS)
Figure 9. (continued) Enhancement of the respiratory burst of peritoneal macrophages 24 hours after (C) cocaine inhalation (168 mg/m3) for 1 hr. Readings were obtained in triplicate for each controlOTcocainetreated animal.
47
A)
800 700 600 COUNTS
500 400 300
200 100 0
4 6 8 TIME (MINS)
B)
500 400 H v>
3
control 1 cocaine 1 control 2 cocaine 2
300 200 100 T
T
T
o o
4 TIME
6 (MINS)
8
10
12
Figure 10. Enhancement of therespiratoryburst of alveolar macrophages (A) 60 minutes and (B) 24 hours after a single 10 mg/kg ip injection of cocaine. Readings were obtained in triplicate for each control OT cocaine-treated animal.
48
1200 1000800(0
control cocaine (25ug^il) cocaine (ICXXig/ml) cocaine (4(XXig/ml)
600-
o o
4002000 0 4 6 8 TIME (MINS)
10
12
Figure 11. Effects of in vitro cocaine on the respiratory burst of peritoneal macrophages 60 minutes ater exposure.
49 A)
5000 400030003 O
control cocaine 5 mg/kg ecg. m.e HCl 5 mg/kg
u
2000 1000-
12 time (mins)
B)
1400 1200 *
m
control cocaine 5 mg/kg ecg. HCl 5mg/kg
1000c 800 600 400 200 0 I I I I I I ' I
1^
3 O
u
8 time (mIns)
10
12
Figure 12. Effects of (A) a 5 mg/kg injection of either cocaineOTecgonine methyl ester HCl and (B) a 5 mg/kg injection of either cocaineOTecgonine HCl on therespiratoryburst of peritoneal macrophages after 60 minutes exposure. Readings were obtained in triplicate for each control or treated animal.
50
8000 control 1 hr control 24 hr 6000 0)
cocaine 1 hr cocaine 24 hr ecg. m. e. 1 hr ecg. m. e. 24 hr
4000 -
o o
2000 -
4 TIME
6 (MIN)
Figure 13. Effects of a 5 mg/ kg injection of either cocaineOTits metabolite, ecgonine methyl ester HCl, on the respiratory burst of peritoneal macrophages, 1 and 24 hr after exposure. Readings were obtained in triplicate f T each controlOTtreated animal. Each experiment was O repeated at least twice and similarresultswere obtained. This figure iUustrates a singlerepresentativeexperiment
51 A)
4000 control 3000(0 I-
benzoylecg ecg HCl ecg m. e. HCl
2000-
O
o
1000-
4 TIME
6 (MIN)
8
B)
10000 8000 0)
* " "
control cocaine norcocaine
0 0 0
/ i
6000 O
O
400020000 ^B
0 0
0
0
-^ A
^ ^j^ ^ ^^
1
**Hr!lBw ""9' . S t 4 6 TIME
I 1 1 1 1
B H 8 10
12
(MIN)
Figure 14. Effects of a single 5 mg/kg ip injection of (A) ecgonine hydrochloride, ecgonine methyl ester hydrochlorideOTbenzoylecgonine and, (B) cocaine or norcocaine, 3 hours after exposure, on therespiratoryburst of peritoneal macrophages. Readings were obtained in triplicate for each control or treated animal.
52 compares the effect of various metabolites of cocaine on the RB after a 3 hr exposure in vivo. It was found that ecgonine and ecgonine metiiyl ester HCl had no effect, whereas benzoylecgonine and norcocaine increased the RB. Norcocaine produced a dramatic increase which was twice as high as that obtained with cocaine. Benzoylecgonine was somewhat less effective than cocaine in the induction of the RB.
Effects of cocaine on phagocytosis, in vitro Studies were done to determine if phagocytosis correlated with the increase in chemiluminescence. Because opsonized zymosan was used as the ligand to study the RB, it was utilized to study phagocytosis in cocaine-injected animals and controls. Table 2 illustrates that there was an increase in phagocytosis in cocaine-treated mice. Phagocytosis of zymosan was increased 25% in peritoneal M0 collected 3 hr following a 5 mg/kg i.p. injection of cocaine. There was no increase noted 24 hr after injection using the same dose. M0frommice injected with 10 or 25 mg/kg cocaine demonstrated a 50-100% increase in phagocytic activity when assayed at both 3 and 24 hr after exposure. This finding correlates with the observed increase in the RB, although the dose of cocaine required to show an increase in phagocytosis was higher.
Effects of cocaine on phagocvtosis. in vivo The effect of cocaine on phagocytosis in vivo was also studied. Five mg/kg cocaine injected i.p.resultedin an inhibition of the uptake of sheep erytiirocytes. Table 3 compares the phagocytosis of sheep erythrocytes by peritoneal M0 in control mice and mice injected i.p. with 5 mg/kg cocaine. It can be seen cocaine that causes decreased phagocytosis in vivo when M0 are harvested 24 hr after exposure to cocaine. This effect persisted for at least 48 hr although the effea was waning. When M0 were harvested one hr after cocaine exposure, there was no significant difference between these cells and comparable controls.
53 Table 2. Effect of cocaine on phagocytosis of opsonized zymosan by peritoneal M0 in vitro.^
Expt#
time (hrs after exposure) 3 3 3 24 24 24
control
cocaine
dose (mg/kg) 5 10 25 5 10 25
p value^
1 2 3 4 5 6
26.89 1.16^ 23.33 1.14 23.11 2.76 15.44 1.00 22.22 1.38 13.22 1.19
34.11 1.14 36.67 3.01 41.112.81 13.11 1.52 38.22 1.96 26.22 1.75
0.002 <0.001 <0.001 N.S. <0.001 <0.001
^ Each averaged value S.E.M. represents groups of three animals which were exposed to either a single i.p. injection of cocaine or saline, 3 or 24 hr prior to harvest. Macrophages were cultured in Lab-Tektissueculture slides f T 2 hours before opsonized O zymosan was added. Slides were then fixed with formalin and stained with acricfine orange and read using afluorescentmicroscope. ^ % of macrophages with ingested opsonized zymosan particles. c p value > 0.05 is not significant (N.S.).
54 Table 3. Effect of cocaine on phagocytosis of erythrocytes by peritoneal macrophages in vivo.^
Expt
#
time (hrs after exposure) 1 24 48
control
cocaine (5 mg/kg)
p value^
1 2 3
20.667 1.333^ 18.333 0.577 17.333 1.764
18.667 1.333 5.0 1.667 10.0 1.732
N.S. 0.002 0.041
^ Mice were injected i.p with PBSOT5 mg/kg cocaine. After the appropriatetimeinterval of 1, 24 or 48 hr, mice were injected i.p. with 0.5 ml PBS containing 1% sheep erythrocytes. After 30 min, peritoneal M0 were harvested and stained. The number of M0 ingesting erythrocytes were determined by microscopic evaluation. Each averaged value S.E.M. represents groups of three animals which were exposed to either PBS or cocaine. ^ All valuesrepresentpercent of macrophages ingesting erythrocytes. ^ p value > 0.05 is not significant (N.S.).
55 Effects of cocaine on macrophage activation Effects of cocaine on macrophage-mediated cvtotoxicitv rMMO Cocaine injected i.p. caused a considerable reduction in the ability of murine peritoneal M0 to kill tumor necrosis factor-resistant, P815 mastocytoma target cells in vitro. Figure 15 describes 4 separate experiments, illustratingtiieeffects of cocaine on MMC after a single injection of 10 mg/kg. It can be seen thattiierewas no effect when M0 were removed and cultured one hr after exposure to cocaine, but at the 3 hr and 24 hr intervals there was a significant reduction in killing when compared with M0 obtainedfromthe saline injected mice. By 48 hr there were no differences observed between M0 from cocaine treated mice and their respective controls. Figure 16 is similar to Figure 15, except that the mice were injected with 25 mg/kg and the effect studied at 1, 3 and 24 hr after exposure. At this dose, there was a reduction in MMC at 1 hr which persisted f T 24 hrs. O Maximum effect was seen in M0 collected 3 hr after cocaine injection. Figure 17 illustrates 3 separate experiments comparing the effects of a single injection of 5, 10 or 25 mg/kg of cocaine administered i.p. 24 hr prior to M0 being harvested There was no effect on target cell killing when 5 mg/kg was used. However, at the 10 and the 25 mg/kg level, a significant reduction (Student's t-test; P< 0.05) in macrophage-mediated cytotoxicity (MMC) was noted when compared withrespectivecontrols. Multiple injections of 5 mg/kg (1/day for 5 days) also failed to reduce MMC, whereas 5 injections of the 10 mg/kg dose markedly reduced the killing of P815 cells (Figure 18). Table 4 summarizes all of the experiments describing the effects of cocaine on MMC. It is apparent that there was no effect on MMC at the low dose of 5 mg/kg cocaine injection. At 10 mg/kg, there was no effect at 1 and 48 hr, but the MMC was decreased when measured 3 and 24 hr following exposure. At the high 25 mg/kg dose, the effect was seen as early as 1 hr following exposure and persisted for at least 24 hours.
56
100-
/ :
Si
80604
iS^'*':= ,"" X-..-.-
28
4020-
iC
0 3
24 48
Hours
Figure 15. Effects of a single ip injection of cocaine on MMC measured at different intervals after exposure. Thisfigureis a composite of 4 experiments. Groups of 3 mice per experiment were injected with 10 m ^ g cocaine or saline. Peritoneal macrophages were removed at various intervals after injection and cultured and evaluated f T cytotoxicity. O
57
.Sic o w
Figure 16. Effects of a single ip injection of cocaine on MMC measured at different intervals after exposure. Thisfigureis a composite of 3 experiments. Groups of 3 mice per experiment were injected with 25 m ^ g cocaine or saline. Peritoneal macrophages were removed at various intervals after injection and cultured and evaluated f T cytotoxicity. O
58
120 n
10080-
28
6040200
10 25
ffr
SiI' <'" >'=:.-
i
Cocaine (mg/kg)
Figure 17. Effects of a single ip injection of cocaine on MMC measured 24 hr after exposure. Thisfigureis a composite of 3 experiments. Groups of 3 mice per experiment were injected with 5,10, or 25 mg/kg cocaine. Matched controls were injected with saline. Peritoneal macrophages wereremoved24 hr after injection, cultured and evaluated for cytotoxicity.
59
100 n
80
28
604020-
0
1^
'^<"
10
Cocaine (mg/kg)
Figure 18. Effects of a one injection of cocaine/day for 5 days on MMC measured 24 hr after the last injection. Thisfigureis a composite of two experiments. Matched controls were injected with saline. Peritoneal macrophages wereremoved24 hr after the last injection, cultured and evaluated for cytotoxicity.
60 Table 4. Effects of Cocaine on Macrophage-mediated Cytotoxicity (MMC)
Ctocaine mg/kg
Time(Hrs)^
24
48
Multiple X 5^
5 10 25
NDC
<->
ND
<->
ND
<-
<-
i i
i i
i
ND
ND
^ Mice were injected with a single IP injection of cocaine and the peritoneal macrophages were harvested after the indicated times. ^ Mice were injected with a single IP injection of cocaine daily for 5 days andtiieperitoneal macrophages were collected 24 hrs after the last injection. c ND - not done; i - decreased; <-^ - no effect
61 Effects of cocaine on TNF secretion The P815 mastocytoma target cells used in the above studies are tumor necrosis factor CI^JF)resistant.It was not apparent whetiier TNF was involved as a mechanism in tiie reduction of MMC by cocaine. To smdy tiie effects of TNF on MMC, a TNF-sensitive cell line, WEHI 164fibrosarcomawas utilized astfietarget cells in the MMC assays. Figure 19 illustrates 2 separate experiments comparing the effects of a single injection of 25 mg/kg cocaine administered i.p. 3 and 24 hr prior to M0 culturing. There was no effect on target cell killing at eitiier interval of exposure (Student's t-test; F^ 0.05). Further direct studies were done to investigate the effects of cocaine on production of TNF by peritoneal M0. Studies were done exposing mice to aerosolized cocaine, removing the peritoneal M0, and challenging them with LPS in vitro (Table 5). These data suggested that M0 from cocaine-treated animals produce less TNF than equivalent controls. Effects of cocaine on production of reactive nitrogen intermediates (RND Since a significant reduction in MMC was noted with M0 from cocaine-injected mice, efforts were focused on the effect of this dmg on the production of RNI. After exposure to a single i.p. injection of cocaine, peritoneal M0 were removed and cultured in the presence of DFN-y and LPS as described in "Materials and Methods." Nitrite (N02") concentrations were measiu^ in culture supematants. It can be seen in Figure 20A that RNI, in the form of NO2'. were significantiy reduced by a single injection of 5 or more mg/kg cocaine when M0 were harvested 3 hr after exposure to the dmg. At 12 hr after exposure (Figure 20B), the levels of N02" were still substantiaUy reduced with the 10 and 25 mg/kg dose but not with the lower 5 mg/kg dose. Figure 21 is a composite of six experiments and represents these same data as a percentage of control. These data indicate that the inhibition of RNI by cocaine was only temporary. By 24 hr the levels of NO2" produced by M0 from the cocaine-treated mice were essentially equivalent to controls. Figure 22 illustrates that even a
62
10O v>^ * * *
l^M
X S /''f
f f f j
80/*"s
^^^f"^
^'*^
s^"
.ac
604020-
^- ^ s '''
*
J^s
^ ;::r ' ,
J s^'s'
'^S
llfl
1
^^fl
U^
o^ ^ o >*
X o
^''' 1 '
;,>' ' % " ' \ ' ^ ^
V''/'"''- s
'
: ^ ^ B
'' 1 "
'9
^H
^= / ^
i:^ * / ^^s ^ ' '^''' ' ^
'
'
^
'
^ ^
fl
s* ^w f J.,
^
' ^9
^
T^L
^ ^ 9
!4<
'
'"
:,C-<
-^:;s
's
^
0-
24
Hours
Figure 19. Effects of a single i.p injection of cocaine on MMC (using WEHI 164 as target cells) measured 24 hr after injection. Thisfigureis a composite of two experiments. Groups of three mice were injected with 25 mg/kg cocaine. Matched controls were injected with saline. Peritoneal macrophages wereremoved3 and 24 hr after the last injection, cultured and evaluated for cytotoxicity.
63 Table 5. Effect of Cocaine Inhalation on Induction of Tumor Necrosis Factor^
LPS 500 ng/ml
LPS 0 ng/ml
Control 180 11.12^ 95 26.96
Cocaine 16 5.41 23 7.34
Control 2.4 0.1 4.1 0.95
Cocaine <2 <2
^ Animals were exposed to air containing cocaine at a concentration of 167 mg/m3 (particle size 1.7 |J.m) for 1 hour or to air without cocaine. The animals were sacrificed 24 hrs later and the peritoneal macrophages wereremovedand cultured Cultures were exposed to either control media or media containing 500 ng/ml of LPS for 6 hrs. After the 6 hr incubation, supematants were harvested and assayed for TNF using WEHI 164 cells. ^ The data are expressed as units of TNF/0.1 ml.
64 A)
40n
30-
o
20-
10-
0
5
0
10
Cocaine (mg/kg)
B)
30 n
It
20-
o
10-
0 5 0
10
0
Cocaine (mg/kg)
Figure 20. Effects of a single 5,10 or 25 mg/kg ip injection of cocaine on the production of RNI (nitrites) by murine peritoneal macrophages. Macrophages wereremoved(A) 3 hours after exposure, (B) 12 hours after exposure.
65
100-
s? ae O ^
ZfcS
80 60 40
20-
0
10 25 5 10
25
Cocaine (mg/kg)
Figure 21. Effects of a single 5,10OT25 mg/kg injection of cocaine on the production ofreactivenitrogen intennediatesrepresentedas a percentage of control. Macrophage wereremoved3 and 12 hours after exposure.
66 low dose 5 mg/kg injection of cocaine was sufficient to reduce RNI productionfiromM0 harvested 3 and 24 hr following exposure. Total NOy + NO2' were also measured using M0 collected 3,12, and 24 hr after mice were injected with 10 mg/kg cocaine. Twenty-four hr after exposure to the activators, IFN-y and LPS, culture supematants were harvested, exposed to E. coli nitrate reductase, and incubated witii Griess reagent Data were essentially similar to those obtained witii N02" alone, intiiatcocaine also inhibited production of total RNI 57 % at 3 hr, 26% at 12 hr, and 11% at 24 hr (figures 23 A and B). Table 6 compares the effects of cocaine on the secretion of RNI at various doses and intervals. Cocaine reduces RNI secretion as early as 3 hr following exposure. However this reduction was short-lived and lasted only 12 hr. After 24 hr, cocaine had no effect on RNI at the levels of cocaine employed. Table 7 compares the effects of cocaine on RNI production and MMC at equivalent doses and intervals. At 5 mg/kg, inhibition was apparent at 3 hr, however, this was not sufficient to affect MMC. Because of the direct correlation between RNI levels and MMC, the data suggests that inhibition of RNI for a minimum of 12 hr was required to reduce MMC activity for upto 24 hr after cocaine exposure.
Effects of cocaine metabolites on production of reactive nitrogen intermediates (RNI) Since some of the metabolites of cocaine caused an increase in the production of reactive oxygen intermediates, their effects on RNI warranted study. Five mg/kg of various cocaine metabolites including norcocaine, benzoylecgonine, ecgonine methyl ester HQ and ecgonine HCl were injected i.p. in groups of three mice. After 3 hr, peritoneal M0 were isolated and incubated with IFN-y and LPS. Supematant fluids from these cultures were evaluated as before. Figure 24 illustrates that nOTCocaine and benzoylecgonine also reduced the RNI production whereas ecgonine HCl and ecgonine methyl ester HCl had no effect
67
30 n
3hr 12 hr 24 hr
o z
3
20-
10-
0 5 0 5 0
Cocaine (mg/kg)
Figure 22. Effects of a single 5 mg/kg injection of cocaine on the production of lutrites by murine peritoneal macrophages. Macrophage were removed 3,12 and 24 hours after exposure.
68 A)
20n
S
i
103hr
12 hr 24 hr
10
0
10
0
10
Cocaine (mg/kg)
B)
80
60It
3hr
40 08
12 hr 24 hr
20
10
0
10
0
10
Cocaine (mg/kg)
Figure 23. Effects of a single 10 mg/kg injection of cocaine, 3,12 and 24 hours after exposure on the production of (A) nitrites and (B) nitrates by murine peritoneal macrophages
69 Table 6, Effects of Cocaine on Production of Reactive Nitrogen Intermediates (RNI)
Cocaine mg/kg 3 5 10 25 ib
Time(Hrs)fl
12
->
24
48 ND
<->
<-^
i i
i i
ND
<-
ND
^ Mice were injected with a single IP injection of cocaine and the peritoneal macrophages were harvested after the indicated time ^ i - decreased; <-^ - no effect; ND - not done
70
Table 7. Effeas of Cocaine on Macrophage-mediated Cytotoxicity (MMC) and Production of Reactive Nitrogen Intermediates (RNI)
Cocaine mg/kg 1 MMC RNI 5
Time (Hrs)^ 3 MMC RNI 12 MMC RNI ND i i N D i N D i i-^ 24 MMC RNI <r^ i i i^ ND 48 MMC RNI ND <r^ ND <r^
l^D^ N D N D i
1 0 < - > N D i 25 i N D i
< - > N D N D
^ - Mice were injected with a single IP injection of cocaine and the peritoneal macrophages were harvested after the indicated times. b. i- decreased; <-^ - no effect; ND - not done.
71
20 n
1
o Z
10-
1 ^
control cocaine norcocaine benzoyl ecg ecgonine ecg me
1 H
z
08
1 ^ 1 ^ 1 ^
5 5 5 5
Treatment (mg/kg)
Figure 24. Effeas of a single 5 mg/kg ip injection of cocaineOTits metabolites on the production of RNI by murine peritoneal macrophages. Macrophage wereremoved3 hours after exposure.
72 Effects of cocaine on macrophage receptors/ surface proteins Flow cytometric analysis was utilised to study the effects of cocaine on various M0 receptors. Figures and tables willrepresentdata acquisitionfromdifferent experiments studying the effects of cocaine, at various doses and intervals of exposure, on receptors involved in phagocytosis and macrophage activation. In all oftiiefigures,tiieX-axis representsfluorescenceintensity and the area under the curves signifies the cell population. A shift to therightin the M0 cell population obtained from cocaine treated mice denoted an increase influorescenceactivity or an up-regulation in receptOT affinity f T the ligand. O Similarly, a shift to the left represented a deaease influorescenceactivityOTa downregulation in receptor affinity. F4/80 is a surface marker found on mature M0. Figure 25 illustrates that F4/80 was down-regulated at 3 and 24 hr following exposure to 25 mg/kg and 10 mg/kg cocaine, respectively. Table 8revealsthat F4/80 was reduced by 52% and 60% at 3 and 24 hr, respectively. The Mac-1 receptor is involved in phagocytosis of particles opsonized by complement. Since the studies involving the RB and in vitro phagocytosis, utilized zymosan opsonized with guinea pig complement, the possibility that the Mac-1 receptor might be altered by exposure to injected cocaine was investigated. Figure 26 shows that the control animals injected with saline display three separate populations of Mac-1 with different affmities for the anti-Mac-1 ligand The populations have been separated by using gates and their percentages calculated in Tables 9 and 10. When M0 were collected 3 hr after exposure to ip injections of 5 and 10 mg/kg cocaine, a shift in the Mac-1 receptOT populations was observed. There was a fourth separate population seen which appeared sandwiched between gates 2 and gate 4 of the control M0. Most of the M0 that shifted appeared to have moved to therightfrom gate 2. This shift represented an increase influorescentactivity. A similar shift was seen in M0 exposed to 25 mg/kg cocaine harvested 3 and 24 hr after
73 injection. There were, however, no differences noted between the control M0 and M0 collected 24 hr after exposure to 10 mg/kg cocaine. Tables 9 and 10 give the percentage of M0 that have undergone up-regulation in their Mac-1 receptOT, 3 and 24 hr after cocaine injection, respectively. Other receptors involved in phagocytosis by M0 are the Fc receptOT and the mannosylfucosyl receptor (MFR). IgG2a (high affinity FcR), IgG2b Gow affinity FcR), and the MFR were studied infigures27 to 29. Minimal changes were observed in these receptors after 3 and 24 hr exposure to cocaine. There was, however, a slight but consistent downregulation seen in all of the three receptors in M0 collected 3 hr after cocaine exposure using doses of 5-25 mg/kg. Similar results were observed in M0 harvested 24 hr following a single 25 mg/kg i.p. injection of cocaine. Macrophages collected 24 hr after a single 10 mg/kg injection of cocaine showed very littieOTno effea on IgG2a. lgG2b or the MFR. la expression is involved during M0 killing and M0 activation. Since cocaine produced a decrease in macrophage-mediated cytotoxicity, study of the effect of cocaine on la expression was necessary. Control M0 stained intensely with 0.5 |ig/100 ^1 mouse, antila, phycoerythrin conjugate. Figure 30 documents a decrease in la expression by 5 and 10 mg/kg ip injeaed cocaine, 3 hr after exposure. There was also a reduction obtained at 24 hr witii the 25 mg/kg injection. A single 10 mg/kg injection of cocaine produced negligible effect 24 hrs after exposure. Tables 11 and 12 display the percentage of M0tiiathave undergone down-regulation in their la protein expression, 3 and 24 hr after cocaine injection, respectively.
74
3i/e91493ei8NFLl-H\FLl-Hei8ht Cocaine SSmg-'kg Shrs F4.'8e FITC 1 Control 0Rg4<g 3hr F4.'80 FITC 1
160
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#3:/e88793815NFLl-H\FLl-Highl Cocait-ie lOmg-kgi 4hr5 F4''8e FITC 1 Ccr.trcl eng'kg 24hrs F4/8e FITC 1
Figure 25. Effects of cocaine on F4/80 surface marker of peritoneal macrophages, 3 and 24 hr after exposure.
75 Table 8. Effects of cocaine on F 4/80 surface marker on peritoneal macrophages (A) Effects of cocaine on F4/80 surface marker of peritoneal macrophages, 3 hours after exposure.
Cell Population
C^ontrol 48.96 50.94
% of cells
Cocaine 25 mg/kg 75.43 24.56
(B) Effects of cocaine on F4/80 surface marker of peritoneal macrophages, 24 hours after exposure.
% of cells Cell Population 1 2 Control 58.95 40.10 Cocaine 10 mg/kg 83.16 16.65
76
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3i^9682938I7\FL1-H\FL1-Height 3"|Cocaine lewg^'kg 3hrs Anti-Macl FITC . 5 Control ftmgxkg 5v-5 wnti-Kcl FITC . 5
S Cocaine -
Smg^'kg 3hrs Anti-Macl FITC . 5 Control r^g^kg 3hrs ftnti-acl FITC . 5
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100
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Figure 26. Effects of cocaine on Mac-1 receptor of peritoneal macrophages, 3 and 24 hr after exposure.
77
Table 9. Effects of Cocaine on Mac-1 receptor 3 hours after exposure.
% of cells Cell Population 1 2 3 4 Control 27.06 53.14 9.87 10.24 Cocaine 5 mg/kg 25.94 38.15 28.34 7.81
% of cells Cell Population 1 2 3 4 Control 27.06 53.14 9.87 10.24 Cocaine 10 mg/kg 26.7 33.6 25.9 14.14
78 Table 10. Effects of Cocaine on Mac-1 receptor 24 hours after exposure.
Cell Population 1 2 3 4
Control 27.06 53.14 9.87 10.24
% of cells Cocaine 10 mg/kg 33.60 46.42 12.65 7.61
% of cells Cell Population 1 2 3 4 Control 26.32 52.70 9.49 11.86 Cocaine 25 mg/kg 27.16 39.70 23.10 10.41
79
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Sn
Cocaine S^gz-kg 3 h r s IgGa RPE 3 C o n t r o l emg^kg 3 hrs IgQga RPE 3
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Figure 27. Effects of cocaine on IgG2a receptor of peritoneal macrophages, 3 and 24 hr after exposure.
80
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, 8-
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Figure 28. Effects of cocaine on IgG2b receptor of peritoneal macrophages, 3 and 24 hr after exposure.
81
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Figure 29. Effects of cocaine on mannose-fucose receptor of peritoneal macrophages, 3 and 24 hr after exposure.
82
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#3:^060293018sFL2-HNFL2-He i gh t C o c a i n e lOmg-kg 3 h r s A n + i - I a RPE . 5 C o n t r o l 6(3/kg 5hrs HnT,i-Ia ftPE . 5
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o_ Cocaine 5mg kg 4hrs Anti-la RPE .5
Coniroi Sma^'kg 24hr Anii-Ia RPE .3
9A
ails.
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Figure 30. Effects of cocaine on la expression of peritomeal macrophages, 3 and 24 hr after exposure.
83 Table 11. Effects of Cocaine on la expression 3 hours after exposure.
Cell population 1 2
Control 16.52 83.45
% of cells Cocaine 5 mg/kg 37.68 62.46
% of cells Cell Population 1 2 Control 16.52 83.45 Cocaine 10 mg/kg 39.53 60.46
84 Table 12. Effects of Cocaine on la expression 24 hours after exposure.
Cell Population 1 2
Control 20.11 80.00
% of cells Cocaine 10 mg/kg 19.52 80.47
% of cells Cell Population 1 2 Control 21.33 78.71 Cocaine 25 mg/kg 36.61 63.19
CHAPTER IV DISCUSSION
PriOT to studying the effects of cocaine on macrophage phagocytosis and activation, it was necessary to establish that cocaine or its metabolites, such as benzoylecgonine, could be detected in sera of mice after parenteral exposure. This was necessary because in the present studies, mice were exposed to cocaine by intraperitoneal injections. It was found that cocaine was rapidly distributed and metabolized within a few minutes after injection. Benzoylecgonine was detected in ng/ml quantities in sera ten minutes after injection of 0.52.5 mg/kg cocaine. This finding was similar to that reported by Kogan et al. in humans. They demonstrated that if 100 mg of cocaine was given intravenously, a plasma peak occurred at five minutes, then declined over a five to six hour period, with a distributional half-life in the plasma of 20-40 minutes (corresponding to the demonstrated psychological effects of the drug). Mean plasma biological half-life was 2.8 hours. In this same study, benzoylecgonine was deteaed in the urine within five minutes of administration of cocaine, and was still detected 24 hr later. This proved that exposing mice to cocaine parenterally would be acceptable for our experimental studies. It has been reported that many of the frequentiy abused drugs affect the immune system through a stress-induced endocrine alteration (Watzl and Watson, 1990). An increase in glucocorticoid production during stress has been implicated in altering the immune system. Glucocorticoids are also known to cause lymphocyte apoptosis. It was hypothesized that cocaine might interfere with the immune system by altering the neuroendocrine system. Corticosterone levels were found to be significantiy higher in cocaine treated mice, just two hr after exposure. Since corticosterone is one of the major glucocorticoids produced by the adrenal cortex, it is highly probable that cocaine produces
85
86 some of its effects on the immune system through its effects on stress-induced endocrine hormones via the classical "stress" pathway.
Effects of cocaine on phagocytosis Phagocytosis is the most important component of the host defense against invading microorganisms once the outer epitiielial surface of tiie body has been breached M0 are strategically located to defend tiie body against invading microOTganisms by lining the blood sinusoids in the liver, spleen, bone marrow, adrenals, etc. They also line the serous cavities such as the pleura and the peritoneum. Optimal phagocytosis requires the participation of opsonins: serum complement and immunoglobulin (mainly of the IgG subclass). In antiserum, IgG is the main opsonic source which can be amplified by complement In antiserum, complement is activated mainly through the classical pathway. In non-immune serum, the alternative pathway of complement is activated by bacterial cell wall components, such as endotoxin and peptidoglycan without the participation of IgG. During activation of complement, C3b, C3bi, and C3d are produced. These activated C3 factors can become fixed to the cell wall of the particle. Only when the particle is loaded with opsonins, does recognition by the phagocytic cell occur followed by phagocytosis. Large particles, such as erythrocytes, coated with C3 fragments alone are usually only bound to the M0; intemalization through complement receptors requires an additional stimulus. MicroOTganisms, by contrast are often efficientiy ingested in the presence of complement alone. In the case of unencapsulated bacteriaOTyeast particles coated with complement only, the extra trigger needed for ingestion is provided by the cell wall components of these microorganisms (yeast B-glucan, LPS). In the present study of therespiratoryburst as a correlate to phagocytic activity, zymosan (yeast cell wall) opsonized with guinea pig complement was used as the ligand.
87 The RB or "oxidative burst" measures the increase in superoxide and other reactive oxygen intermediates (ROI). The capacity to generate increased amounts of ROI is a consistent biochemical marker of metabolic activity. This occurstiiroughmetabolism of large quantitities of glucose by way of tiie hexose monophosphate shunt, and an increased oxygen consumption. This respiratory burstresultsin altered activity of the membranebound oxidase complex and the reduction of molecular oxygen to superoxide (Babior, 1984). The superoxide thus generated is rapidly converted to hydrogen peroxide and hydroxy] radicals, which provide most of the microbicidal oxidative activity both within the phagosome and in the extracellular environment. The molecular oxygen is ultimately reduced to water, but the superoxide anion, hydrogen peroxide, and the hydroxy] radicals are referred to as thereactiveoxygen intermediates. O2 -> 02" ---> H2O2 > OH- -> H2O Water
Oxygen
Superoxide anion
Hydrogen peroxide Hydroxy] radical
Chemiluminescence is a laboratory technique that measures photon emission by phagocytic cells while ingesting microorganisms and other particles (Easmon et al., 1980). Light emission is triggered by perturbation of the M0 membrane whichresultsin activation of the hexose monophosphate shunt. Activation of this biochemical pathway causes the production of high energy oxygen compounds (ROI) which are involved in the bactericidal activity of the cell. Since, phagocytosis by M0 is usually accompanied by a large increase in the production of superoxide ions, the change in RB correlates strongly with phagocytic activity (Stjemholm et al., 1973). One of the advantages of measuring the RB by chemiluminescence is that the sensitivity of the system is extremely high and can detect 10"^ to 10-^6 M levels of oxygen radicals (Whitehead et al., 1979). Intiiepresent study, as littie as 1.25 mg/kg of intraperitoneally injected cocaine was sufficient to affect the RB when measured 24 hr after exposure. However, a dose of 5 mg/kg cocaine did not affect the observed phagocytic
88 activity 24 hr after injeaion. This is probably a reflection of tiie difference in sensitivities between the two methods. Cocaine levels used in humans range from 0.5 mg/kg to 5 mg/kg per day. However, there have beenreportedcases of doses as high as 30 mg/kg in few habitual users. In tiie present studies, cocaine was administered by the intraperitonealroutein most of the experiments. Cocaine at levels of 1.25 to 25 mg/kg enhancedtiieRB. These doses were a littie higher than those used commonly by most drug addicts. Effeas were discernible as early as 1 hr after injection and persisted up to 48 hr. The effects were found to be both dose and time dependent Tolerance to cocaine injections was not developed within five days. Multiple exposure to cocaine of four, daily, consecutive injections also produced a similar increase in the RB. Further studies, using daily consecutive injections for 1 week to 1 month need to be performed. This would better mimic the effects of cocaine which might occur in the clironic user of cocaine. There was an increase in the RB using otherroutesof cocaine administration. Exposure to cocaine via the intramuscular, intravenous, or intraperitonealrouteall resulted in an elevated RB. There was also an increase in the RB noted after cocaine was administered via the inhalationroute.In this study, cocaine was finely ground to a particle size of 1.7 |i.m and aerosolized in special chambers. Mice were exposed to cocaine at a concentration of 167 mg/m3 for one hr and the RB measured 24 hr later. We also observed an increase in the RB of alveolar murine M0 after a single i.p. injection of cocaine. Fewer alveolar M0 (2.5 x 10^)tiianperitoneal M0 (1 x 10^) were used for the chemiluminescence assay because of the 10 fold lower yields of alveolar M0 per mouse. Even after factoring in the difference in M0 numbers used the alveolar M0 were found to require higher levels of cocaine (10 mg/kg) which induced a lower response when compared with peritoneal M0. It is also possible that the brief exposure to xylocaine which was used during collection of M0, could have reduced the response of alveolar M0.
89 Anotiier explanation might be that the intraperitonealrouteof exposure to cocaineresultsin direa exposure of peritoneal M0 to cocaine which is nottiiecase witii the alveolar M0. This mightresultin direct synthesis ofreactivecocaine intermediates by the exposed M0, which could accumulate to extremely high levels in the peritoneal cavity. It has beenreportedby others that cultured human peripheral blood mononuclear cells, exposed directiy to cocaine for 48 hr, caused a suppression of 02" production (Chao et al., 1990). However, we were not able to detect any changes in the RB when 25-400 M-g/ml cocaine was incubated directiy with M0 for up to 2 hr. The disparate observations obtained from these two studies couldreflectspecies differencesOTthe types of mononuclear phagocytes under study (peritoneal M0 versus blood monocytes). Further, Chao et al. exposed the monocytes for 48 hr to cocaine, whereas we exposed the peritoneal M0 f T O only 2 hr owing to the limitations of the chemiluminescence assay in measuring the RB. In addition, the lymphocyte population in peripheral blood mononuclear cell preparations may be a source of suppression (e.g., through production of immunosuppressive cytokine(s) by these cells). However, it is possible that cocaine itself does not directiy cause the increase in the RB. Our finding seems to suggest that cocaine may either act through its "active" metabolitesOTit may act secondarily through the induction of other pharmacologically active compounds. Otiiers have postulated that the effect of cocaine on the immune response is neuroendocrine-mediated (Watzl and Watson, 1990). Cocaine is known to alter the metabolism of various neurotransmitters, including dopamine, serotonin, norepinephrine and acetylcholine (Jones, 1984; Gold Washton and Dackis, 1985). Cocaine has also been observed to induce ACTH, B-endorphin and corticosterone in plasma after i.p. injection (Moldow and Fischman, 1987). We had earlier shown tiiat cocaine injected in mice caused an increase in corticosterone levels. The next question addressed was whether the metabolites of cocaine could cause a change in tiie RB similar to that obtained witii cocaine. The metabolites ecgonine HCl and
90 ecgonine methyl ester HCl, which are formed from ester hydrolysis of cocaine, were used initially in tiiese studies. These metabolites are known to have a longer half-life (8-16 hr) tiian cocaine (Ambre, 1982) and are generally considered inactive and nontoxic. These metabolites did not affea tiie RB when tested 1 or 24 hr after a 5 mg/kg injection. It should be emphasized that the half-life of cocaine is only 30-60 minutes and the observed effeas on the RB persisted fOT up to 48 hr. Since tiie ester hydrolytic metabolites, ecgonine HCl and ecgonine methyl ester HCl, did not affect on tiie RB; it was considered possible that benzoylecgonine, or the N-oxidative metabolites norcocaine and N-hydroxynorcocaine may be involved. Norcocaine and benzoylecgonine have half-lives of 30 minutes and 5 hr, respectively. These metabolites are known to cause increased hepatotoxicity and/or cerebral toxicity (Williams, 1992; Thompson, Shuster and Shaw, 1979). Norcocaine andNhydroxynorcocaine are produced from oxidation of the tropane nitrogen. The initial step in the theorized activation of the cocaine molecule is the A^-demethylation of cocaine to norcocaine by either of theflavinadenine dinucleotide-containing monooxygenases of the cytochrome P-450 system. Norcocaine can then undergo arelativelyrapid N-hydroxylation to A^-hydroxynorcocaine (Boyer and Petersen, 1992). The hepatotoxicity of both norcocaine and N-hydroxynorcocaine has been demonstrated and appears to require a functional cytochrome P-450 system as inhibition of this system in vivoresultsin elimination of the hepatotoxicity of these metabolites (Thompson, Shuster and Shaw, 1979). The exaa metabolite(s) ultimatelyreponsiblef T the hepatotoxicity of cocaine has O not been established but has been suggested to be the one-electron oxidation product of Nhydroxynorcocaine: norcocaine nitroxide (Rauckman, Kloss, and Rosen, 1982). It has also been suggested that the ultimate toxic species may be the nonxx:aine nitroso species (Chaikoudian and Shuster, 1985). A single 5 mg/kg injection of eitiier benzoylecgonine or norcocaine were found to enhance the RB when measured 3 hr after exposure. NOTCocaine was found to enhance the RB even more than cocaine, whereas benzoylecgonine was less
91 effective than cocaine. Hence, it was observed that the metabolites of cocaine known to cause toxicity in otiier systems were also able to interfere witiitiieRB. Because of the effects on the RB, the next step was to consider whether cocaine would affect the phagocytic activity of murine M0. To compare RB with phagocytosis, the same ligand, zymosan opsonized with guinea pig complement, as described previously, was used Cocaine caused an increase intiiephagocytic activity of M0. However, higher concentrations of cocaine were required to inaease phagocytosis by murine M0. This could reflect differences in sensitivity between the two assays. However, it should be emphasized that a strong correlation was found between in vitro RB and in vitro phagocytic activity of peritoneal M0 from mice injected with cocaine. Results obtained with phagocytosis of sheep erytiirocytes in vivo differed from those obtained using zymosan in vitro. The findings of other investigators were confirmed in that cocaine deaeases in vivo phagocytic activity of peritoneal M0 (Ou et al., 1989). A possible explanation for the difference between in vivo and in vitro phagocytic activity of murine M0 ftom cocaine-treated animals could be because of the different ligands and receptors involved, in the two methods used. For the in vitro assay, uptake of zymosan was primarily through the C receptor present on the M0. Uptake of sheep erythrocytes was probably through the mannose-fucose receptor and other receptors which bind the sugar moieties present on the erythrocyte surface. Another possiblereasonfor the disparity, could be the production of different metabolites of cocaine under in vivo and in vitro conditions. Norcocaine is probably produced very rapidly and in far greater quantities when cocaine is metabolized in tiie body. The CR3 (Mac-1), FcR (IgG2a and IgG2b) as well as the mannose-fucose receptOT (MFR) have been studied and will be discussed later, to further elucidate tiie mechanisms by which cocaine affects phagocytosis. Finally it should be noted that under in vivo conditions, induction of corticosterone could also inhibit
92 phagocytosis. Phagocytosis by M0 in vitro would not be subject to changes in steroid levels by cocaine. Previousreportshave indicated that cocaine could have both enhancing or depressing effects on tiie immuneresponse.Increased ROI production by M0 suggests that both microbicidal activity and phagocytosis would be increased The implications of this includes possible effects on the immune function which couldresultin significant effects onresponsesto disease agents. Further studies are required to ascertain if these effects relate to changes in hostresistanceto disease.
Effects of cocaine on macrophage activation (macrophagemediated cytotoxicity) "Macrophage activation" is the acquisition of enhanced competence to complete complex functions, such as destruction of microbes, lysis of tumor cells, processing and presentation of antigen,OTregulation of hematopoiesis. Extensive evidence indicates that interferon-y (IFN-y) is the principal priming or macrophage activating factor both in vivo and in vitro, and it seems clear that many of the forms of M0 activation, in regard to enhanced destructive capacity, can be attributed in good part to IFN-y. The prototype second signal has been shown to be the Upid A component of bacterial lipopolysaccharide (LPS) (Adams, 1992). Macrophage-mediated cytotoxicity (MMC) is one of the methods of measuring M0 activation. MMC is the acquisition oftiiecompetence to destroy neoplastic cells in tiie absence of antibody. Russell (1983) showed tiiat tiie induction of such a function was tiie resuh of a multistep cascade of events. Treatment of M0 witii IFN-y, induced a state which was termed "primed." Primed M0, altiiough not cytolytictiiemselves,readily became so when pulsed witii traces (i.e., ng/ml) of LPS. These cytolytic M0 were termed "fully activated" MMC depends upon two cellular capacities: (1)tiieability to capture and tiien bind vigorously neoplastic cells; and (2)tiieability to secrete cytolytic factors including
93 reactive nitrogen intermediates (RNI), a novel cytolytic proteinase (CP), and tumor necrosis factor a (TNF-a). The fmdingtiiatcocaine inhibits MMC by peritoneal M0 has not been reported previously. In the present studies, activated peritoneal M0frommice injected i.p. with cocaine exhibited a reduced cytotoxicresponsetoward P815 target cells. P815 is a tumOT ncCTOsis factor resistant, mouse mastocytoma cell line. It was also observed that there was no reduction in the cytotoxicresponsetowards WEHI 164 target cells. WEHI 164 is a tumOT necrosis factOT sensitive,fibrosarcomacell line. Thesefindingssuggested that reduction in tumor necrosis factor was not the major cause in the inhibition of MMC. The main factor responsible for monocyte- and macrophage-mediated cytolysis of P815 cells has been shown to bereactivenitric oxide (NO) produced during metabolism of L-arginine to N02'/N03- (Higuchi et al., 1990; Stuehr and Natiian, 1989; Keller, Geiges and Keist, 1990). The stable products of the L-arginine pathway, N02" and N03-, were found to be incapable of causing cytostasis under culture conditions. N02" only became cytostatic after mild acidification, which would favOT its transformation into nitrogen oxides which have greater toxicreactivity(Stuehr and Nathan, 1989). The reduced cytotoxic activity of M0 correlated with their reduced ability to secrete N02' following exposure to IFN and LPS. The reduced level of N02" from M0 obtainedfiiommice injected witii > 10 mg / kg cocaine persisted f T 12 hr and correlated witii a diminished ability to kill P815 cells. This O time frame parallels the time required f Ttiiekilling of target cells by activated M0 which O peaks 4-9 hr after exposure to lymphokines with maximumreleaseof ^^Cr 12-16 hr after contact (Adams and Marino, 1984; Meltzer, Occhionero and Ruco, 1982). The short-term inhibition of RNI lasting only a few hrs which was induced by 5 mg/kg of cocaine was not sufficient to inhibit MMC under the experimental conditions utilized in this study. Several hypotheses can be proposed to explain the effects of cocaine on the reduction of RNI. One possible explanation is that cocaine induces TGF-B. This cytokine has been
94 reported to be released by isolated human monocytes exposed to cocaine in vitro (Peterson et al., 1991). Induction of TGF-B, in inhibiting tiie production of RNI (Ding, Natiian and Stuehr, 1990) cannot be ruled out Another hypothesis is an interference between ROI and RNI, both of which are produced via separate patiiways (Ding et al., 1988). Nitric oxide has an extremely short half-life in the presence of ROI (Lancaster, 1992; Natiian, 1992). It is well established that superoxide is continuously inactivated by nitric oxide even under normal conditions (Rubanyi and Vanhoutte, 1986), and so inhibition of nitric oxide could conceivably permit superoxide to increase. In other studies (Vaz, Lefkowitz and Lefkowitz, 1993), both alveolar and peritoneal M0fromcocaine-injeaed mice demonstrated a significant increase in therespiratoryburst suggesting an increase in superoxide production. This increase was not demonstrated after in vitro exposure of M0 to cocaine. Nitric oxide (NO) has various other functions besides its involvement in killing foreign invaders in the immuneresponse.The molecule has been found to be essential to activities that range from digestion to blood pressureregulation.NO also carries important information in the nervous system. In males, it is the messenger that translates sexual excitement into an erea penis. In the brain, NO may be a long sought memory molecule that aids in learning andremembering.As a defensive weapon, NO appears to work in at least two ways: by inhibiting key metabolic pathways to block growth, and by killing cells outright. In the first mode, NO attacks susceptible iron groups in certain enzymes, including those that synthesize DNA and help cellsrespire.When those enzymes are crippled, cells cannot grow and divide;tiusmay be an important part of NO's antitumOT function. Secondly, NO has been shown to be able to combine witii oxygen to eventually pixxiuce potent cellular assassins such as tiie hydroxyl radical, OH, and nitrogen dioxide. This patiiway may be behind NO's antibacterial properties. The possibilitytiiatNO is a primitive defense against a whole range of microbes is under intense investigation. The findingtiiatcocaine in vivo, decreases RNI secretion has important ramifications in
95 explaining certain of the effects of cocaine on different systemic functions. Besides helping to understand tiie reduction in MMC by cocaine, study of RNI may illuminate its effects on cardiovascular and central nervous systems. It was necessary to determine whetiiertiieseeffects are directiy caused by cocaine OT an intermediateOTby-product of cocaine metabolism. Studies described earlier, by the present investigators (Vaz et al., 1993) indicatedtiiatcertain metabolites of cocaine including ecgonine HCl and ecgonine methyl ester HCl, did not enhance the respiratory burst as compared to cocaine. Therefore, the intermediates of cocaine which have been reported to cause hepatotoxicity (Roth et al., 1992) and cerebral toxicity, such as norcocaine and benzoylecgonine,respectively,were investigated for their capability in altering M0 functions. In these studies, norcocaine when injected in vivo was shown to dramatically increase the RB as well as reduce the production of RNI of isolated peritoneal M0. The effect with norcocaine was even greater than that obtained with cocaine. Benzoylecgonine was also effective in increasing the RB and deaeasing RNI production. Its effect, however, was less potent than cocaine. These two metabolites may be the intermediates through which cocaine affeas most of the macrophage functions.
Effects of cocaine on macrophage receptors/ surface proteins Discovery of cell surface receptors involved in cell-cell, cell-substrate, or cell-soluble ligand binding has been a key factor in understanding tiie mechanisms underlying inflammatory and immune phenomena. Engagement of various surface receptors on M0 can transduce a signal leading to cellular events that change the phenotype, movement, gene expression, or activation of the cell. In effect, tiiey determine the control of activities, such as growth, differentiation, activation, recognition, endocytosis, migration, and seaetion. Phagocytosis is a dynamic process in which bacteria are attached to the M0 membrane in preparation for ingestion. This attachment step is mediated by specific M0 receptors and
96 is dependent upon the nature of the bacterial surface. Opsonization of bacteria with complement and/or Ig permits M0 and otiier phagocytic cells to recognize, by a limited number of receptors, a wide array of microrganisms with broad heterogeneity of surface characteristics. The principal M0 phagocytic receptors recognize the breakdown products of complement component 3 (C3b and C3bi) andtiieFc portion of IgG (Silverstein et al., 1989; Horwitz, 1982). The function of both is to promote the uptake of antibodies- and/ or complement-coated molecules. Both receptors can act in synergy to increase, by several hundredfold, the rate of uptake of an opsonized particle. In addition to phagocytosis via opsonic receptors, M0 are well equipped for non-opsonic phagocytosis (Sharon, 1984; Ofek and Sharon, 1988). Although described as "non-specific" phagocytosis, the involvement of specific receptors in the process has been demonstrated recentiy (Speert et al., 1989; Wright and Jong, 1986). The mannose-fucose receptor (MFR) is perhaps the best characterized of these non-opsonic receptors. In addition to its role in recycling mannosylated macromolecules from the circulation (Stahl et al., 1980), it appears to be involved in the ingestion of certain bacteria, fungi and parasites (Kan and Bennett, 1988; Blackwellm et al., 1985). Other lectin-like interactions have been described, but the specific receptors involved remain to be characterized. Binding of bacteria to M0 via la antigen has also been described (Stewart, Glass, and Weir, 1982). The F4/80 antigen is a sensitive and efficient marker of mature M0 in most adult and fetal murine tissues, except in T-lymphocyte-dependent areas, and has been used to describe the ontogeny of M0. The F4/80 monoclonal antibody provides a tool to map cell distribution, to identify microheterogeneity among maaophages witiiin a single organ, and torevealaccumulation of newly recruited monocytes at sites of injury and their adaptation within a specific tissue environment. The functional significance of the F4/80 antigen is unknown at this time. Cocaine down-regulated expression of F4/80 by peritoneal M0 at 3 and 24 hr after exposure. This finding might indicate that there were more immature M0
97 populating tiie peritoneal cavity after cocaine injection; since expression of F4/80 is downregulated by inflammatory stimuli, which induce the recruitment of immature cells (Gordon, 1986). At tiiis time, we cannot be sure that this down-regulation might have any relevance to M0 function. Complement receptors ontiieM0 are involved in the binding and ingestion of opsonized particles. The Mac-1 molecule (CDl lb/CD 18) recognizes C3bi and is equivalent to the complement type 3 receptOT. Mac-1 is an cx/p heterodimeric glycoprotein which consists of non-covalentiy linked chains. Mac-1 is a member of the integrin supergene family of adhesion molecules. Mac-1 and FcR are key surface molecules for the clearance frx)m blood of microorganisms and soluble immune complexes. Mac-1 may be the most essential for in vivo clearance, inasmuch as they have specificity for split products of complement, such as iC3b, which are not normally found in significant amounts in blood. The Mac-1 receptor was up-regulated 3 and 24 hr after exposure to cocaine. This finding complemented earlierresultsobtained studying the effects of cocaine on the RB and phagocytosis in vitro. In both those studies, zymosan opsonized with complement was used as the ligand which putatively was phagocytized via the Mac-1 or CR3. Since, both the RB and phagocytosis in vitro showed an increase after cocaine injections, it was not surprising to find that the Mac-1 receptOT was similarly up-regulated. The FcRs are heterogenous and show different affinities for Ig. The human FcR comprises three distinct receptors. FcRI binds to monomeric and polymeric IgG with high affinity (Ka of approximately 10^ M). It shows subclass preference, in decreasing OTder IgGl > IgG3 > IgG4 > IgG2. It is expressed constitutively in the M0 and increases in numbers after treatment with IFNy. Two other FcR receptors termed II and in have lower affinity for IgG. The mouse M0 also has a high-affinity receptortiiatpreferentially binds to IgG2a and a low-affinity receptortiiatbinds IgGl, IgG2a. and IgG2b- The low affinity receptors II of both mouse and human are also found on other cells, for example, B cells.
98 Mouse M0 also have a poOTly characterized receptor for IgG3. The effects of cocaine on the high-affmity IgG2a as well as tiie low-affinity IgG2b Fc receptors were studied. Cocaine exposure caused a slight down-regulation of botii receptOTS when studied 3 and 24 hr after injection. It must be noted that cocaine also caused a decrease in phagocytosis of sheep erythrocytes in vivo, which probably involves the Fc receptor and the mannose-fucose receptor. Many of the cell-molecule and cell-cell interactions involving M0 are oligosaccharide mediated. Complex carbohydrates have long been suggested as ideal biomolecules f T O specific recognition because the enormous variability found in their structre provides a lexicon for specific and selective interactions. The mannose-fucose receptor (MFR) is a 165 kD membrane glycoprotein that recognizes mannose chains present on the ligand It is found on almost all mononuclear phagocytes, but most macrophage-like cell lines do not express the receptor or do so at very low levels. The receptor has served as an excellent model for for tiie study of endocytosis in M0 (Stahl et al., 1980). It is constitutively i...