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Alcohol and Drug Abuse Research Center, McLean Hospital, Harvard Medical School (J.H.H., M.B.S., N.K.M., J.H.M., A.J.S.), Belmont, Massachusetts 02478; and Departments of Psychiatry (J.H.H.) and Orthopedic Surgery (J.G.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: John H. Halpern, M.D., Alcohol and Drug Abuse Research Center, McLean Hospital, 115 Mill Street, Belmont, Massachusetts 02478-9106. E-mail: john_halpern{at}hms.harvard.edu.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cocaine’s effects on the HPA axis may also result in alterations in immune function. Experimental animal and in vitro studies report cocaine-induced changes in immune reactivity, antibody formation, lymphocyte subset profile, lymphocyte proliferation, and cytotoxic activation of macrophages, natural killer cells, and cytotoxic T lymphocytes (12, 13, 14). These factors may contribute to the increased rate of infections, including human immunodeficiency virus (HIV), noted in cocaine abusers (15, 16, 17).
Cytokines are involved in the regulation of the HPA axis, immunity, and inflammation (18, 19, 20, 21). Infection, foreign bodies, and traumatic tissue damage activate a cascade of responses that include the local synthesis and secretion of cytokines from a variety of cells. These agents will then bind to specific cytokine surface receptors and soluble receptors to induce a local inflammatory response; they also circulate to the central nervous system and induce further neuroendocrine effects, some of which are counterregulatory (22). The synthesis and release of cytokines change as the immune response proceeds and normal physiological homeostasis is achieved. Cytokines have been described as the "hormones of the immune system" (23).
The proinflammatory cytokine, IL-6, in particular, is involved in antibody production, differentiation of B cells, activation of T cells, hematopoiesis, pyrogenesis, CRF stimulation, induction of acute phase proteins in the liver, as well as other neuroendocrine changes (24, 25, 26). IL-6 stimulation of the HPA axis serves to control the inflammatory response. Thus, IL-6 also has significant indirect antiinflammatory properties (27). Elevated levels of IL-6 have been documented in numerous clinical conditions and in states of physical and/or psychological stress (28, 29, 30, 31).
Cytokines may be involved in cocaine-induced changes in immunity. In vitro exposure to cocaine decreases IL-2 and interferon-
(IFN-) secretion from mouse spleen cells (32), IL-1 and TNF release from mouse peritoneal macrophages (33), and IL-2, IL-8, and IFN- release from human peripheral blood lymphocytes (14). Peripheral blood lymphocytes collected from 47 polydrug users with a history of cocaine use were shown to secrete higher levels of IL-2, but not IFN- or IL-1, when challenged with antigen in the absence of cocaine (34).
Serum levels of IL-6 also increase when sampled through a chronic indwelling venous catheter rather than via direct venipuncture (35). This inflammatory response at the site of catheter placement thus offers a model for assessing acute release of IL-6 in the presence or absence of cocaine (injected via direct venipuncture into the opposite arm). One purpose of the present study was to evaluate the relationships among blood levels of cocaine, ACTH, cortisol, DHEA, and IL-6. A second objective was to determine whether cocaine’s effects on the HPA axis and IL-6 differed in men and women or in women as a function of menstrual cycle phase.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Sixteen healthy adult women and 14 healthy adult men between the ages of 21–35 yr, all reporting a history of illicit cocaine use, including at least one episode of use within the past month, provided informed consent for participation in this study. All subjects reported using cocaine exclusively by intranasal insufflation during the prior month. The study was approved by the institutional review board of the McLean Hospital. Volunteers with a history of cocaine dependence or with any lifetime DSM-IV Axis I disorder (other than cocaine abuse, nicotine dependence, and caffeine dependence) were excluded from participation. Women using oral contraceptive medications were also excluded. Subjects who met DSM-IV criteria for cocaine abuse did not differ across gender for age and body mass index (see Table 1
); all reported ingesting cocaine on 2–6 d of the preceding month. All subjects were in good physical health and had normal medical and laboratory screening examinations, negative pregnancy tests (see below), and negative urine screens for drugs of abuse (see below). Female subjects were required to be at either the midfollicular or midluteal phase of the menstrual cycle, defined as 5–9 or 18–22 d from the onset of menses, respectively. Progesterone and estradiol levels were determined on the day of the study to verify menstrual cycle phase.
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Enrolled subjects participated in a study conducted on the clinical research unit and received either 0.4 mg/kg cocaine or saline placebo. Subjects were randomized to cocaine or placebo injections such that equal numbers of subjects of each gender were assigned to the two treatments. The challenge dose was iv administered via direct venipuncture into the antecubital vein of the dominant arm over a 1-min period by a trained physician, certified in cardiopulmonary resuscitation and advanced cardiac life support, who remained present during each study. All subjects were studied in a semisupine position; continuous cardiovascular monitoring commenced 10 min before infusion and extended for 4 h postinfusion. A cardiac defibrillator and appropriate emergency treatment medications were also present on the unit. A Kowarski-Cormed thrombo-resistant blood withdrawal butterfly needle and tubing set (Dakmed, Inc., Buffalo, NY) was inserted into the antecubital vein of the opposite arm 30 min before the infusion. Samples for analysis of plasma cocaine, ACTH, and cortisol were collected at baseline (20 min before infusion), immediately before infusion, and at 2, 4, 8, 12, 16, 20, 30, 40, 60, 80, 120, 180, and 240 min postinfusion. DHEA samples were collected at baseline and 8, 20, 30, 40, 80, 120, 180, and 240 min postinfusion. Samples for analysis of plasma IL-6 were collected at baseline and 30, 60, 120, 180, and 240 min postinfusion for men. Samples for analysis of plasma IL-6 for women were collected at baseline and at 240 min postinfusion; interim time point samples proved insufficient for IL-6 analysis. Aliquots for cocaine analysis were transferred to Vacutainer tubes (Becton Dickinson and Co., Franklin Lakes, NJ) containing sodium fluoride and acetic acid (to prevent the hydrolysis of cocaine). Samples were immediately centrifuged, and plasma was removed and frozen at -70 C for cocaine, ACTH, DHEA, cortisol, and IL-6 analyses.
Pregnancy test
Serum pregnancy tests were completed in the morning before cocaine or placebo administration to ensure that no woman had become pregnant since the prescreening exam. The Stanbio QuPID Plus Test (Stanbio Laboratory, Inc., San Antonio, TX) is a qualitative immunoassay for the detection of human chorionic gonadotropin (hCG; C6 subunit) in serum. The test uses a combination of monoclonal and polyclonal antibody reagents to selectively detect elevated levels of hCG. The Stanbio QuPID Plus Test for Pregnancy detects hCG concentrations of 20 mIU/ml or more in serum.
Urinary drug screens
It is important for subject safety as well as to avoid confounding of dependent variables to ensure that subjects have not used any drugs before administration of iv cocaine. On the morning of each study day, urine samples were collected and analyzed with a Triage screen. The Triage Panel for Drugs of Abuse (Biosite Diagnostics, San Diego, CA) is a rapid multiple immunoassay system for the qualitative detection of the major metabolites of these drugs of abuse in urine at the following cut-off concentrations (as recommended by the Substance Abuse and Mental Health Services Administration): phencyclidine, 25 ng/ml; benzodiazepines, 300 ng/ml; cocaine, 300 ng/ml; amphetamines, 1000 ng/ml; tetrahydrocannabinol, 50 ng/ml; opiates, 300 ng/ml; and barbiturates, 300 ng/ml.
Cocaine hydrochloride and placebo preparation
Cocaine hydrochloride was acquired from the NIDA in powder form and was dissolved in sterile water for iv injection. Sterility was ensured by filtration through a 0.22-µm filter (Millipore Corp., Bedford, MA) and verification with the limulus amebocyte lysate test (BioWhittaker Bioproducts, Walkersville, MD) for detection of Gram-negative bacterial endotoxins. A commercial preparation of 0.9% saline (1 ml) in sterile vials was used for the placebo injection.
ACTH RIA procedures
Plasma ACTH concentrations were measured in duplicate with an immunoradiometric assay (Nichols Institute Diagnostics, Allegro, CA) sensitive to 0.15 pmol/liter and displaying intra- and interassay coefficients of variance of 2.9% and 8.2%, respectively.
Plasma cocaine analysis
Plasma cocaine concentrations were measured in duplicate with a solid phase extraction method described by SPEC Instruction Manual by Ansys with a gas chromatograph (model 5890 series, Hewlett-Packard Co., Palo Alto, CA) equipped with a capillary column and a mass selection detector (5971 series, Hewlett-Packard Co.). The assay sensitivity was 10 ng/ml, and the intra- and interassay coefficients of variance were 2.0% and 2.5%, respectively.
Cortisol assay
Plasma cortisol concentrations were determined in duplicate with GammaCoat RIA kits (DiaSorin, Inc., Stillwater, MN). The assay sensitivity was 16.55 nmol/liter, and the intra- and interassay coefficients of variance were 5.0% and 8.4%, respectively.
IL-6 assay
Serum IL-6 was quantified at 1:1 and 1:4 dilutions with the High-Sensitivity Interleukin-6 Kit purchased from Endogen, Inc. (Cambridge, MA). The assay sensitivity was 0.16 pg/ml, and the intra- and interassay coefficients of variance were 5.1% and 3.4%, respectively.
DHEA assay
Plasma concentrations of DHEA were measured in duplicate with a coated tube RIA (Diagnostic Systems Laboratories, Inc., Webster, TX). The assay sensitivity was 0.02 ng/ml, and intra- and interassay coefficients of variance were 6.5% and 9.0%, respectively.
Estradiol assay
The serum estradiol concentration was determined in duplicate with direct, double antibody RIA kits (Diagnostic Products, Los Angeles, CA). The assay sensitivity was 1.1 pg/ml, and the intra- and interassay coefficients of variation were 2.0% and 7.8%, respectively.
Progesterone assay
The serum progesterone concentration was determined in duplicate with Coat-A-Count RIA kits (Diagnostic Products). The assay sensitivity was 0.06 ng/ml, and the intra- and interassay coefficients of variation were 4.0% and 5.1%, respectively.
Data analysis
Plasma ACTH, cortisol, and cocaine values for subjects were analyzed using a 2 (group) x 14 (time) repeated measures ANOVA. DHEA values were analyzed using a 2 (group) x 9 (time) repeated measures ANOVA. For the entire sample of 30 subjects, IL-6 values were available only at baseline (-20 min) and at the end point (240 min); therefore, differences between the cocaine and placebo groups in IL-6 response (defined as end point minus baseline levels) were determined by one-way ANOVA. For the subgroup of 14 male subjects, however, we also had IL-6 values at 30, 60, 120, and 180 min. Therefore, we compared the area under the curve (AUC) for IL-6 levels in the 7 men administered cocaine vs. the 7 given placebo. The AUC was calculated using the trapezoidal rule approximation, unequal intervals, with PHARM/PCS version 4.2 software (Microcomputer Specialists MCS, Philadelphia, PA), which is based upon the Manual of Pharmacologic Calculations with Computer Programs (36). The AUC values for men in the two treatment groups were then compared by one-way ANOVA. Estradiol and progesterone baseline values in the female subjects were analyzed by one-way ANOVA.
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Results |
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Placement of the indwelling catheter in the antecubital vein was followed by increased serum concentrations of IL-6. This elevation of IL-6 was significantly blunted 240 min after cocaine administration to all subjects (3.85 ± 0.49 pg/ml) vs. placebo (11.64 ± 2.21 pg/ml; P = 0.0019). In the 14 men, IL-6 was 68% lower after cocaine than after placebo (4.21 ± 0.73 vs. 12.94 ± 3.68 pg/ml; P = 0.038). Examination of the serial IL-6 levels in the 7 men receiving placebo suggested that IL-6 typically rose sharply beginning between 150 and 210 min after catheter placement (corresponding to 120–180 min postinjection). In the 7 men receiving cocaine, a much more modest rise in IL-6 occurred, also typically 150–210 min after catheter placement and approximately 80–140 min after peak cortisol release (see below). The AUC for the 7 men who received placebo was more than twice as great as that in the 7 men who received cocaine (1317 ± 1153 vs. 540 ± 303), but did not reach statistical significance (T = 1.72; df = 12; P = 0.11). This difference did not reach statistical significance, perhaps due to the small numbers in this comparison. In the 16 women, IL-6 was 66% lower after cocaine than after placebo (3.53 ± 0.68 vs. 10.50 ± 2.80 pg/ml; P = 0.030; Fig. 2). No significant differences were observed across gender for IL-6 release 240 min after administration of cocaine (P = 0.60) or saline placebo (P = 0.60).
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Plasma levels of ACTH, cortisol, and DHEA did not change upon iv injection of saline placebo (Fig. 1). Each of these significantly increased from baseline in both men and women after an iv dose of 0.4 mg/kg cocaine (P < 0.002). No gender differences occurred over the sampled time course for plasma cocaine, ACTH, or DHEA after administration of 0.4 mg/kg cocaine, iv. Males did have significantly higher cortisol levels vs. females at 12, 15, 20, and 30 min after 0.4 mg/kg cocaine, iv (P < 0.05). For males and females combined, peak plasma concentrations of cocaine (243 ± 17 ng/ml), ACTH (21.9 ± 4.0 pmol/liter), cortisol (483 ± 44 nmol/liter), and DHEA (17.1 ± 1.3 ng/ml) occurred at 8, 12, 40, and 20 min, respectively. In males alone, peak plasma concentrations of cocaine, ACTH, cortisol, and DHEA were 234 ± 20 ng/ml, 26.2 ± 4.6 pmol/liter, 572 ± 46 nmol/liter, and 15.4 ± 2.1 ng/ml. In females alone, peak plasma concentration of cocaine, ACTH, cortisol, and DHEA were 250 ± 27 ng/ml, 18.1 ± 6.2 pmol/liter, 406 ± 63 nmol/liter, and 17.6 ± 2.1 ng/ml (Fig. 1). All of these substances returned to baseline values within 180 min postinfusion, whereas IL-6 release increased, as noted above.
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Eight (50%) of the 16 women were in the follicular phase and 8 were in the luteal phase of the menstrual cycle on the basis of self-reports of last onset of menses. As expected, average estradiol and progesterone levels were significantly lower in the women in the follicular phase than in those in the luteal phase [estradiol, 40.7 ± 7.7 vs. 86.4 ± 11.3 pg/ml (P = 0.005); progesterone, 0.62 ± 0.10 vs. 6.60 ± 1.28 ng/ml (P = 0.0004)]. There were no significant differences over the sampled time course in plasma cocaine, ACTH, cortisol, and DHEA and serum IL-6 responses between the women studied in the follicular or luteal phase of the menstrual cycle, so these data were combined in Figs. 1
and 2.
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Discussion |
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The results of the present study suggest that cocaine blunts IL-6 release via cocaine’s stimulation of the HPA axis, which results in the release of glucocorticoids such as cortisol. Approximately 2.5 h after peak cortisol release in the cocaine-administered group, IL-6 release is blunted. Under placebo test conditions, ACTH, cortisol, and DHEA remain close to baseline values, and IL-6 release increases, with the highest values noted 4.5 h after catheter placement. Increased cortisol levels in humans are associated with decreased peripheral production of IL-6 (40, 41) as well as with other immunosuppressive effects (42, 43), and CRF inhibits endotoxin-evoked IL-6 production from human mononuclear cells (reversed with a specific CRF receptor antagonist) (44). It is difficult to quantitate the clinical significance of IL-6 levels in this or other studies; of note, however, is a recent report that elderly men have higher sensitivity to glucocorticoids than young men, resulting in suppressed proinflammatory cytokine production (such as lower lipopolysaccharide-stimulated IL-6 release from monocytes) (45) and hence perhaps increasing susceptibility of the elderly to infections. Similarly, in his recent review of the relationships among cytokines, neural circuits, and the HPA axis, Rivest (21) notes that "inappropriate plasma levels of glucocorticoids may play a crucial role contributing to deviant regulation of the immune response, indicating the importance to identify and characterize the mechanisms through which inflammatory molecules interact with, and depend on, the neuroendocrine system."
There has been considerable recent interest in determining whether cocaine has different effects on men and women or in women as a function of phase of the menstrual cycle. There is evidence of gender and menstrual cycle phase differences in some behavioral effects of cocaine (46) and in the degree to which cocaine stimulates LH release (10). Plasma concentrations of cortisol were significantly lower in women compared with men at 12, 15, 20, and 30 min after cocaine infusion (P = 0.05). The impact such differences may have upon immunity and other neuroendocrine functions is not clear, especially as no gender differences were observed at other time points for cortisol (including the time of peak concentration) or for plasma cocaine, ACTH, or DHEA. Overall, then, these findings appear consistent with prior reports that the pharmacokinetic properties of cocaine do not appreciably differ between men and women, or between women in the follicular or luteal phase of the menstrual cycle (47). Because rates of HIV infection appear to be increasing in heterosexual women at a higher rate than in heterosexual men (48), we were interested to learn whether cocaine had different effects on one measure of immune competency, IL-6, as a function of gender. We also compared cocaine’s effects on IL-6 in women at two phases of the menstrual cycle to determine whether there were detectable differences in susceptibility. Our results indicate that the decrease in IL-6 release by cocaine is not gender specific and suggest that women do not differ in IL-6 release when cocaine or saline is administered in either phase of the menstrual cycle. The power of these comparisons between women in the two phases of the cycle is limited, however, by the small number of subjects (eight in each phase), so that a type II error cannot be excluded.
The principal limitation of the present study is the use of an indwelling venous catheter as a model for immune/inflammatory stress. Although well documented (35), it is not certain that this model correlates to the immune responses that follow challenge by viruses, other natural antigens, or inflammatory agents. This question could be resolved by assessing whether cocaine suppresses IL-6 in response to natural agents in the same manner that it did in our indwelling venous catheter model. This study also does not address the effects of long-term cocaine exposure on HPA axis activation. Nevertheless, the benefits of this model include a minimally invasive, easily reproducible method by which rapid results can be obtained in intact humans on a wide variety of cytokines and other indexes of immunological and neuroendocrine functioning. Indeed, our results suggest a mechanism by which cocaine abuse in humans leads to compromised host defenses.
Several strategies should be considered in further investigations. First, it is possible that cocaine has a less significant impact on IL-6 release when ingested by intranasal insufflation as opposed to the iv route used in our study; this possibility should be tested. Second, our study measured only one cytokine; subsequent studies should examine a broad array of cytokines, such as proinflammatory IL-1, TNF, and IFN-. Finally, it would also be desirable to compare ambient IL-6 levels in users and nonusers of cocaine to better elucidate the persistence of any immune deficits/alterations from cocaine use.
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Acknowledgments |
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Footnotes |
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Abbreviations: AUC, Area under the curve; CRF, corticotropin-releasing factor; DHEA, dehydroepiandrosterone; hCG, human chorionic gonadotropin; HIV, human immunodeficiency virus; HPA, hypothalamic-pituitary-adrenal; IFN-, interferon-.
Received May 24, 2002.
Accepted November 20, 2002.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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production. Cell Immunol 172:217–223[CrossRef][Medline]
after acute psychological stress, exercise, and infused isoproterenol: differential effects and pathways. Psychosom Med 62:591–598This article has been cited by other articles:
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