Cocaine administration increases CD4/CD8 lymphocyte ratio in peripheral blood despite lymphopenia and elevated corticosterone

International Immunopharmacology 10 (2010) 1229–1234

Contents lists available at ScienceDirect

International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p

Cocaine administration increases CD4/CD8 lymphocyte ratio in peripheral blood despite lymphopenia and elevated corticosterone Maciej M. Jankowski a,⁎, Bogna Ignatowska-Jankowska a, Wojciech Glac a, Artur H. Swiergiel a,b a b

Department of Animal Physiology, University of Gdansk, Gdansk, Poland Department of Animal Behavior, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, Poland

a r t i c l e

i n f o

Article history: Received 21 January 2010 Received in revised form 1 July 2010 Accepted 2 July 2010 Keywords: Cocaine Immune system Intravenous Lymphocyte subset Peripheral blood Rat

a b s t r a c t The CD4/CD8 lymphocyte ratio in peripheral blood is used in the diagnosis of HIV infection, autoimmune disorders or susceptibility to infections. The present experiment aimed to evaluate the lymphocyte subsets, their distribution and CD4/CD8 ratio in blood after repeated, intravenous administration of cocaine. Adult male Wistar rats received three daily, in 30 min intervals, intravenous infusions of cocaine hydrochloride (5 mg/kg) or saline for 14 consecutive days. After each infusion the locomotor-activating effects of cocaine were assessed. Blood samples were collected 30 min after the last daily infusion on the 1st, 7th and 14th day of treatment. Total leukocyte numbers, percentages of leukocyte subpopulations, and T, B, NK, T CD4+, and T CD8+ lymphocyte subsets, IFN-γ, and plasma corticosterone were determined. Repeated cocaine treatment resulted in an increase in neutrophil numbers and a significant decrease in total leukocyte and lymphocyte numbers involving a significant reduction in numbers of T, B, and NK lymphocyte subsets. T CD4+ and T CD8+ lymphocyte numbers were reduced but with a considerably smaller decrease in T CD4+ number. Cocaine treatment altered proportions between the lymphocyte subsets by decreasing the percentages of T CD8+, B, and NK cells but increasing a percentage of T CD4+ cells. Destabilization in proportions between T CD4+ and T CD8+ was manifested as an elevated CD4/CD8 ratio that occurred despite increased plasma corticosterone and the lymphocytopenia. Cocaine did not affect the concentration of IFN-γ. The results suggest that although cocaine induced lymphopenia, it did not suppress the overall immune activity in terms of the CD4/CD8 ratio. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Cocaine is a potent stimulant of the central nervous system (CNS) [1] but it is also known to affect immune function [2–5]. In comparison with other intravenous drug abusers, cocaine users display increased risk for human immunodeficiency (HIV) and hepatitis C virus (HCV) infection [5]. Addicts show faster progression of HIV infection and increased incidence of acquired immunodeficiency syndrome (AIDS) [6] suggesting that cocaine use results in a functional impairment of the immune system. Increased susceptibility for HIV infection, which selectively involves T CD4+ lymphocyte subset [7], suggests that cocaine may affect peripheral blood lymphocyte subsets. The CD4/CD8 lymphocyte ratio in blood is used in the diagnosis of HIV infection [8–10], evaluation of pathogenesis of HCV infection [11] and autoimmune disorders such as rheumatoid arthritis or vitiligo [12,13]. The present study aimed to evaluate changes in the numbers and proportions of the peripheral blood B, NK, T CD3+, T CD4+ and T CD8+ lymphocyte subsets, and CD4/CD8 ratio following repeated ⁎ Corresponding author. Department of Animal Physiology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. Tel.: +48 585236305; fax: +48 585236422. E-mail address: [email protected] (M.M. Jankowski). 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.07.003

intravenous cocaine administration in rats. Concentration of IFN-γ in blood plasma was measured as a marker of basal and, possibly, cocainerelated activity of T CD4+, T CD8+ and NK cells in peripheral blood, and susceptibility to infection. Plasma corticosterone concentration was aimed to evaluate alterations in the activity of hypothalamic-pituitaryadrenal (HPA) axis after repeated ‘binge’ cocaine administration. To assure the effectiveness of the cocaine dose and characterize any habituation to cocaine during the 14 days of treatment, locomotor activity during and after cocaine administration was monitored.

2. Materials and methods 2.1. Animals Adult male Wistar rats (250–300 g) were obtained from a licensed breeder (Grabowski Laboratory Animals Breeding, Gdansk, Poland). All rats were housed individually under standard temperature and humidity conditions under a 12/12 h light/dark cycle (lights on at 6:00 a.m.). Animals had free access to water and standard rodent feed except for the time of cocaine infusions and behavioral measurements. They were acclimated to the new environment and experimenter one week prior to the onset of the study. All procedures were

1230

M.M. Jankowski et al. / International Immunopharmacology 10 (2010) 1229–1234

performed with permission of the Local Ethical Committee for Animal Experiments (No 34/2008). 2.2. Jugular cannulation Animals were anesthetized with pentobarbital (60 mg/kg, ip, Vetbutal, Biowet Pulawy, Poland) and atropine sulfate (0.25 mg/kg, sc, Warsaw Pharmaceutical Company Polfa, Poland). The scalp and neck were shaved and disinfected, and a jugular vein was exposed over a collarbone on the right side of the neck. In the ligated vein a small incision was made and a sterile catheter (Standard Silicone Tubing, Helix Mark TM, inside diameter 0.51 mm, outside 0.94 mm) inserted. The free end of the catheter had a stainless steel connector to enable attachment of the catheter to a polyethylene (PE) tube connected to a sterile syringe used to flush the cannula. A 2 cm long incision was made along the mid-saggital line of the skull and four holes for mounting screws were drilled in the cranium. The connector was attached with dental acrylic (Duracryl Plus, Spofa Dental, Prague, Czech Republic) to the screws and skull. Catheter patency was verified and the incisions were treated with penicillin (Penicillinum procainicum, 1,200,000 IU, Polfa Tarchomin, Poland) and closed with collagenous sutures (Sutura No 5, LorcaMarin, Spain). 2.3. Recovery after surgery Rats were allowed to recover for at least 14 days prior to experimentation. During the first three days after surgery, every 12 h, each rat received 9.5 mg of ampicillin (Ampicillin 500 mg, Polfa Tarchomin, Poland) dissolved in 0.2 ml of 0.9% sterile NaCl to prevent inflammation. Catheters were flushed daily with sterile heparin (Heparinum natricum, Warsaw Pharmaceutical Company Polfa, Poland) solution (80 IU/ml) in a volume of 0.3 ml and then filled with 0.3 ml sterile streptase (Streptase 1,500,000 IU, ZLB Behring GmbH, Marburg, Germany) solution (8000 IU/ml). Beginning on the 7th day after the surgery catheters were flushed every three days with 0.4 ml of 0.9% sterile saline. Sterile, single use syringes were used to flash the catheters. All solutions used during the experiment were prepared in a laminar flow unit using sterilized plastic test tubes.

Fig. 1. Experimental arena used for simultaneous measurements of locomotor activity and repeated intravenous cocaine infusions in a freely moving rat. During infusion a sterile syringe with cocaine solution was attached to the free end of the PE tube leading outside of the chamber through a ventilation hole. 1, actometer; 2, plexiglas chamber; 3, cover; 4, PE tube; 5, washer; 6, bead; 7, stainless steel plug.

2.6. Leukocytes and leukocyte subpopulations Peripheral blood (10 μl) was diluted 20 fold with Türk solution and leukocytes were counted twice in a Neubauer hemocytometer. Leukocyte subpopulations were assessed by microscopic examination of peripheral blood smears stained in a centrifuge (Aerospray Slide Stainer, 7120 Wescor, USA) by the May–Grünwald and Giemsa methods. Two smears of each blood sample were prepared and the percentages of leukocyte subpopulations assessed on 200 cells per smear. Total numbers of lymphocytes or neutrophils were calculated by multiplying the total leukocyte number and percentage of lymphocytes or neutrophils. 2.7. Cytofluorographic analysis

2.4. Cocaine administration Cocaine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% sterile saline (5 mg/ml). Control rats were infused with saline. The infusions were performed in plexiglas chambers (43 × 43 × 20 cm) for measuring locomotor activity (Columbus Instruments, Opto-Varimex Minor, Columbus, OH, USA) (Fig. 1). For four consecutive days preceding cocaine administration the rats were placed in the chambers for the 90-min sessions to habituate to a new environment and to establish baseline locomotor activity assessed as a number of the horizontal infra red beam breaks. For 14 consecutive days following the habituation period, each rat received three intravenous infusions of cocaine (3 × 5 mg/kg/day, iv) or saline (3×1 ml/kg/day, iv) in 30 min intervals during the 90-min session performed every day between 11:00 and 16:00. Each infusion lasted about 15 s. Dose and route of cocaine administration were chosen based on our previous experiments with the same strain of rats and also in accordance with research reports from other laboratories. 2.5. Blood sampling Blood samples (0.5 ml) were collected by catheter 30 min after the last daily cocaine infusion on the 1st, 7th and 14th day of treatment. Blood was drawn using a sterilized PE tube connected to a sterile, single use syringe containing 20 μl of 10% EDTA.

Three color flow cytometry of blood samples was performed using the following reagents: IOTest CD3-FITC/CD45RA-PC7/CD161a-APC and CD3-FITC/CD4-PC7/CD8-APC (Beckman Coulter) for the determination of T, B, NK, and T CD4+ and T CD8+ lymphocytes, respectively. According to the manufacturer's instructions, each antibody (25 μl) was added to the separate whole blood samples (25 μl), mixed and incubated for 20 min in the darkness. After the first incubation erythrocytes were lysed (1 ml of Versalyse, Beckman Coulter) and the lymphocytes fixed (25 μl of Fixative Solution, Beckman Coulter), mixed and incubated for 10 min in the darkness. Flow cytometry was performed in a model FC500 cytometer (Beckman Coulter, Inc., Brea, CA, USA). During the assay, the percentage of tested cell phenotypes was assessed. Total numbers of lymphocyte subsets were calculated by multiplying the total lymphocyte number and percentage of each lymphocyte subset. 2.8. IFN-γ measurements Blood samples (0.4 ml) were centrifuged and plasma samples for determinations of IFN-γ and corticosterone concentrations were stored at −60 °C. Quantitative determination of IFN-γ concentration in rat plasma was performed using a specific commercial cytokine ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA). Assay procedure was performed according to the manufacturer's instructions and IFNγ concentrations were determined using a DTX 880 Multimode

M.M. Jankowski et al. / International Immunopharmacology 10 (2010) 1229–1234

Detector (Beckman Coulter, Inc., Brea, CA, USA) set to 450 nm. The assay sensitivity was 6 pg/ml. 2.9. Corticosterone measurement Quantitative determination of corticosterone concentration in rat plasma was performed using a commercial radioimmunoassay kit (ICN Biochemicals, Inc., Costa Mesa, CA, USA) and Wizard 1470 gamma counter (Pharmacia LKB, Turku, Finland). 2.10. Statistical analysis One- or two-way repeated measures analysis of variance (ANOVA), with treatment and day as factors, was used to analyze data. 3. Results Intravenous infusions of cocaine, in comparison to the control infusions of saline, significantly decreased the total leukocyte number in peripheral blood during the 14-day treatment [F (1,42) = 49.2, p b .0001] but no effect of the treatment time (day) was observed [F (2,42) = 0.33, p b .73] (Table 1). The decline in the total leukocyte number was mainly caused by the decrease in lymphocyte number [F (1,42) = 140, p b .0001]; no effect of time was observed [F (2,42) = 0.68, p b .52]. Neutrophil number was slightly but significantly elevated [F (1,42) = 5.49, p b .05] with no significant effect of treatment time [F (2,42) = 0.04, p b .96] (Table 1). Alterations in leukocyte and lymphocyte number were at comparable levels on the 1st, 7th and 14th day of cocaine treatment as opposed to the elevation of neutrophil number which was delayed and exhibited only on the 7th [F (1,14) = 10.1, p b .01] and 14th [F (1,14) = 7.8, p b .05] day of treatment. Cytofluorographic analysis revealed that lymphocyte depletion involved all examined lymphocyte subsets. Repeated intravenous cocaine significantly reduced the total number of T CD3+ lymphocytes in comparison to the control infusions [F (1,42)=71.4, p b .0001] with no effect of time [F (2,42)= 0.85, p b .44] (Table 1). Within T CD3+ lymphocyte subpopulation its two major subsets, T CD4+ and T CD8+ lymphocytes, were significantly reduced but with a considerably smaller decrease in T CD4+ number (T CD4+ percentage was increased while T CD8+ percentage was decreased). T CD4+ lymphocyte number was decreased [F (1,42)=36.2, pb .0001] and number of T CD8+ lymphocyte subset was also depleted [F (1,42)=105, pb .0001]. Time of treatment

1231

failed to affect T CD4+ [F (2,42)=0.71, pb .5] and T CD8+ [F (2,42)=0.28, pb .76] lymphocyte numbers during 14 days of cocaine administration (Table 1). Cocaine treatment also decreased the number of B (CD45RA+) lymphocytes [F (1,42)=77, pb .0001] with no significant time effect [F (2,42) = 1.31, p b .28]. NK (CD161a+) cell subpopulation number was also significantly decreased [F (1,42) = 26.7, p b .0001] independent of treatment day [F (2,42) = 0.05, p b .95] (Table 1). Repeated administration of cocaine increased the proportion of T CD3+ lymphocyte subset in peripheral blood [F (1,42) = 105, p b .0001] and the time of treatment altered that effect [F (2,42) = 4.11, p b .05] (Table 1). The increase in percentage of T CD3+ lymphocytes was induced by the significant increase in percentage of T CD4+ lymphocytes [F (1,42) = 91.6, p b .0001] despite the decreased T CD8+ subset [F (1,42) = 7.99, p b .01] (Table 1). Administration of cocaine induced destabilization in proportions between peripheral blood T CD4+ and T CD8+ lymphocyte subsets manifested as an elevated CD4/CD8 ratio [F (1,42) = 30.5, p b .0001] which remained unchanged through 14 days of cocaine treatment [F (2,42) = 0.19, p b .83] (Fig. 2). The time of cocaine treatment failed to affect T CD4+ and T CD8+ percentage [F (2,42) = 1.18, p b .32] and [F (2,42) = 0.007, p b .99], respectively. Additionally, intravenous cocaine induced a decrease in percentage of B (CD45RA+) lymphocytes during the cocaine treatment period in comparison to the control [F (1,42) = 18.2, p b .001] and, again, no effect of treatment time was observed [F (2,42) = 1.14, p b .33] (Table 1). Moreover, a slight but significant decrease in percentage of NK (CD161a+) cells was observed in cocaine treated animals [F (1,42) = 5.21, p b .05] independent of time [F (2,42) = 0.2, p b .82] (Table 1). IFN-γ concentration in rat plasma reached detectable level only in four samples from three control rats (6–13 pg/ml) and in six samples from four cocaine treated animals (6–78 pg/ml) on different days of procedure. In the remaining 29 samples IFN-γ concentration was below 6 pg/ml. Plasma corticosterone concentration significantly increased after cocaine treatment [F (1,42) = 12, p b .01] with a significant time effect [F (2,42) = 6.2, p b .01]. Comparisons made for each day separately revealed that cocaine was effective only on the 1st day of treatment [F (1,14) = 6.3, p b .05] (Fig. 3). Ambulatory activity in response to cocaine increased on average 40 times in comparison to the saline control [F (1,196) = 946, p b .0001] and time of treatment failed to alter that response [F (13,196) = 0.23, p b .99] (Fig. 4A). Subsequent responses to intravenous cocaine infusions measured also separately for each daily infusion were

Table 1 Effect of repeated intravenous saline or cocaine infusions (3 × 5 mg/kg/day, iv, at 30 min intervals) on the immune cell numbers and percentages in peripheral blood of rats. Results are expressed as means ± SEM; n = 8 for saline (control) or cocaine groups. Significant differences from saline controls are indicated, where ***p b .001, **p b .01 and *p b .05. Time of treatment (day) 1st day

7th day

14th day

Control

Cocaine

Control

Cocaine

Control

Cocaine

13,271 ± 666 9345 ± 365 3743 ± 862

8892 ± 535*** 4904 ± 288*** 3688 ± 350

12,563 ± 396 9157 ± 406 3182 ± 265

9866 ± 551*** 5021 ± 432*** 4482 ± 311**

12,849 ± 593 9598 ± 560 3132 ± 262

10,205 ± 614*** 5495 ± 510*** 4550 ± 434*

Numbers of cells of lymphocyte subsets CD3+ 5456 ± 235 CD4+ 3886 ± 180 + CD8 1752 ± 193 + CD45RA (B) 2149 ± 191 CD161a+ (NK) 420 ± 88

3440 ± 203*** 2601 ± 167*** 814 ± 74*** 847 ± 86*** 174 ± 30***

4942 ± 239 3541 ± 253 1767 ± 127 2379 ± 253 399 ± 54

3407 ± 307*** 2595 ± 226*** 786 ± 74*** 928 ± 88*** 161 ± 32***

5381 ± 250 3853 ± 266 1870 ± 116 2559 ± 321 394 ± 61

3587 ± 298*** 2802 ± 223*** 843 ± 66*** 1076 ± 120*** 175 ± 45***

Percentage of cells of lymphocyte subsets CD3+ 58.5 ± 1.8 CD4+ 41.8 ± 1.9 + CD8 18.8 ± 2.1 + CD45RA (B) 23.2 ± 2.2 CD161a+ (NK) 4.5 ± 0.9

70.2 ± 0.7*** 52.9 ± 0.8*** 16.5 ± 1.0** 17.2 ± 1.3*** 3.5 ± 0.5*

54.0 ± 1.4 38.4 ± 1.7 19.7 ± 1.8 25.7 ± 2.1 4.5 ± 0.7

67.7 ± 1.0*** 51.7 ± 0.8*** 15.7 ± 0.8** 18.8 ± 1.3*** 3.0 ± 0.5*

56.5 ± 1.9 40.2 ± 2.0 20.0 ± 1.8 26.0 ± 2.2 4.1 ± 0.6

65.6 ± 1.1*** 51.4 ± 1.4*** 15.7 ± 1.0** 19.7 ± 1.5*** 3.0 ± 0.5*

Cells/μl: Leukocytes Lymphocytes Neutrophils

1232

M.M. Jankowski et al. / International Immunopharmacology 10 (2010) 1229–1234

Fig. 2. Effects of repeated cocaine infusions (3 × 5 mg/kg/day, iv, at 30 min intervals) on CD4/CD8 ratio in rat peripheral blood. Cocaine significantly decreased T CD4+ and T CD8+ lymphocyte numbers but with a considerably smaller decrease in T CD4+ number (T CD4+ percentage was increased while T CD8+ percentage was decreased). Destabilization in proportions between T CD4+ and T CD8+ cell subsets manifested as an elevated CD4/CD8 ratio. Each group represents the mean ± SEM of 8 animals and significant differences from saline controls are indicated, where ****p b .0001.

comparable suggesting that this dose of cocaine (5 mg/kg) may be maximal because no habituation was evident. Body weight gain was reduced in cocaine treated animals [F (1,196) = 8.79, p b .01] and during 14 days of cocaine administration the effect was more pronounced [F (13,196) = 4.84, p b .0001] (Fig. 4B).

4. Discussion Repeated intravenous cocaine infusions resulted in a significant decrease in total leukocyte and lymphocyte numbers, an increase in neutrophil numbers and a significant reduction in total numbers of T, B, NK, and both T CD4+ and T CD8+ lymphocyte subsets in peripheral blood. Furthermore, cocaine treatment altered the proportions between lymphocyte subsets by increasing T CD3+ and T CD4+, and decreasing T CD8+, B and NK cell percentages. Destabilization in proportions between T CD4+ and T CD8+ was manifested as an

Fig. 3. Effects of repeated cocaine infusions (3 × 5 mg/kg/day, iv, at 30 min intervals) on plasma corticosterone concentration. Cocaine treatment significantly increased corticosterone concentration in comparison to control with a significant time effect. Comparisons made for each day separately revealed that cocaine was effective only on the 1st day of treatment. Each group represents the mean ± SEM of 8 animals and significant differences from saline controls are indicated, where *p b .05.

elevated CD4/CD8 ratio. Cocaine increased plasma corticosterone concentration and did not affect the secretion of IFN-γ. The reasons for increased susceptibility for HIV or HCV infections [5], faster progression of HIV infection and increased incidence of AIDS [6] in cocaine addicts are still unknown. Some experimental data suggest that cocaine or its metabolites can alter the functional capacity of specific and nonspecific immune cell responses. For example, cocaine affects lymphocyte proliferation, cytokine production, NK cell cytotoxicity, and phagocytic activity of macrophages and neutrophils [14–22]. Therefore, the current study was conducted to assess whether modulation of immune system induced by cocaine can be explained, in part, by changes in the numbers and proportions between lymphocyte subsets in peripheral blood. We were also interested whether alterations in lymphocyte subsets distribution can be presented as a change in a simple T lymphocyte derived measurement — CD4/CD8 ratio. To test this hypothesis we applied a rodent model of chronic ‘binge’ cocaine administration which can mimic the pattern often seen in human heavy cocaine addicts who make repeated intravenous administrations at short time intervals [23–26]. Also, the repeated daily cocaine administration was aimed to extend the duration of cocaine action on the immune system and to counteract its rapid metabolism to pharmacologically inactive metabolites [27]. The dose of cocaine (5 mg/kg) used in the experiment results in high concentrations of plasma cocaine [28] and elicits robust and persistent behavioral responses. Under these conditions, we have demonstrated that cocaine induces a destabilization in lymphocyte subsets in rat blood. In contrast, Ruiz et al. [29] and Chao et al. [30] showed that the distribution of peripheral blood lymphocytes in human patients positive for cocaine or its metabolites was not affected by cocaine. However, those results might be attributed to the uncontrolled dose range, duration, frequency, and route of administration of cocaine, or multi-drug use in tested patients. Hypothalamic-pituitary-adrenal (HPA) axis activation by cocaine and increased secretion of glucocorticoids [31–36] are major pathways through which the CNS modulates functions of the immune system [37,38]. Therefore, activation of the HPA axis may be involved in the regulation of immune cell distribution and function following cocaine exposure. It was reported that chronic cocaine administration in rodents resulted in fluctuations of HPA axis activation manifested as attenuation in the release of adrenocorticotropic hormone (ACTH) and corticosterone after 14 days of ‘binge’ cocaine treatment [26]. In the present experiment similar effects have been noticed with the lowest plasma corticosterone concentration detected on the 7th day of ‘binge’ cocaine administration. Attenuated increase in plasma corticosterone concentration on the 7th and 14th day corresponded with increased neutrophil numbers on the 7th and 14th day of treatment. In contrast, on the 1st day plasma corticosterone was significantly elevated and there were no changes in neutrophil number. Earlier occurrence of the attenuated increase in plasma corticosterone after repeated cocaine than reported by Zhou et al. [26] may be due to different routes of cocaine administration, dose and intervals between the subsequent doses. Elevation in neutrophil number on the 7th and 14th day suggested a possibility of inflammation but IFN-γ was not increased. IFN-γ was chosen because of its antiviral properties [39] and as a marker of basal and, possibly, cocaine-related activity of T CD4+, T CD8+ and NK cells in peripheral blood. Undetectable IFN-γ concentration suggests that although cocaine induced lymphopenia it did not affect the secretory activity of T CD4+, T CD8+ and NK cells in peripheral blood. Experimental data suggest that cocaine can alter the immune response and the number of immune cells but the effects seem to depend on conditions under which the drug was administered. The results obtained in the present study are consistent with reports of the decreased numbers of T CD3+, T CD4+, T CD8+, B, or NK cells after cocaine administration [4,21,40–42]. Reduction in number of all tested lymphocyte subpopulations may be due to increased cell death or migration to peripheral lymphoid tissues. However, alterations in lymphocyte subset distributions, particularly the elevation of

M.M. Jankowski et al. / International Immunopharmacology 10 (2010) 1229–1234

1233

Fig. 4. Effects of repeated cocaine infusions on ambulatory activity and body weight gain. (A) Locomotor-activating effects of cocaine were measured in 30 min intervals during and after each of the three daily infusions. Cumulative 90 min (3 × 30 min) activity score and activities during the 30 min consecutive periods are shown. (B) Percentage body weight gain in cocaine and saline treated animals. The plots include data obtained during the habituation period which preceded cocaine or saline treatment.

percentage portion of T CD4+ and CD4/CD8 ratio with a concomitant decrease in the number of all tested lymphocyte subsets are reported for the first time. There is some evidence that alterations in activation and proportion of T CD4+ lymphocytes may change the susceptibility to HIV-1 infection [43–45]. The results suggest that although cocaine induces secretion of glucocorticoids and lymphopenia, it does not necessarily suppress the overall immune activity in terms of the CD4/ CD8 ratio. Acknowledgments This research was supported by the Polish Ministry of Science and Higher Education Predoctoral Research Grants NN303 417137 and NN303 394036 to M. Jankowski and B. Ignatowska-Jankowska. Editorial assistance of Mr. Brandon Pearson of the University of Hawaii, Honolulu, is highly appreciated. References [1] Prakash A, Das G. Cocaine and the nervous system. Int J Clin Pharmacol Ther Toxicol 1993;31:575–81. [2] Pillai R, Nair BS, Watson RR. AIDS, drugs of abuse and the immune system: a complex immunotoxicological network. Arch Toxicol 1991;65:609–17. [3] Pacifici R, Di Carlo S, Bacosi A, Zuccaro P. Macrophage functions in drugs of abusetreated mice. Int J Immunopharmacol 1993;15:711–6. [4] Pacifici R, Fiaschi AI, Micheli L, Centini F, Giorgi G, Zuccaro P, et al. Immunosuppression and oxidative stress induced by acute and chronic exposure to cocaine in rat. Int Immunopharmacol 2003;3:581–92. [5] Pellegrino TC, Dunn KL, Bayer BM. Mechanisms of cocaine-induced decreases in immune cell function. Int Immunopharmacol 2001;1:665–75. [6] Cabral GA. Drugs of abuse, immune modulation, and AIDS. J Neuroimmune Pharmacol 2006;1:280–95.

[7] Seligmann M, Pinching AJ, Rosen FS, Fahey JL, Khaitov RM, Klatzmann D, et al. Immunology of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. An update. Ann Intern Med 1987;107:234–42. [8] Pahwa S, Read JS, Yin W, Matthews Y, Shearer W, Diaz C, et al. CD4+/CD8+ T cell ratio for diagnosis of HIV-1 infection in infants: Women and Infants Transmission Study. Pediatrics 2008;122:331–9. [9] Butt FM, Vaghela VP, Chindia ML. Correlation of CD4 counts and CD4/CD8 ratio with HIV-infection associated oral manifestations. East Afr Med J 2007;84: 383–8. [10] Shearer WT, Pahwa S, Read JS, Chen J, Wijayawardana SR, Palumbo P, et al. CD4/ CD8 T-cell ratio predicts HIV infection in infants: the National Heart, Lung, and Blood Institute P2C2 Study. J Allergy Clin Immunol 2007;120:1449–56. [11] Viso AT, de Castro Barbosa T, Yamamoto L, Pagliari C, Fernandes ER, Brasil RA, et al. Portal CD4+ and CD8+ T lymphocyte correlate to intensity of interface hepatitis in chronic hepatitis C. Rev Inst Med Trop São Paulo 2007;49:371–8. [12] Hussein MR, Fathi NA, El-Din AM, Hassan HI, Abdullah F, Al-Hakeem E, et al. Alterations of the CD4(+), CD8 (+) T cell subsets, interleukins-1beta, IL-10, IL17, tumor necrosis factor-alpha and soluble intercellular adhesion molecule-1 in rheumatoid arthritis and osteoarthritis: preliminary observations. Pathol Oncol Res 2008;14:321–8. [13] Pichler R, Sfetsos K, Badics B, Gutenbrunner S, Berg J, Auböck J. Lymphocyte imbalance in vitiligo patients indicated by elevated CD4+/CD8+ T-cell ratio. Wien Med Wochenschr 2009;159:337–41. [14] Di Francesco P, Pica F, Croce C, Favalli C, Tubaro E, Garaci E. Effect of acute or daily cocaine administration on cellular immune response and virus infection in mice. Nat Immun Cell Growth Regul 1990;9:397–405. [15] Vaz A, Lefkowitz SS, Lefkowitz DL. Effects of cocaine on the respiratory burst of murine macrophages. Adv Exp Med Biol 1993;335:135–42. [16] Phillips DL, Tebbett IR, Masten S, Shiverick KT. Stimulatory effects of cocaine and its metabolites on IM-9 human B-lymphoblastoid cells. Int J Immunopharmacol 1995;17:57–63. [17] Stanulis ED, Jordan SD, Rosecrans JA, Holsapple MP. Disruption of Th1/Th2 cytokine balance by cocaine is mediated by corticosterone. Immunopharmacology 1997;37:25–33. [18] Xu W, Bai F, Tummalapalli CM, Miller DD, Middaugh L, Boggan WO. The interactive effects of cocaine/gender on immune function in mice. An observation of in vivo acute cocaine exposure. Int J Immunopharmacol 1997;19:333–40.

1234

M.M. Jankowski et al. / International Immunopharmacology 10 (2010) 1229–1234

[19] Gan X, Zhang L, Newton T, Chang SL, Ling W, Kermani V, et al. Cocaine infusion increases interferon-gamma and decreases interleukin-10 in cocaine-dependent subjects. Clin Immunol Immunopathol 1998;89:181–90. [20] Mukunda BN, Callahan JM, Hobbs MS, West BC. Cocaine inhibits human neutrophil phagocytosis and phagolysosomal acidification in vitro. Immunopharmacol Immunotoxicol 2000;22:373–86. [21] Colombo LL, López MC, Chen GJ, Watson RR. Effect of short-term cocaine administration on the immune system of young and old C57BL/6 female mice. Immunopharmacol Immunotoxicol 1999;21:755–69. [22] Irwin MR, Olmos L, Wang M, Valladares EM, Motivala SJ, Fong T, et al. Cocaine dependence and acute cocaine induce decreases of monocyte proinflammatory cytokine expression across the diurnal period: autonomic mechanisms. J Pharmacol Exp Ther 2007;320:507–15. [23] Fischman MW, Schuster CR. Cocaine self-administration in humans. Fed Proc 1982;41:241–6. [24] Fischman MW, Schuster CR, Javaid J, Hatano Y, Davis J. Acute tolerance development to the cardiovascular and subjective effects of cocaine. J Pharmacol Exp Ther 1985;235:677–82. [25] Siegel RK. New patterns of cocaine use: changing doses and routes. NIDA Res Monogr 1985;61:204–20. [26] Zhou Y, Spangler R, Schlussman SD, Ho A, Kreek MJ. Alterations in hypothalamicpituitary-adrenal axis activity and in levels of proopiomelanocortin and corticotropin-releasing hormone-receptor 1 mRNAs in the pituitary and hypothalamus of the rat during chronic ‘binge’ cocaine and withdrawal. Brain Res 2003;964:187–99. [27] Morishima HO, Whittington RA, Iso A, Cooper TB. The comparative toxicity of cocaine and its metabolites in conscious rats. Anesthesiology 1999;90:1684–90. [28] Hutchaleelaha A, Sukbuntherng J, Mayersohn M. Simple apparatus for serial blood sampling in rodents permitting simultaneous measurement of locomotor activity as illustrated with cocaine. J Pharmacol Toxicol Meth 1997;37:9–14. [29] Ruiz P, Berho M, Steele BW, Hao L. Peripheral human T lymphocyte maintenance of immune functional capacity and phenotypic characteristics following in vivo cocaine exposure. Clin Immunol Immunopathol 1998;88:271–6. [30] Chao C, Jacobson LP, Tashkin D, Martínez-Maza O, Roth MD, Margolick JB, et al. Recreational drug use and T lymphocyte subpopulations in HIV-uninfected and HIV-infected men. Drug Alcohol Depend 2008;94:165–71. [31] Calogero AE, Gallucci WT, Kling MA, Chrousos GP, Gold PW. Cocaine stimulates rat hypothalamic corticotropin-releasing hormone secretion in vitro. Brain Res 1989;505: 7–11.

[32] Borowsky B, Kuhn CM. Monoamine mediation of cocaine-induced hypothalamo-pituitary-adrenal activation. J Pharmacol Exp Ther 1991;256:204–10. [33] Levy AD, Li QA, Kerr JE, Rittenhouse PA, Milonas G, Cabrera TM, Battaglia G. Alvarez Sanz MC, Van de Kar LD. Cocaine-induced elevation of plasma adrenocorticotropin hormone and corticosterone is mediated by serotonergic neurons. J Pharmacol Exp Ther 1991;259:495–500. [34] Sarnyai Z, Bíró E, Telegdy G. Cocaine-induced elevation of plasma corticosterone is mediated by different neurotransmitter systems in rats. Pharmacol Biochem Behav 1993;45:209–14. [35] Baumann MH, Gendron TM, Becketts KM, Henningfield JE, Gorelick DA, Rothman RB. Effects of intravenous cocaine on plasma cortisol and prolactin in human cocaine abusers. Biol Psychiatry 1995;38:751–5. [36] Bayer BM, Mulroney SE, Hernandez MC, Ding XZ. Acute infusions of cocaine result in time- and dose-dependent effects on lymphocyte responses and corticosterone secretion in rats. Immunopharmacology 1995;29:19–28. [37] Ader R, Felten D, Cohen N. Interactions between the brain and the immune system. Annu Rev Pharmacol Toxicol 1990;30:561–602. [38] Madden KS, Felten DL. Experimental basis for neural-immune interactions. Physiol Rev 1995;75:77–106. [39] Maher SG, Romero-Weaver AL, Scarzello AJ, Gamero AM. Interferon: cellular executioner or white knight? Curr Med Chem 2007;14:1279–89. [40] Klein TW, Matsui K, Newton CA, Young J, Widen RE, Friedman H. Cocaine suppresses proliferation of phytohemagglutinin-activated human peripheral blood T-cells. Int J Immunopharmacol 1993;15:77–86. [41] Lopez MC, Watson RR. Effect of cocaine and murine AIDS on lamina propria T and B cells in normal mice. Life Sci 1994;54:147–51. [42] Duncan R, Shapshak P, Page JB, Chiappelli F, McCoy CB, Messiah SE. Crack cocaine: effect modifier of RNA viral load and CD4 count in HIV infected African American women. Front Biosci 2007;12:1488–95. [43] Patterson BK, Landay A, Siegel JN, Flener Z, Pessis D, Chaviano A, et al. Susceptibility to human immunodeficiency virus-1 infection of human foreskin and cervical tissue grown in explant culture. Am J Pathol 2002;161:867–73. [44] Koning FA, Otto SA, Hazenberg MD, Dekker L, Prins M, Miedema F, et al. Lowlevel CD4+ T cell activation is associated with low susceptibility to HIV-1 infection. J Immunol 2005;175:6117–22. [45] Bégaud E, Chartier L, Marechal V, Ipero J, Léal J, Versmisse P, et al. Reduced CD4 T cell activation and in vitro susceptibility to HIV-1 infection in exposed uninfected Central Africans. Retrovirology 2006;3:35.