Effects of cocaine administration to influenza virus-immunized mice on cytokine profiles of individual splenic CD4+ and CD8+ T cells

Abstract

We have analysed the effects of cocaine, administered to mice during the in vivo differentiation of effector T cells stimulated by antigen (influenza virus) recognition, on the frequency of IL-2-, IL-4- and interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ T cells. Each animal was injected intraperitoneally with 10 mg/kg of cocaine 6, 24, 48 and 72 h after immunization with A/PR8 influenza virus (PR8). This enabled the determination of the pharmacological effects of cocaine on T cells during the initial step of the immune response, which is characterized by the production of large amounts of immunoregulatory cytokines. The distribution of IL-2-, IL-4- and IFN-gamma-producing CD4+ and CD8+ T cells was assayed on unseparated PR8-immune spleen cells, obtained from mice treated with cocaine or vehicle, and restimulated in vitro with UV-inactivated PR8 virus. The frequency of T cells singly or co-expressing the above three cytokines was determined at single-cell level by simultaneous flow cytometric analysis of intracellular cytokines and surface antigen expression. In parallel, the levels of IL-2, IL-4 and IFN-gamma in the culture supernatants were quantified by ELISA. The results showed that cocaine, administered during the in vivo virus-induced differentiation of T cells, caused an increase of both the frequencies of CD8+ T cells singly and co-expressing IL-2 and IFN-gamma and the levels of these cytokines in virus-restimulated spleen cell culture supernatants, compared with those of untreated controls. In contrast, no effect was found on IL-4-positive CD8+ T cells and on IL-2-, IFN-gamma- and IL-4-positive CD4+ T cells. Our findings suggest that the immunomodulatory effects of cocaine may be due to the up-regulation of the production of IL-2 and IFN-gamma by CD8+ T cells with a type 0 cytokine profile.

Fig. 1

Effects of cocaine administration to influenza virus-immunized mice on the frequency of IL-2-, IL-4- or IFN-γ-expressing CD8⁺ and CD4⁺ T cells, as determined by two-colour flow cytometry analysis. The percentage of cytokine-producing cells was evaluated on in vitro PR8-restimulated spleen cells derived from PR8-immunized animals treated (▪) or not (□) with cocaine. All the samples were analysed for their CD4 or CD8 surface antigen expression versus intracellular IL-2 (a), IFN-γ (b) and IL-4 (c) expression. Samples were analysed by first gating on the CD4⁺ or CD8⁺ population. The threshold for detection of positive cytokine-producing cells was set at 1% above the negative control consisting of cells treated with an isotype-specific antibody. Results represent the mean + s.e.m. for each group. The spleen cells from 19 and 20 individual mice were used for the control and the cocaine-treated group, respectively. Statistical analysis (Student's t-test) was performed comparing cocaine-treated animals with untreated animals. ***P < 0.001. NS, Not significant.

Fig. 2

Effects of cocaine administration to influenza virus-immunized mice on the frequency of CD8⁺ T cells expressing one or more of the cytokines IL-4, IL-2 and IFN-γ, as determined by three-colour flow cytometry analysis. The percentage of cytokine-producing cells was evaluated on in vitro PR8-restimulated spleen cells derived from PR8-immunized animals treated (▪) or not (□) with cocaine. All the samples were analysed for single and combinatorial (IFN-γ/IL-2 (a), IL-2/IL-4 (b) and IFN-γ/IL-4 (c)) cytokine expression. Samples were analysed by first gating on the CD8⁺ population. The threshold for detection of positive cytokine-producing cells was set at 1% above the negative control consisting of cells treated with an isotype-specific antibody. Results represent the mean + s.e.m. for each group. The spleen cells from six and eight individual mice were used for the control and the cocaine-treated group, respectively. Statistical analysis (Student's t-test) was performed comparing cocaine-treated animals with untreated animals. *P < 0.05; **P < 0.01. NS, Not significant.

Fig. 3

Effect of cocaine administration to influenza virus-immunized mice on in vitro production of cytokines by spleen cells. The levels of IL-2 (a), IFN-γ (b), and IL-4 (c) were determined by ELISA in culture supernatants of PR8-restimulated spleen cells derived from cocaine-treated or untreated PR8-immunized animals. Results represent the mean + s.e.m. for each group. The spleen cells from 12 individual mice were used for each group. Statistical analysis (Student's t-test) was performed comparing cocaine-treated animals with control animals. *P < 0.05. NS, Not significant.

Fig. 4

Immunophenotypic analysis of splenic T cells derived from PR8-immunized animals treated (▪) or not (□) with cocaine. The immunophenotypes were determined by FACS analysis and expressed as percentages of total lymphocytes. All the samples were analysed for both CD4⁺ and CD8⁺ expression. Results represent the mean values + s.e.m. for each group. The spleen cells from 12 individual mice were used for each group. Statistical analysis (Student's t-test) was performed comparing cocaine-treated animals with untreated animals. NS, Not significant.