Regional heterogeneity and diversity in cytokine and chemokine production by astroglia: differential responses to HIV-1 Tat, gp120, and morphine revealed by multiplex analysis

Abstract

HIV-infected individuals who abuse opiates show a faster progression to AIDS and higher incidence of encephalitis. The HIV-1 proteins Tat and gp120 have been shown to cause neurodegenerative changes either in vitro or when injected or expressed in the CNS, and we have shown that opiate drugs can exacerbate neurotoxic effects in the striatum through direct actions on pharmacologically discrete subpopulations of mu-opioid receptor-expressing astroglia. Opiate coexposure also significantly enhances release of specific inflammatory mediators by astroglia from the striatum, and we theorize that astroglial reactivity may underlie aspects of HIV neuropathology. To determine whether astroglia from different regions of the central nervous system have distinct, intrinsic responses to HIV-1 proteins and opiates, we used multiplex suspension array analyses to define and compare the inflammatory signature of cytokines released by murine astrocytes grown from cerebral cortex, cerebellum, and spinal cord. Results demonstrate significant regional differences in baseline secretion patterns, and in responses to viral proteins. Of importance for the disease process, astrocytes from all regions have very limited inflammatory response to gp120 protein, as compared to Tat protein, either in the presence or absence of morphine. Overall, the chemokine/cytokine release is higher from spinal cord and cortical astroglia than from cerebellar astroglia, paralleling the relatively low incidence of HIV-related neuropathology in the cerebellum.

Figure 1

Basal levels (pg/ml) of individual chemokines/cytokines in medium from untreated astroglial cultures from three different brain regions. All values are expressed as the mean ± S.E.M. N=5 independent cultures from each brain region. Statistical analysis (one-way MANOVA) revealed significant differences between the three brain regions for GM-CSF, IFN-γ, RANTES, MCP-1, IL-9, and TNF-α; post hoc Bonferroni's test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 2

Regional heterogeneity of cytokine secretion: Main effects and Brain Region × Tat interactive effect. N=5 independent cultures from each brain region. A. Significant brain region effects were observed for all cytokines, except for IL-1β (see Table 1). ¹Post hoc analyses revealed significantly higher expression of all analytes in cortical as compared to cerebellar cultures (p < 0.001), except for IL1-β, IFN-γ, and KC. ²Post hoc analyses revealed significantly higher expression in spinal cord as compared to cerebellar cultures for IFN-γ (p ≤ 0.001), KC (p ≤ 0.001), MIP-1α (p ≤ 0.023), RANTES (p ≤ 0.001), and MCP-1 (p ≤ 0.01). ³Post hoc analyses revealed significantly higher expression in cortical as compared to spinal cord cultures for IFN-γ (p ≤ 0.01), TFN-α (p ≤ 0.001), IL-6 (p < .001), MIP-1α (p ≤ 0.001), eotaxin (p ≤ 0.001), MIP-1β (p ≤ 0.001), MCP-1 (p ≤ 0.019), and IL-9 (p ≤ 0.001). B. Significant Tat treatment effects were observed for all analytes (**TFN-α, p ≤ 0.01, ***all other cytokines, p ≤ 0.001; see Table 1). Tat treatment increased chemokine/cytokine levels compared to control conditions. C-E. A significant Brain Region × Tat interaction effect revealed significance [F(24, 262) = 8.42, p < .001], except for IFN-γ (see Table 1). Post hoc analyses for the Brain Region × Tat interaction effect, using Bonferroni as a correction factor, are indicated in Table 3. The largest effect of Tat was observed in the cortical cultures. Cerebellar cultures were least affected.

Figure 3

Measurements of viral protein and opiate effects on 12 chemokine/cytokines in cultures from each brain region. To accommodate the wide range of values for individual analytes, three graphs (A-C) represent different levels of expression for each brain region. Statistical analysis (3-way MANOVA) revealed significant Tat effects for each brain region. Table 2 gives the F values and significance level for each chemokine/cytokine. Figure 1 gives the actual values (pg/ml) of individual chemokines/cytokines. M = morphine; Nal = naloxone. Error bars represent S.E.M. N=5 independent cultures from each brain region. ¹Significant Tat effect for all 12 chemokines/cytokines (see Table 2 for F values and significance level) ²Significant Tat effect for all chemokines/cytokines, except for IFN-γ and TNF-α (see Table 2 for F values and significance level) Cortex: Tat significantly increased expression of all 12 analytes. The Tat effect was not altered by concurrent gp120 and/or morphine treatment. Significance values are given in Table 2. Cerebellum: Tat increased the expression of 10 out of 12 analytes. The exceptions were IFN-γ and TNF-α, whose levels were unchanged. The Tat effect was not altered by concurrent gp120 and/or morphine treatment. Significance values are given in Table 2. Spinal Cord: Tat significantly increased expression of all 12 analytes. The Tat effect was not altered by concurrent gp120 and/or morphine treatment. Significance values are given in Table 2.

Figure 4

Analysis of [Ca²⁺]_i activation in astrocytes after exposure to morphine or gp120. Cultures of enriched primary astrocytes derived from cerebral cortex were grown to 80-90% confluence, then loaded with 10μM fura-2/AM. Ratiometric Ca²⁺ measurements were made before (7.3 sec, 5 timepoints) and after (∼5 min) injection of morphine (500 nM, final concentration; filled circles) or gp120 (500 pM, final concentration; filled squares). Results are reported as the mean 340/380 ratio, and represent N = 6 samples per treatment group ± S.E.M. A two-way mixed ANOVA [treatment and time] showed that either morphine or gp120 significantly increased (p ≤ 0.001 and p ≤ 0.05, respectively) the 340/380 ratio compared to cells treated with vehicle (open circles). Morphine effects were noted immediately after exposure and remained stable over the 5 min recording period, while gp120 effects reached significance after 2 min.