CCL5/RANTES gene deletion attenuates opioid-induced increases in glial CCL2/MCP-1 immunoreactivity and activation in HIV-1 Tat exposed mice

Abstract

To assess the role of CC-chemokine ligand 5 (CCL5)/RANTES in opiate drug abuse and human immunodeficiency virus type 1 (HIV-1) comorbidity, the effects of systemic morphine and intrastriatal HIV-1 Tat on macrophage/microglial and astroglial activation were assessed in wild-type and CCL5 knockout mice. Mice were injected intrastriatally with vehicle or Tat and assessed after 7 days. Morphine was administered to some Tat-injected mice via time-release implant (5 mg/day, s.c. for 5 days) starting at 2 days post injection. Glial activation was significantly reduced in CCL5(−/−) compared to wild-type mice at 7 days following combined Tat and morphine exposure. Moreover, the percentage of 3-nitrotyrosine immunopositive macrophages/microglia was markedly reduced in CCL5(−/−) mice injected with Tat ± morphine compared to wild-type counterparts, suggesting that CCL5 contributes to nitrosative stress in HIV-1 encephalitis. In CCL5(−/−) mice, the reductions in Tat ± morphine-induced gliosis coincided with significant declines in the proportion of CCL2/MCP-1-immunoreactive astrocytes and macrophages/microglia compared to wild-type counterparts. In knockout mice, neither Tat alone nor in combination with morphine increased the proportion of CCL2-immunoreactive astrocytes above percentages seen in vehicle-injected controls. Macrophages/microglia differed showing modest, albeit significant, increases in the proportion of CCL2-positive cells with combined Tat and morphine exposure, suggesting that CCL5 preferentially affects CCL2 expression by astroglia. Thus, CCL5 mediates glial activation caused by Tat and morphine, thereby aggravating HIV-1 neuropathogenesis in opiate abusers and non-abusers. CCL5 is implicated as mediating the cytokine-driven amplification of CCL2 production by astrocytes and resultant macrophage/microglial recruitment and activation.

1. Introduction

Opiate abuse can exacerbate the pathogenesis of human immunodeficiency type 1 (HIV-1) encephalitis (HIVE) through intrinsic actions largely at µ-opioid receptors (MOR) (Gurwell et al., 2001; Hauser et al., 2006; Hauser et al., 2007). There is considerable evidence that many of the deleterious effects of opiates are mediated through the modulation of immune function and opiates have been proposed as potential cofactors in the pathogenesis of AIDS (Donahoe and Vlahov, 1998). Not only are opiates reported to modulate HIV propagation in immune cells (Peterson et al., 1990; Carr and Serou, 1995; Carr et al., 1996; Sharp et al., 1998; Nyland et al., 1998; Wetzel et al., 2000; McCarthy et al., 2001), they can intrinsically modulate the function of macrophages and lymphocytes (Hemmick and Bidlack, 1991; Adler et al., 1993; Sharp, 2006; Eisenstein and Hilburger, 1998; Bidlack, 2000; Rahim et al., 2003). Opioids affect immune function through a variety of direct and indirect mechanisms. In fact, opioids and chemokine receptors undergo heterologous, bidirectional cross-desensitization (Rogers et al., 2000; Rogers and Peterson, 2003), which highlights just one of many reported mechanisms by which opioids modulate immunity (Bidlack et al., 2006; Machelska and Stein, 2006; Roy et al., 2006). Importantly, the effects of opiates are not restricted to a particular cell type, and chemokine ligands and functional chemokine receptors are expressed by a wide variety of neural (neurons, microglia, and astroglia) and non-neural cells (lymphocytes and monocytes/macrophages) (Rogers and Peterson, 2003; Bidlack et al., 2006; Turchan-Cholewo et al., 2008).

Chemokines are involved in a wide-variety of disorders in the CNS and their actions contribute to reactive glial changes and neuronal injury in a spectrum of diseases (Glass et al., 2003; Ransohoff et al., 2007; Allen et al., 2007; Balistreri et al., 2007), with neurotrauma (Mahad and Ransohoff, 2003), and HIV encephalitis (Nath, 1999; Miller and Meucci, 1999; Kaul et al., 2001). CC-chemokine ligand 5 [CCL5, also known as regulated on activation normal T-cell expressed and secreted (RANTES)], in particular, attracts and activates mononuclear phagocytes, as well as several other leukocyte types, to sites of injury or infection (Miller and Krangel, 1992; McManus et al., 2000). CCL5 is dramatically increased in the CNS of HIV infected individuals (Kelder et al., 1998; Sanders et al., 1998; Vago et al., 2001) and with SIV infection (Sasseville et al., 1996; Westmoreland et al., 1998; Klein et al., 1999). CCL5 preferentially activates its cognate receptor, CCR5, which is a significant co-receptor in HIV-1 pathogenesis and whose activation may alter HIV and simian immunodeficiency virus (SIV) pathogenesis (Sasseville et al., 1996; Kitai et al., 2000). CCL5 and/or CCR5 are expressed by astrocytes and are upregulated in response to a variety of CNS insults (Ghirnikar et al., 1996; Sun et al., 1997; Dorf et al., 2000; Boutet et al., 2001; Dong and Benveniste, 2001; Luo et al., 2002; Zhang et al., 2002; Lee et al., 2005). Besides its role as a chemokine, CCR5 is a cofactor for HIV entry into cells and can modulate HIV/SIV infectivity in the CNS and elsewhere (Edinger et al., 1997; Westmoreland et al., 1998; Albright et al., 1999; Klein et al., 1999; Kitai et al., 2000; Overholser et al., 2003).

Although the cellular mechanisms by which opiates exacerbate the neuropathogenesis of HIV-1 are incompletely understood, there is emerging evidence that astrocytes are the principal cellular site where opioid and HIV protein signals converge and the origin of synergistic increases in macrophage recruitment/microglial activation, inflammation, and neuronal injury caused by opiate abuse (Hauser et al., 2007). Exposure to HIV-1 Tat (Conant et al., 1998; Kutsch et al., 2000; El-Hage et al., 2005), or intact virions (Janabi et al., 1998), markedly increases the production of chemokines by astrocytes. In addition to their response to HIV-1 proteins, subsets of astroglia express functional µ opioid receptors (Eriksson et al., 1991; Stiene-Martin and Hauser, 1991; Hauser et al., 1996; Stiene-Martin et al., 1998; Stiene-Martin et al., 2001), and opiates markedly increase the production of CCL5, CCL2, and IL-6 by HIV-1 Tat exposed astrocytes (El-Hage et al., 2005). Furthermore, opiates increase CCR5 expression in U373 astrocytoma/glioblastoma cells (Mahajan et al., 2005). As noted, the chemokines produced can potentially interact with opioids and vice versa (Rogers and Peterson, 2003), and CCR5 undergoes heterologous sensitization with µ opioid receptors (Szabo et al., 2002; Rogers and Peterson, 2003; Szabo et al., 2003; Chen et al., 2004).

Despite findings that CCR2 deletion largely abolished HIV-1 Tat and opioid drug-induced glial activation (El-Hage et al., 2006a), we questioned whether alternative chemokines might contribute to reactive gliosis and CNS inflammation. This was prompted by findings that (1) opiates potentiate the production of CCL5 in HIV-1 Tat-exposed astrocytes, (2) Tat morphine-exposed CCL2 null astrocytes still retain significant chemokine activity (El-Hage et al., 2006b), and evidence that (3) CCL5 regulates and amplifies CCL2 production by astrocytes (Luo et al., 2002; Luo et al., 2003). For these reasons and because of the established importance of CCL5 in inflammation in a variety of systems, we examined cellular changes in CCL2 and glial activation in the striata of wild type and CCL5 null mice exposed to morphine and/or HIV-1 Tat. Because CCL5 can activate CCR1 and CCR3 in addition to CCR5 (Luo et al., 2002; Allen et al., 2007), knockout mice lacking the CCL5 ligand rather than the receptor were used to assess the role of CCL5 on Tat ± morphine-induced inflammation and reactive gliosis. The results suggest that CCL5 contributes significantly to increases in astroglial CCL2 and to the activation of macrophages and glia seen within the brains of HIV-1 infected drug abusers and non-abusers.

2. Materials and Methods

To generate CCL5 null mice, a targeting vector disrupting exon I of the CCL5 gene was transfected into 129P2 embryonic stem cells and transferred into C57Bl/6 embryos (Makino et al., 2002). CCL5(−/−) offspring were crossed and subsequently backcrossed at least 10 generations to C57BL/6J background strain mice (Makino et al., 2002). Wild type [CCL5(+/+)] and CCL5(−/−) mice obtained from the Jackson Laboratory (Bar Harbor, ME) (strain B6.129P2-^Ccl5tm1Hso/J), were bred and treated according to local IACUC guidelines regarding the humane care and use of animals.

Vehicle (placebo implant), morphine (25 mg), and/or naltrexone (30 mg) pelleted implants (NIDA, Rockville, MD, USA) were used to continuously deliver drugs for 5 days. In a paradigm established in previous studies, pellets were surgically implanted subcutaneously under the subscapular skin using anesthesia and aseptic conditions (El-Hage et al., 2006b). Intrastriatal HIV-1 Tat_1–72 (25 µg) or saline vehicle was injected at coordinates AP = +0.7 mm, ML = 2.0 mm and DV = −4.0 mm from bregma. Drug implants were administered 2 days after stereotaxic surgery and mice were euthanized by anesthesia overdose after 5 days of continuous drug exposure (El-Hage et al., 2006b). Treatment groups consisted of mice receiving: (1) placebo implant with intrastriatal vehicle; (2) placebo implant with intrastriatal Tat; or (3) morphine implant (25 mg/kg) with intrastriatal Tat.

Tissues were fixed in Zamboni’s modified, neutral phosphate-buffered paraformaldehyde (3%). Glial fibrillary acidic protein (GFAP), F4/80, MOR, and CCL2 were detected by indirect immunofluorescence in frozen sections (10–18 µm thick) as described previously (El-Hage et al., 2006b), with the exception that CCL5 immunoreactivity was additionally colocalized with GFAP or F4/80 in alternative sections. CCL5 primary antibodies were obtained from Santa Cruz Biotechnology (1:100 dilution; Santa Cruz, CA). GFAP and MOR primary antibodies were purchased from Chemicon (Chemicon International Inc., Temecula, CA), rabbit anti-3-NT was obtained from Upstate (1:500 dilution; Upstate, Charlottesville, VA), and rat anti-mouse F4/80 antibodies were obtained from Serotec (1:500 dilution, Serotec, Raleigh, NC). Sections were counterstained with Hoechst 33342 (15 µg/ml in 0.1% BSA in PBS for 15 min at room temperature; Molecular Probes) to detect cell nuclei.

A computer imaging system [Zeiss Axio Vision (version 4.6), Carl Zeiss Inc., Thornwood, NY] and fluorescent microscope (Zeiss Axio Observer Z1) equipped with a motorized X-Y and Z-axes stage controller were used to systematically, but arbitrarily, sample cells near (300 ± 100 µm) the site of Tat injection within the striatum as previously described (El-Hage et al., 2006a; El-Hage et al., 2006b). The proportion of GFAP and F4/80 immunoreactive astroglia and macrophages/microglia, respectively, with or without CCL2 immunoreactivity were assessed as previously described (El-Hage et al., 2006a; El-Hage et al., 2006b). To determine whether the inflammatory cascade involving CCL5 contributes to a neurotoxic phenotype in microglia, 3-NT immunoreactivity was evaluated in macrophages/microglia following intrastriatal Tat injection with or without morphine in wild type and CCL5(−/−) mice as described before (Bruce-Keller et al., 2008). Five to six mice were assessed for each experimental condition. Digital photomicrographs were acquired and cell-specific marker and chemokine immunofluorescence colocalized using a Zeiss Axio Observer Z1 microscope aided in some instances by using structured illumination microscopy (ApoTome, Zeiss) to acquire and reconstruct z-stacks in 3-dimensions (Bruce-Keller et al., 2008). Statistical differences were assessed by ANOVA and multiple comparisons assessed post-hoc using Duncan’s test.

3. Results

CCL5 gene deletion attenuates Tat ± morphine-induced increases in reactive macrophages/microglia and astrocytes

In wild type mice, Tat and morphine in combination significantly increased the proportion of F4/80 or GFAP immunoreactive macrophages/microglia or astroglia, respectively, 7 days after exposure and at loci 300 ± 100 µm distant from the Tat injection site [*P<0.05 vs. all other groups in wild type or CCL5(−/−) mice], while Tat alone did not (Fig. 1A–B). In contrast to observations in wild type mice, CCL5 gene deletion prevented combined Tat and morphine-induced increases in the proportions of F4/80 immunoreactive macrophages/microglia (Fig 1A) or GFAP immunoreactive astrocytes (Fig. 1B).

Figure 1.

CCL5 gene deletion significantly reduced the proportion of F4/80 immunoreactive macrophages/microglia (A) or GFAP immunoreactive astrocytes (B) following combined Tat and morphine exposure. The proportion of F4/80-immunoreactive macrophages/microglia (A) and glial fibrillary acidic protein (GFAP)-immunopositive astrocytes was significantly reduced in the striatum of CCL5 knockout mice exposed to HIV-1 Tat and opiates compared to wild type mice [*P<0.05 vs. all other groups in CCL5(+/+) or CCL5(−/−) mice].

CCL5 gene deletion reduces Tat ± morphine-induced increases in CCL2 immunoreactive macrophages/microglia and astrocytes

To characterize the role of CCL5 in regulating CCL2 production by macrophages/microglia and astroglia exposed to HIV-1 Tat ± opiates, wild type and CCL5 null mice were injected with HIV-1 Tat intrastriatally in the presence of placebo or morphine implants (Fig. 2). In wild type mice, CCL2 immunoreactivity was significantly increased in macrophages/microglia following Tat ± morphine treatment and in astrocytes after combined treatment with Tat and morphine [*P<0.05 vs. other treatments in wild type or CCL5(−/−) mice]. By contrast, CCL5 deletion significantly reduced the proportion of CCL2 immunoreactive F4/80+ macrophages/microglia (Fig. 2A) or GFAP+ astroglia compared to wild type mice exposed to combined Tat and morphine (Fig. 2B) (^#P<0.05 vs. wild type mice).

Figure 2.

CCL5 deletion significantly reduced the proportion of CCL2 immunoreactive macrophages/microglia (A) and astrocytes (B), respectively, in which F4/80 or GFAP immunoreactivity could be colocalized in response to Tat and/or combined Tat and morphine exposure (^#P<0.05 vs. equivalent treatment in wild type mice). In wild type mice, Tat and/or Tat plus morphine administration markedly increased the percentages of macrophages/microglia and astrocytes possessing CCL2 immunoreactivity [*P<0.05 vs. other groups in wild type or CCL5(−/−) mice; ^bP < 0.05 vs. vehicle- or Tat plus morphine-treated wild type mice], while in CCL5 null mice significant increases in CCL2 immunoreactivity were only seen in macrophages/microglia co-exposed to Tat and morphine (^§P<0.05 vs. vehicle injected CCL5 knockout mice).

Tat ± morphine exposure increases the proportion of CCL5 immunoreactive astroglia

CCL5 immunoreactivity (red immunofluorescence) is associated with GFAP-positive astrocytes (arrows; green immunofluorescence) in wild type (Fig. 3A–D) but not CCL5 null (Fig. 3E–F) mice. In wild type mice, Tat alone or in combination with morphine significantly increased the proportion of cells in which CCL5 was colocalized with GFAP at 7 days post injection compared to ipsilateral (ipsi), sham vehicle-injected or non-injected contralateral (contra) controls (Fig. 3G). We previously found that the primary injury associated with injection alone increased the proportion of CCL2 immunopositive astroglia compared to the proportion in the uninjected contralateral striatum at 7 days post injection (El-Hage et al., 2006a), and surmised that the percentage of CCL5 immunoreactive astrocytes might also be increased in the present study. Although the proportion of CCL5 immunoreactive astrocytes tended to increase from vehicle injection alone compared to the uninjected, contralateral striatum, the increase was not significant (Fig. 3G).

Figure 3.

CCL5 and 3-NT-immunoreactivity following intrastriatal Tat injection ± morphine in wild type and CCL5 null mice. CCL5 was colocalized within a subset of glial fibrillary acidic protein (GFAP) immunoreactive astrocytes (arrows) in the striatum of wild type (WT) mice receiving intrastriatal placebo (vehicle) (A,D) or Tat injections (B,E), but not CCL5 knockout (CCL5 KO) mice receiving Tat injections (C,F) at 7 days following Tat ± morphine exposure. In wild type mice, HIV-1 Tat ± morphine exposure dramatically increased the proportion of CCL5 immunoreactive astroglia compared to astrocytes in vehicle-injected controls or in the contralateral (uninjected) striatum (G) (*P < 0.05). The proportion of F4/80-immunoreactive macrophages/microglia co-expressing 3-NT was increased significantly following intrastriatal Tat injection (*P < 0.05), while coadministering morphine did not increase the percentage of 3-NT reactive macrophages/microglia beyond proportions seen with Tat alone (Fig. 3H). Importantly, deletion of the CCL5 gene significantly attenuated Tat or combined morphine and Tat-induced increases in 3-NT reactive macrophages/microglia (^#P<0.05 vs. equivalent treatment in wild type mice) (Fig 3H). Despite the absence of CCL5, 3-NT levels were nevertheless increased by Tat in knockout mice, suggesting factors besides CCL5 contribute to increases in the proportion of 3-NT macrophages/microglia (^§P<0.05 vs. vehicle injected CCL5 knockout mice).

CCL5 gene deletion limits Tat-induced increases in the proportion of 3-NT immunoreactive macrophages/microglia

We previously found that morphine treatment alone significantly increased the proportion of 3-NT-immunopositive Mac1-labeled macrophages/microglia in inducible Tat transgenic mice, while Tat induction alone had little effect (Bruce-Keller et al., 2008). Interestingly, however, Tat induction potentiated the effects of morphine. In combination, 3-NT was increased significantly above levels seen with morphine exposure alone (Bruce-Keller et al., 2008). In the present study, the proportion of F4/80-immunoreactive macrophages/microglia co-expressing 3-NT was increased significantly following intrastriatal Tat injection, while coadministering morphine did not increase the percentage of 3-NT reactive macrophages/microglia (Fig. 3H). Importantly, deletion of the CCL5 gene significantly attenuated Tat or combined morphine and Tat-induced increases in 3-NT reactive macrophages/microglia (Fig 3H). The findings indicate that CCL5 is a key proinflammatory signal mediating free radical production caused by HIV-1 Tat. Lastly, 3-NT immunoreactivity was not detected when non-specific staining was evaluated (data not shown).

The proportion of MOR immunoreactive astrocytes was unaffected by CCL5 deletion

To verify that patterns of expression of MOR were not dramatically altered in CCL5 knockout mice, MOR was assessed immunocytochemically. The pattern and intensity of MOR immunoreactivity was similar to that seen in wild type striatum, and appeared to be distributed similarly among neurons and glia. A brief assessment of the proportion of MOR immunoreactive (GFAP+) astrocytes yielded similar values in wild type (20.1 ± 5.6%) and CCL5 (−/−) (18.0 ± 5.3%) mice at locations 300 ± 100 µm distant from the Tat injection site at 7 days. Thus, patterns of MOR expression appeared to be qualitatively similar in wild type and CCL5 knockout mice.

4. Discussion

Our data indicate that CCL5 contributes to reactive astrogliosis and the recruitment and activation of macrophages or microglia, respectively, seen with HIV-1 Tat and combined morphine and Tat exposure. Importantly, in CCL5 null mice, the astroglial and macrophage/microglial responses to Tat ± morphine were eliminated indicating that CCL5 is necessary for certain aspects of glial reactivity. CCL5 gene deletion prevented Tat ± morphine-induced increases in astroglial CCL2, while significantly attenuating similar increases in CCL2 in macrophages or microglia. This suggests that CCL5 regulates CCL2 expression in astrocytes exposed to HIV-1 Tat ± opiates, which is responsible for reactive increases in the proportion of GFAP-positive astrocytes and macrophage recruitment/microglial activation. Thus, our findings implicate CCL5 as a critical intermediate in inflammation caused by Tat ± opiates (Fig. 4).

Figure 4.

Summary of proinflammatory events associated with HIV-1 Tat and opiate interactions in astrocytes. CCL5 (RANTES) gene deletion significantly reduced the proportion of Tat ± morphine-exposed macrophages/microglia and astroglia compared to wild type mice, indicating that CCL5 is involved in reactive gliosis and inflammation. Moreover, CCL5(−/−) mice lacked Tat ± morphine-induced increases in astroglial CCL2, while significantly attenuating similar increases in CCL2 (MCP-1) in macrophages or microglia. CCL5 release is thought to be triggered by cytokines including TNF-α and IL-1β (and perhaps IFN-γ or other cytokines; see text), which can be produced by astrocytes themselves and triggered by exposure to HIV-1 Tat alone. Our earlier reports and studies in progress suggest that opiates appear to have few effects on Tat-induced cytokine production early during the cascade (El-Hage et al., 2005; El-Hage et al., 2006a; El-Hage et al., 2006b); instead, opiates appear to exacerbate chemokine release at later stages during the proinflammatory process. Some cytokines, such as TNF-α, may regulate multiple steps of the cascade. Thus, the present findings suggest that HIV-1 Tat ± opiates cause CNS inflammation, in part, through a cascade involving CCL5-dependent increases in CCL2 production by astrocytes. Additional factors have been proposed to mediate the proinflammatory cascade but these are less well substantiated (?). Moreover, autocrine/paracrine feedback amplification is also thought to be important (dashed arrow) and likely occurs at multiple points within the cascade (not shown).

In wild type mice, the proportion of CCL2 immunoreactive astroglia increased in response to morphine and HIV-1 Tat co-administration, but was not significantly increased with Tat treatment alone. This differed from a prior study in which incremental increases in CCL2 were observed following exposure to morphine or Tat alone in wild type C57/Bl6 mice (El-Hage et al., 2006a), but agrees with studies using identical treatments and measures in ICR mice (El-Hage et al., 2006b). We speculate that we are near the threshold of Tat effects, which are likely to be dependent on the duration of exposure and to differ among mouse strains. Studies in progress using an inducible Tat transgenic mice demonstrate profound increases in macrophage/microglial activation within 2 days after Tat induction or morphine treatment and somewhat more limited responses after 10 days (Bruce-Keller et al., 2008), suggesting that peak reactive glial changes may occur much earlier than 7 days post treatment.

Cell culture studies in isolated astrocytes suggest that inflammatory signaling by astrocytes is tightly controlled by intercellular cascades involving autocrine and paracrine cytokine signals (Chung and Benveniste 1990; Norris et al. 1994; Benveniste et al. 1995; Benveniste and Benos 1995; Barnes et al. 1996; Luo et al. 2000, 2002, 2003; Zhang et al. 2002; Kim et al. 2004). Collective evidence suggests that the innate astroglial “host” response to microbial pathogens involves the rapid production of key proinflammatory cytokines including interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and perhaps interferon gamma (IFN-γ) in mice or IFN-α in human astrocytes. The proinflammatory cytokines subsequently trigger the expression and release of chemokines such as CCL5 (Luo et al. 2003) and CCL2 (Barnes et al. 1996; Conant et al. 1998; Guo et al. 1998; Oh et al. 1999; Luo et al. 2003), which signal the recruitment of macrophages and activation of microglia. The signature increases in TNF-α, IL-1β, and IFN-γ and resultant increases in chemokines have been described as part of a generalized “inflammatory transcriptome” underlying reactive astrogliosis, which is similar for a variety of insults (Falsig et al. 2006). There are additional hypothesized regulatory steps within this general scheme. CCL5, for example, stimulates TNF-α, which in turn reportedly regulates CCL2 production and release (Luo et al. 2000, 2002, 2003). Murine keratinocyte chemoattractant (KC) chemokine CXCL1, a homologue of human GRO-α, also appears to stimulate RANTES, CCL2, and IL-6 production (Luo et al. 2000). KC itself is activated by TNF-α, IL-1β, or MIP-1α, but not by RANTES or CCL2, while deletion of the cognate receptor for KC, CXCR2, prevents increases in RANTES and CCL2, which suggests that KC controls the production of both chemokines (Luo et al. 2000). Moreover, findings that combined Tat and opiates no longer increase the number of CCL2-immunoreactive astrocytes in CCL5(−/−) mice implicate increases in RANTES production by astrocytes as a key site of opiate-HIV-1 Tat protein interactions. Other investigators similarly describe the coordinated actions of IL-1β and IFN-β as necessary for CCL5 expression by astrocytes including human astrocytes (Oh et al. 1999; Kim et al. 2004).

Although considerable evidence suggests that RANTES may be an important intermediary in astroglial inflammation, it has been reported that CCL5 by itself is insufficient to cause leukocyte chemotaxis across the blood brain barrier (Eugenin et al., 2006). By contrast, CCL2 appears to be able to induce trafficking of HIV-1 infected cells into the brain (Eugenin et al., 2006). Together with our earlier findings that CCR2 deletion largely eliminated Tat ± morphine–induced gliosis (El-Hage et al., 2006a), we hypothesized that astrocyte-derived CCL5 could modulate Tat ± opiate-induced increases in CCL2 in astrocytes that were essential for glial activation. The present findings support this hypothesis by demonstrating that (1) glial activation was markedly attenuated and (2) the proportion of CCL2 immunoreactive astroglia in Tat ± morphine exposed CCL5 null mice were similar to their wild type counterparts—suggesting CCL5 regulates Tat-induced increases in CCL2 by astrocytes. CCL2 immunoreactivity was evident in a small proportion of astrocytes and macrophages/microglia in placebo controls suggesting that baseline levels of CCL2 expression are not under CCL5 control in either cell type. Interestingly, the proportion of CCL2 immunoreactive macrophages/microglia still increased significantly in CCL5(−/−) mice after Tat and morphine co-administration. Taken together, the findings suggest that CCL5 regulates Tat and morphine-induced increases in CCL2 in astrocytes, but not in microglia, and that baseline levels of CCL2 are regulated independently of CCL5. Additional studies, including a more detailed time-course, are needed to confirm or deny this notion.

Thus, CCL5 acts together with other signals to induce leukocyte trafficking. As noted, we and others previously found that MCP-1-CCR2 interactions are important for glial activation following Tat and/or morphine exposure. CCL5 is reportedly responsible for initiating CCL2 production in astrocytes (Luo et al., 2002), while CCL2 does not increase RANTES (Luo et al., 2002), inferring directionality in the inflammatory cascade. Our results generally support this concept, and in fact suggest that CCL5 regulates CCL2 production in astrocytes. In the absence of CCL5, HIV-1 Tat ± morphine-induced proportional increases in CCL2-positive astroglia are absent, and astrogliosis and macrophage recruitment/microglial activation are prevented. Lastly, CCL2 expression may be regulated differently in astroglia than in macrophages/microglia. Unlike astrocytes, CCL2 expression increased markedly (albeit significantly less than wild type controls) in macrophages/microglia despite CCL5 gene deletion. Despite some elevations in CCL2 in macrophages/microglia, reactive glial changes were not evident in CCL5 knockout compared to wild type mice at 7 days. A speculative idea is that the differential regulation of CCL2 production by astrocytes compared to macrophages/microglia might have different consequences for HIV-1 encephalitis.

We additionally demonstrated that Tat alone or in combination with systemic morphine increased the proportion of CCL5-immunopositive astrocytes near the site of injection. The increases in CCL5 positive astrocytes confirm and extend in vitro observations indicating that morphine and Tat synergistically increase CCL5 mRNA and protein products in astrocytes (El-Hage et al., 2006b). Based on immunocytochemical measures of the number of CCL5-immunopositive astroglia alone, we cannot infer the extent to which CCL5 levels increase. Assuming that concurrent morphine and Tat exposure causes sustained increases in CCL2 release by astrocytes that are similar to the several fold increases seen in vitro (El-Hage et al., 2005), then the local tissue concentrations in CCL5 are likely to be much greater than the levels predicted by the increased proportion of CCL5 cells seen in the present study.

Immunoreactivity for 3-NT has been used as a biomarker to identify cells capable of inducing significant oxidative stress (Ryu and McLarnon, 2006; Shavali et al., 2006). Recently, evidence for increased nitrosative stress has been reported in the cerebrospinal fluid of individuals infected with HIV that display active dementia, and of some relevance to the present study, in patients with a history of IV drug use (Li et al., 2008). Our data agree with these findings and indicate that CCL5 contributes to nitrosative stress, because CCL5 deletion limits the generation of 3-NT-modified proteins in macrophages/microglia. However, this observation should be tempered by the fact that despite significant attenuation of nitrosative stress in CCL5 knockout mice, the proportion of 3-NT-positive macrophages/microglia nevertheless increased with Tat injection, suggesting factors in addition to CCL5 contribute to increases in reactive nitrogen species. Moreover, CCL5 gene deletion also markedly reduced combined Tat and morphine-induced increases in 3-NT in macrophages, although in the present study, Tat and morphine together did not increase the proportion of 3-NT reactive cells beyond percentages seen with Tat alone. An earlier study using inducible Tat transgenic mice found that morphine treatment alone was sufficient to increase the proportion of 3-NT and Mac1-immunopositive macrophages/microglia, while Tat induction by itself did not increase the proportion of 3-NT-positive cells (Bruce-Keller et al., 2008). The reasons for the discrepancy are uncertain but may result from differences in rodent models of HIVE (Tat-induction vs. intrastriatal Tat injection), differences in the duration of morphine exposure, or Tat induction (2 vs. 5 days), or dissimilarities between Mac1 and F4/80-immunoreactive subpopulations of macrophages/microglia (Lloyd et al., 2008). For example, if cells become tolerant to continuous morphine exposure, or if oxidative stress modifies opioid signaling as has been speculated for some experimental situations (Kielstein et al., 2007; Muscoli et al., 2007), increases in 3-NT may be short-lived and decline after a few days, which may limit or preclude interactions with Tat.

In summary, the findings indicate that CCL5 contributes significantly to the activation of macrophages and astroglia in the CNS of HIV-1 infected drug abusers and non-abusers, and further suggest that CCL5 may regulate, and possibly amplify, CCL2 production by astrocytes and potentially by other cell types. Because CCL2 levels positively correlate with neurocognitive deficits accompanying HIV-1 or SIV infection (Sevigny et al., 2004; Mankowski et al., 2004; Avison et al., 2004; Chang et al., 2004), therapeutic strategies that act upstream of CCL2 and interfere with CCL5 signaling would likely be beneficial to HIV-1 infected individuals.