Multiple mechanisms have been proposed for HIV-1-induced neuroinflammation. In order to address the issue properly, it is critical to determine the potential mediators as well as the pathways involved in the induction or generation of such mediators. In the present study, gp120-mediated induction of IL-6 has been demonstrated to be dependent upon the NF-κB pathway. Interleukin-6 (IL-6) is a classical pro-inflammatory cytokine, which has been shown to be involved in response to various stimuli 
. In our study using a human astrocyte cell line we observed that IL-6 mRNA expression reached a peak 6 h after transfection with a plasmid encoding gp120. In both types of cells IL-6 mRNA reached a peak at 1 h after the initiation of treatment with gp120 protein. These results are consistent with the previous studies by Ronaldson and Bendayan 
who have found similar kinetics with respect to IL-1β, TNF-α and IL-6 after treatment of rat astrocytes with gp120. These results are also consistent with those obtained by Li et al, 
, who demonstrated that binding of HIV-1 to human fetal astrocytes resulted in increased production of IL-6. The authors confirmed the involvement of gp120 in mediating IL-6 over-expression by utilizing a truncated mutant virion, VSV-G pseudotype NL4-3, which was incapable of expressing gp120. The wild type VSV/NL4-3 was capable of inducing IL-6 in human fetal astrocytes, whereas the mutant failed to do so. In our experiments, as expected, there was a lag phase between the peak of mRNA and protein expressions and the results clearly demonstrated that gp120 increased the expression of IL-6 in SVGA astrocytes and in human primary astrocytes at the levels of both mRNA and protein. Our work demonstrates for the first time that gp120 induces IL-6 protein and mRNA in human astrocytes. In addition to CXCR4, astrocytes are reported to express CCR5 
. We showed differential expression levels of IL-6 in response to various strains of gp120. gp120IIIB was used as an X4 strain of gp120, while gp120 CN54, gp120 CM and gp120Bal were used as R5 stains. Our results clearly demonstrate that in addition to the gp120IIIB (X4 Strain), gp120 CN54, gp120CM and gp120Bal significantly altered IL-6 expression to various extents.
Using siRNA targeted against gp120, as well as transfections with empty vector, we have definitively shown that the increase in IL-6 observed is due to the presence of gp120. Interestingly, we observed differential regulation of IL-6 mRNA and protein expression after siRNA knockdown of gp120. Although gp120 siRNA 1 was the most effective siRNA in blocking expression of IL-6 mRNA, gp120 siRNA 4 was the most effective siRNA in blocking IL-6 expression at the level of protein. The reason for this discrepancy could be attributed to the fact that mRNA expression was monitored within 6 hrs after transfection whereas protein was monitored 48 hours after transfection and these siRNAs might have required more than 6 hour to exhibit full effect.
Nuclear Factor kappa B (NF-κB) has been shown to be involved in a wide array of cellular responses. The pathways in which NF-κB has been determined to be a key mediator include cell death, apoptosis and inflammation 
. In rat astrocytes, induction of both MCP-1 and MCP-3 has been shown to be dependent upon NF-κB 
. Ronaldson, et al. showed that pretreatment of astrocytes with a peptide inhibitor of NF-κB, SN-50 dramatically reduced the level of TNF-α elicited by gp120 treatment 
. IL-6 production has also been found to be mediated though the NF-κB pathway in patients with rheumatoid arthritis 
, retinal microglia 
, lung pericytes 
and mice splenocytes treated with LPS 
. In our study, we sought to address whether NF-κB activation is involved in mediating gp120-induced IL-6 expression. We demonstrated that there is increased phosphorylation of IκBα along with significant translocation of p50 from the cytoplasm to the nucleus that is dependent upon gp120. This is similar to a report by Saha et al. 
that demonstrates that gp120 activates the NF-κB pathway and leads to nuclear translocation of p50. Results obtained from pharmacological inhibitors and siRNA approach also confirmed that induction of IL-6 expression was dependent upon the NF-κB pathway. Taken together, the evidence presented in this paper along with results from other laboratories provides strong support for the involvement of NF-κB in mediating gp120 induction of IL-6. Along with its role in inducing inflammatory cytokines, NF-κB has also been demonstrated to be involved in regulating the responses to oxidative stress in astrocytes 
. Oxidative stress has been widely demonstrated as an important mechanism through which gp120 affects astrocytes 
. Thus, activation of NF-κB by gp120 could be an important mechanism by which the cell protects itself from the oxidative stress associated with viral infection of the CNS.
Our data on the induction of IL-6 by gp120 are consistent with previously reported results. It should be noted that the results that we have observed using the two models of astrocyte exposure to gp120 (i.e. intracellular production of gp120 vs. extracellular administration of the protein) are somewhat different from each other in terms of kinetics and levels of IL-6 induction observed. These differences can be attributed to 2 differences in the models; one model utilizes extracellular exposure of primary astrocytes and the other model utilizes transfection of an immortalized cell line. We have utilized both models in order to determine the effects of gp120 on astrocytes regardless of the route of exposure. Our results not only confirm what other laboratories have reported with regard to extracellular exposure of astrocytes to gp120, but we have also shown that non-productive infection of astrocytes by HIV-1 may be a significant and persistent source of IL-6 in the CNS. It is important to note that the work presented here is the first demonstration that IL-6 is induced in human astrocytes in response to gp120. All previous reports in the literature have used either rat astrocytes or human mixed glial cultures.
This is an especially important finding because previously reported evidence suggests that IL-6 may be involved in the regulation of other cytokines such as TNF-α and IL-1β 
. Taken together, this suggests that gp120-mediated activation of the NF-κB pathway may be critical therapeutic target for the treatment of HIV-related neuroinflammation. One approach that could be explored would be to try to interfere with the interaction between NF-κB and its binding site in the IL-6 promoter in astrocytes. Recent advances suggest that small RNAs or miRNA might be a useful tool for silencing promoters/enhancers 
, and such an approach might be applied to the IL-6 promoter in astrocytes. Also, recently Kim et al. have employed a novel approach to target specific cell types using antibody-based strategies 
. Using their approach they were able to efficiently target delivery of siRNA specifically to T-cells via the CD67 receptor. Similarly, Wu et al. used microRNA delivery to astrocyte specific promoter as a tool for cancer therapy 
. Such an approach might be further exploited in astrocytes by targeting these cells using GFAP to deliver siRNA/miRNA for NF-κB or IL-6 promotor. As successful antiretroviral treatment has extended the lifespan of those infected with HIV, the importance of finding novel treatments for the HIV-associated morbidities caused by chronic inflammation and oxidative stress, such as HAND, has become increasingly critical. Identification of a critical therapeutic target, as has been presented in this study, is an important step towards the development of more effective therapeutic regimens for neuroAIDS.