Macrophage-Derived Factors with the Potential to Contribute to Pathogenicity of HIV-1 and HIV-2: Role of CCL-2/MCP-1.

Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that ß-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.

Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

BACKGROUND: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. RESULTS: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. CONCLUSIONS: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes.

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.

Platelet factor 4 (CXCL4) facilitates human macrophage infection with HIV-1 and potentiates virus replication.

Platelet factor 4 (CXCL4), a member of the CXC chemokine subfamily released in high amounts by activated platelets, has been identified as a monocyte survival factor that induces monocyte differentiation into macrophages. Although CXCL4 has been shown to have biological effects unique to chemokines, nothing is known about the role of CXCL4-derived human macrophages or CXCL4 in human immunodeficiency virus (HIV) disease. In this study, CXCL4-derived macrophages are compared with macrophage-colony stimulating factor (M-CSF)-derived macrophages for their ability to support HIV-1 replication. We show that CXCL4-derived macrophages can be infected with macrophage-tropic HIV-1 that uses either CC-chemokine receptor 5 (CCR5) or CXC-chemokine receptor 4 (CXCR4) as a co-receptor for viral entry. We also find that M-CSF and the chemokines, monocyte chemoattractant protein 1 (MCP-1; CCL2) and macrophage-inflammatory-protein-1-alpha (MIP-1alpha; CCL3) are produced upon R5- and X4-tropic HIV-1 replication in both M-CSF- and CXCL4-derived human macrophages. In addition, CXCL4 added to M-CSF-derived macrophages after virus adsorption and maintained throughout the infection enhances HIV-1 replication. We thus propose a novel role for CXCL4 in HIV disease.

APOBEC3G-independent reduction in virion infectivity during long-term HIV-1 replication in terminally differentiated macrophages.

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. In the case of HIV, the antiviral activity of APO3G is counteracted by the viral Vif protein. Monocyte-derived macrophages (MDM) are terminally differentiated, non-dividing cells susceptible to HIV infection. Human MDM are known to express APO3G and HIV replication in these cells is dependent on Vif. Here we analyzed the correlation between HIV-1 replication and APO3G expression in MDM. Replication of wild type HIV-1 induced a gradual 4-5-fold reduction in APO3G expression. The efficiency of APO3G downregulation correlated with the efficiency of virus replication. Interestingly, despite downregulation of APO3G, the relative infectivity of viruses rapidly declined during the course of infection and was already reduced approximately 90% prior to peak virus production. Cell-free virus preparations showed increased levels of a 41 kDa MA-CA processing intermediate. Sequence analysis around the MA-CA cleavage site and the protease and LTR regions did not reveal deaminase-induced hypermutation of the viral genome, suggesting that APO3G activity is not responsible for the incomplete Gag processing. Thus, the loss of infectivity of HIV-1 viruses produced from long-term infected primary macrophages is due to an APO3G-independent mechanism.

HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro.

We report a novel mechanism, involving up-regulation of the interleukin (IL)-7 cytokine receptor, by which human immunodeficiency virus (HIV) enhances its own production in monocyte-derived macrophages (MDM) in vitro. HIV-1 infection or treatment of MDM cultures with exogenous HIV-1 Tat(86) protein up-regulates the IL-7 receptor (IL-7R) alpha-chain at the levels of steady-state RNA, protein, and functional IL-7R on the cell surface (as measured by ligand-induced receptor signaling). This IL-7R up-regulation is associated with increased amounts of HIV-1 virions in the supernatants of infected MDM cultures treated with exogenous IL-7 cytokine. The overall effect of IL-7 stimulation on HIV replication in MDM culture supernatants is typically in the range of one log and greater. The results are consistent with a model in which HIV infection produces the Tat protein, which in turn up-regulates IL-7R in a paracrine manner. This results in increased IL-7R signaling in response to the IL-7 cytokine, which ultimately promotes early events in HIV replication, including binding/entry and possibly other steps prior to reverse transcription. The results suggest that the effects of IL-7 on HIV replication in MDM should be considered when analyzing and designing clinical trials involving treatment of patients with IL-7 or Tat vaccines.

The CXC chemokine NAP-2 mediates differential heterologous desensitization of neutrophil effector functions elicited by platelet-activating factor.

During early inflammation, the chemoattractants neutrophil-activating peptide-2 (NAP-2), platelet-activating factor (PAF), and complement component C5a are rapidly generated within the vasculature and potently induce effector functions in neutrophils, such as chemotaxis and degranulation. We investigated whether these mediators would cross-desensitize each other's activities on isolated neutrophils. We demonstrate that NAP-2 and C5a desensitize degranulation of neutrophils in response to PAF. However, whereas C5a-mediated desensitization correlated with the downregulation of PAF binding sites on neutrophils, NAP-2 did not regulate PAF receptors, nor did it modulate their calcium signaling. In further contrast to C5a, which desensitized PAF-induced neutrophil chemotaxis, NAP-2 did not affect the chemotatic response to PAF. These observations indicate that NAP-2 mediates selective desensitization of PAF-induced neutrophil degranulation by a mechanism downstream from PAF receptors, still allowing migration toward PAF. Thus, a role for NAP-2 may be to limit PAF-dependent vascular tissue damage by preventing degranulation of neutrophils without affecting their migration into the inflamed tissue.