Viral Related Proteins in Immune Dysfunction Associated with AIDS


The mechanisms of pathogenesis associated with infection with human immunodeficiency virus (HIV), initially discovered in 1983,1,2 and the development of acquired immune deficiency syndrome (AIDS) are complex and are undiscovered or poorly understood. Perhaps for no disease in history has there been such a focused research effort to understand exactly how a known etiological agent effects its pathogenesis.

Much progress has been made since the identification of HIV as the human retrovirus responsible for AIDS. However, many of the earlier concepts regarding the mechanisms of immune dysfunction, initially widely accepted, have come under closer scrutiny as we learn more about this virus and its lentivirus relative, simian immunodeficiency virus (SIV) , Some of those concepts included the idea that HIV infects only CD4+ lymphocytes and that CD4 was the only receptor for the virus. Both of those early concepts have now been clearly disproved. Another assumption now recognized as erroneous is that only a very small percentage of lymphoid cells are infected in HIV-infected individuals and that at any one time there is very little virus in the body. We now know that HIV infects a wide variety of cells and is often sequestered in sites such as the lymph nodes, and that viral replication is extensive, with a turnover rate of 109-1010 viral particles per day. The demonstration that certain chemokines can suppress viral infec-

GEORGE J. CIANCIOLO • Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.

Human Retroviral Infections, edited by Kenneth E. Ugen et al. Kluwer Academic / Plenum Publishers, New York, 2000.

tivity and the identification of chemokine receptors as coreceptors for HIV has resulted in reexamination of our early concepts regarding viral binding and entry to infected cells. Furthermore, while early studies focused on the cytopathogenic potential of HIV infection of T lymphocytes, much effort is now being directed toward understanding the role of the host response in disease pathogenesis and, in particular, in the role of cytokine dysregulation in the immune dysfunction so characteristically associated with HIV infection.

Infection with different viruses has been thought to produce immuno-logic dysfunctions, such as immunodeficiency or autoimmune disorders, in the hosts. It has been generally assumed that such dysfunctions were related to the direct effects, such as infection, of whole virions on their target cells. This chapter seeks to examine other potential mechanisms of HIV-mediated immune dysfunction, ones that have received much less attention than many of the much more traditional mechanisms associated with viral patho-genesis. It will focus on the potential roles of soluble proteins, encoded by the HIV genome and shed from virions or infected cells, in the initiation or propagation of immune dysfunction associated with HIV infection and AIDS. These proteins may act directly on target immune cells to suppress their function or they may act indirectly by inducing soluble suppressive factors from the hosts' own cells.

The research herein reviewed is essentially in its infancy in that most of the studies describe the immunomodulatory effect on human immune cells of HIV proteins added to in vitro cell cultures. In the past, such studies were sometimes cursorily dismissed because the concentrations used in vitro appeared to be physiologically irrelevant to what one might expect to occur in vivo. However, with the recent evidence suggesting that the number of viral particles produced per day can be extraordinarily high and that virus may be concentrated in certain tissues, what exactly constitutes a physiologically relevant concentration remains undefined. As yet, however, only occasionally does one find published descriptions from patient studies of anecdotal evidence supporting the hypothetical mechanisms proposed from these in vitro investigations. Perhaps the proposition of these often intriguing, yet unproven hypotheses will prompt additional investigators to initiate new, confirmatory studies. Such studies might examine the fluids and tissues of patients not only for the presence of potentially immunoregulatory concentrations of these proposed mediators, but for the presence or absence of immune correlates which might serve to prove or disprove the hypothetical role proposed.

A review of the published HIV research literature would undoubtedly identify at least several reports demonstrating in vitro effects on immune cell function by any of the HIV structural, regulatory, or accessory proteins. This chapter, however, will focus only on those four proteins for which the most data seem to have been accumulated: the regulatory protein Tat, the structural proteins gp120 and gp41, and the regulatory protein Nef.


An important point to be considered in this discussion of the potential role of HIV-encoded soluble proteins in immune dysfunction is that it was recognized early in the AIDS epidemic that the destruction and loss of CD4+ cells does not explain all of the immunopathogenic effects of HIV infection.3 For example, the loss of T helper lymphocyte function in asymptomatic individuals, as evidenced by the ability of patients' lymphocytes to respond to soluble antigen in vitro, occurs even before any appreciable decline in CD4 counts is seen.4-6 However, in most cases, the responses of patients' lymphocytes to mitogens remain normal.


Tat, a potent viral transcriptional trans-activator protein which mediates its effects by the Tat-response (TAR) element, is a 16-kDa, 86-amino acid protein with several distinct domains: residues 22-37 comprise a cysteine-rich domain which can bind cadmium and zinc, residues 37-48 constitute the core domain, and residues 48-57 contain basic residues required for nuclear and nucleolar targeting and RNA binding. Tat is a potent enhancer of viral replication and considered important since replication does not proceed in its absence. Its actions do not appear to be restricted to HIV replication, but also involve pleiotropic effects on the immune, vascular, and central nervous systems of HIV-infected individuals. Tat is known to be secreted extracellularly by infected cells and thus is readily available to interact with uninfected bystander cells.

As summarized in Table I, soluble HIV Tat protein has been reported to have a wide range of potentially deleterious effects on human immune cells which could play a role in the immune dysfunction associated with AIDS. One of the first studies reporting the ability of Tat to inhibit in vitro immune functions of human cells was that of Viscidi et al. in 1989.7 They demonstrated that recombinant HIV-1 Tat (amino acids 1-72), expressed in Esche-richia coli, inhibited the proliferation of human peripheral blood mono-nuclear cells (PBMCs) in response to stimulation by tetanus toxoid (66-


Potential Immunomodulating Activities Reported for HIV-1 Tat Protein

Activity References

In human PBMCs or lymphocytes

Increases Ig production 9, 10

Increases IL-6 release 9, 10

Upregulates Bcl-2 expression 19

Inhibits antigen-induced proliferation 7, 13, 14

Inhibits mitogen-induced proliferation 13

Inhibits anti-CD3-induced proliferation of CD4+ and CD8+ T cells 12, 14, 16

Induces apoptosis in uninfected T cells 18, 20, 21 In human monocytes or endothelial cells

Induces chemotaxis and inflammatory mediator release by monocytes 22, 23

Increases chemotactic activity or monocytes 22

Increases IL-6 in endothelial cells 11 In human bone marrow cultures

Increases TGF-P release 8

Inhibits CFU-E, CFU-M, CFU-GM 8

Enhances CFU-GM (HIV-2 Tat) 17

97%) or Candida antigen (75-91%). The IC50 for inhibition of tetanus toxoid-stimulated responses was approximately 0.5 ^g/ml (ca. 50 nM). The effects of Tat did not appear to be due to toxicity since the proliferative responses of PBMCs in response to mitogens such as phytohemagglutinin (PHA), concanavalin A (ConA), or pokeweed mitogen (PWM) were unaffected. A synthetic peptide, representing amino acids 1-58, also inhibited tetanus toxoid-stimulated proliferation, although this inhibition required concentrations ten times higher than required with Tat 1-72. A second bacterially expressed Tat, representing amino acids 1-86,was as active as Tat 1-72 at inhibiting proliferative responses. The specificity of the induction of immunosuppression by Tat was demonstrated with antibody to Tat; however, the authors could not rule out that another molecule was the actual effector.

Several years later Zauli et al.8 reported that recombinant Tat protein, when added to enriched normal bone marrow (BM) macrophages, induced the production of a factor that inhibited the in vitro growth of CD34+ cells in liquid cultures and the growth of colony-forming units (CFU)-erythroid, CFU-megakaryocytic, and CFU-granulocyte/macrophage in semisolid agar. The suppressive factor was identified by use of neutralizing antibodies such as those to transforming growth factor P1 (TGF-P1) and subsequent experiments demonstrated that recombinant Tat increased the Ievels of both active and latent forms of TGF-P1 from BM macrophage cultures. The ability of Tat to upregulate the expression and release ofTGF-P1 was con firmed by this same group9 using human peripheral blood monocytes and they also demonstrated upregulation of IG6, which they postulated was at least partially responsible for the upregulation ofTGF-01. Rautonen et al.10 also reported increased expression of interleukin-6 (IL-6) from exogenous recombinant Tat-stimulated uninfected PBMCs. Furthermore, they reported increased production of immunoglobulin (Ig) from their cultures. The optimal concentration of Tat was 100 ng/ml (10 nM), but they saw effects at concentrations as low as 1 ng/ml (100 pM) . Anti-IL-6 antibodies and IL6 antisense oligonucleotides could block some of the Tat-induced IgG and IgA synthesis, but not the IgM synthesis, suggesting that at least some of the effects on increased Ig synthesis were secondary to the increased synthesis of IL-6. Increased synthesis of IL-6 by Tat stimulation had also been reported the previous year by Hofman et al.11 in studies on human endothelial cells (EC) . In addition to increased release of IL-6 from EC, they also reported increased expression of E-selectin on EC exposed to Tat.

In 1992, contrasting data to those published by Viscidi et al.1 were reported by Meyaard et al.12 They found that proliferation of purified T cells to anti-CD3 monoclonal antibodies was inhibited by up to 70% by 5 ^g/ml of Tat protein. Surprisingly, however, they did not observe suppression when accessory cells were present in the cultures and they could not demonstrate any inhibition of responses to recall antigen. These results led the authors to conclude that Tat did not have a significant role in the immunosuppression associated with HIV infection. However, in 1993 a study by Benjouad et al.13 seemed to add to the confusion by both confirming and conflicting with the Viscidi et al. data. The authors reported that synthetic Tat (amino acids 286) inhibited not only in vitro antigen-induced peripheral blood lymphocyte proliferation, but mitogen-induced proliferation as well. The IC50 for inhibition of antigen-induced proliferation was 0.9 ^M, which was substantially higher than that reported by Viscidi et al., and the IC50 for mitogen-induced proliferation was 8 ^M. They suggested that the inhibition of proliferation was due to lymphocyte cytotoxicity by Tat and that such cytotoxicity was associated with the basic region of amino acids 49-57.

Another study published that same year suggested another mechanism for Tat-induced anergy. Subramanyam et al.14 reported that blockage of DP IV (CD26) by Tat partially inactivated antigen and anti-CD3 stimulated lymphocyte proliferation. As in the initial studies by Viscidi et al., they found no effect on mitogen-stimulated proliferative responses. The antigen-specific inhibition they observed could be overcome by addition of exogenous IL-2 or by costimulation of PBMCs via CD28. The same group of investigators subsequently reported studies characterizing the binding of Tat to CD26.15 The affinity of Tat for DP IV varied from 20 pM to 11 nM as the NaCl concentration was varied from 0 to 140 mM.

Although Benjouad et al.13 had suggested that the inhibitory activity of

Tat might be related to its cytotoxic effects on target cells, a 1995 report by Chirmule et al.16 demonstrated inhibition by Tat peptides of anti-CD3-stimulated proliferative responses of both purified CD4+ and CD8+ T cells. They reported that there was no effect on cell viability, as measured by trypan blue dye exclusion. The Tat concentrations tested ranged from 100 to 300 ng/ml. Another somewhat conflicting report was that of Calenda and Chermann.17 Their 1995 study demonstrated that the HIV-2 Tat gene product enhanced growth of CFU-GM in agar, a result which contrasted with the earlier report of Zauli et al.8 which showed inhibition of CFU-GM by HIV-1 Tat in CD34+ cell cultures. Whether these apparently contradictory effects reflect true differences in the HIV-1 and HIV-2 Tat proteins remains to be determined.

Another potential mechanism for the immunosuppressive effects on T cells was introduced in 1995 with one of the first reports for Tat-mediated T cell apoptosis. In this study Li et al.18 showed that Tat protein could induce apoptotic cell death in both a T cell line and in cultured PBMCs from un-infected donors. This apoptotic effect, observed initially in serum-deprived cells, was reversed to some extent by the inclusion of serum in the cultures, suggesting a protective effect for growth factors. In PBMC cultures, Tat induced apoptosis in T cells, but not in monocytes, and CD4+ T cells did not appear to be more sensitive than CD8+ T cells. Exogenous Tat added to PBMCs was found to markedly enhance total cyclin-dependent kinase (Cdk), activity and treatment of Tat-transfected Jurkat T cell lines with antisense oligonucleotides corresponding to the highly conserved regions of human cyclins A, B, and E blocked Tat-associated apoptosis. These results led the authors to propose that T cells in HIV-1-infected individuals are stimulated by Tat in lymphoid tissue, prematurely activating Cdks and preventing the cells from returning to a quiescent state. Later, when the cells are stimulated by antigen they undergo apoptosis and are depleted.

In contrast to the results of Li et al.,18 who reported that Tat-induced apoptosis had no effect on Bcl-2 levels, Zauli et al.19 reported that picomolar concentrations of native or recombinant Tat upregulated Bcl-2 in both Jurkat T cell lines and in primary PBMCs. They also demonstrated inhibition of apoptosis in serum-deprived Jurkat cells in which Tat had been transfected, and correlated this inhibition with Tat expression.

Katsikis et al.20 reported that Tat protein had no effect on spontaneous apoptosis, but did enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat did not enhance Fas-induced apoptosis of either CD4+ or CD8+ T cells. Also in 1997, McCloskey et al.21 published data which supported dual roles for Tat as both an inducer of, and a protector against, apoptosis. They demonstrated that while exogenous Tat induced apoptosis in uninfected T cells, T cell clones stably expressing Tat protein were protected from activation-induced apoptosis. These pleiotropic effects for Tat seem to support both of the previously described conflicting results of Li et al.18 and Zauli et al.19 and suggest that much remains to be learned regarding the actual role of Tat in HN-1-associated T cell apoptosis.

Although the majority of studies have focused on the effects of Tat on T cell activation or death, several studies have suggested possible roles in regulating other cells of the immune system. For instance, in a 1996 study, Lafrenie et al.22 reported that treatment with Tat at a concentration of 10 ng/ml enhanced both chemokinesis (migration in the absence of a stimulus) and chemotaxis (directed migration) to the chemotactic peptide FMLP. Tat itself was chemotactic for both treated and untreated monocytes. Pretreatment of monocytes with a similar concentration of Tat for 24 h increased their ability to invade reconstituted extracellular membrane (Matrigel)-coated filters by fivefold. The authors postulated that Tat might play a role in the recruitment of monocytes into extravascular tissues and thus contribute to the destruction of such tissues in patients with AIDS. The following year these results were confirmed by Mitola et al.23 in studies in which they demonstrated that Tat induced monocyte chemotaxis at sub-nanomolar concentrations and that such chemotaxis could be inhibited by cell preincubation with vascular endothelial growth factor-A (VEGF-A). Furthermore, the soluble form of VEGFR-1 could block Tat-induced mono-cyte chemotaxis, and specific binding of radiolabeled Tat to monocyte surface membranes was blocked by an excess of either unlabeled Tat or VEGF-A.


The viral envelope protein of HIV-1 is synthesized as a 160-kDa precursor protein, gp160, which is eventually cleaved to gp120 surface (SU) and gp41 transmembrane (TM) proteins. The gp120 is subdivided into five variable (V) loops; four of these loops are bounded by disulfide-linked cysteine residues. The regions between the variable domains are designated constant (C) domains. The V3 loop is a major determinant of cell tropism, and a region of gp120, including the V2 loop and the C4 domain, is involved in CD4 interactions.

Like HIV-1 Tat, gp120 is shed from virus or virus-infected cells and is found in the plasma of HIV-infected individuals, making it a reasonable candidate to examine as a potential soluble suppressor of immune functions. Some of the immunomodulatory activities ascribed to gp120 are listed in Table II. One of the earliest studies examining gp120 was that of Shalaby


Potential Immunomodulating Activities Reported for HIV-1 gp120/gp160 Protein



In human PBMCs or lymphocytes

Inhibits antigen-stimulated proliferation Inhibits mitogen-stimulated proliferation Inhibits CD3-stimulated proliferation Inhibits apoptosis Increases Fas antigen expression

Reduces expression of protooncogene Bcl-2 in CD4+ cells Induces IL-10, IFN-a, Y, TNF-a, IL6, IL-1a, P Inhibits PHA-induced IFN-y and IL2 secretion Increases PHA-induced IL-4 secretion

Induces anergy in T helper lymphocytes stimulated with IL-2, IL-4, IL6, anti-CD2, anti-CD3, or PMA Inhibits upregulation of CD40L In human monocytes

Stimulates release of TNF-a, IL-1P, IL6, and GM-CSF

Impairs chemotaxis

Induces release of IL-1P, PGE2


Reduces accessory cell function Inhibits upregulation of B7-1 In human bone marrow cultures Inhibits CFU-GM

Enhances growth of myeloid hematopoietic progenitors Increases TGF-P

33 50

et al.24 in which they demonstrated that recombinant gp120 (rgpl20), at concentrations of 1-20 ^g/ml, inhibited tetanus toxoid-stimulated proliferation of human PBMCs. At 5 ^g/ml, rgp120 also inhibited by 70% the number of immunoglobulin-secreting cells in PWM-stimulated PBMC cultures. Previous work by Sandstrom et al.25 had suggested the possible existence of a soluble suppressor factor based on the observation that HIV-infected H9 cells could suppress the responses of uninfected PBMCs to Con A. However, studies published the same year as those of Shalaby et al.24 by Mann et al.26 seemed to both support and conflict with the previously described studies. They found inhibition of PHA-induced blastogenesis, but no effect on Con A-, PWM-, or alloantigen-induced proliferation. The following year, however, Chirmule et al.21 demonstrated that HIV-1 gp120 could inhibit both antigen-specific and anti-CD3-stimulated proliferation of normal human lymphocytes. In that same year Krowka et al.28 reported that recombinant gp120, at concentrations s®10 ^g/ml, could inhibit the prolif-

erative responses of peripheral blood lymphocytes to UV-inactivated cyto-megalovirus (CMV) and that this immunosuppression could be abrogated by recombinant IL-2.

Over the next 10 years additional studies29-32 confirmed these initial reports of direct immunosuppressive effects of gp120, although the concentrations required for activity remained relatively high. Of interest, a study by Di Rienzo et al.32 demonstrated that rgpl20 could induce anergy in human T helper lymphocytes stimulated to proliferate with a variety of stimuli (IL-2, IL-4, IL-6, anti-CD2, anti-CD3 and PMA [phorbol 12-myristate 13-acetate]), but lymphocytes from chimpanzees, which are susceptible to HIV-1 infection, but do not easily develop immunodeficiency, were resistant to rgpl20-associated anergy.

Much interest has focused on potential mechanisms by which gp120 exerts its effects on T helper cells. One report33 described the production of prostaglandin E2 and IL-1 from normal human monocytes exposed to low concentrations of gp120 purified from HIV-1. Since PGE2 is known to suppress a variety of immune functions and the authors found a 12-fold increase in PGE2 with gp120 concentrations of 200-400ng/ml, these studies offered a plausible mechanism for gp120-mediated suppression. However, they were unable to demonstrate an effect with recombinant gp120 fragments, thus offering no explanation for the effects seen by others using rgpl20.

The 1991 studies by Terai et al.34 suggested that the direct cytopatho-logic effect of HIV-1 in T cells might be due to apoptosis and implicated gp120 in this mechanism. They reported that acute HIV-1 infection of MT2 lymphoblasts and activated PBMCs induces apoptosis and that addition after infection of anti-gpl20 neutralizing antibody permitted sustained high levels of infection, but blocked apoptosis and cell death. The following year Banda et al.35 described studies in which cross-linking of bound gp120 on human CD4+ cells, followed by antigen signaling through the T cell receptor resulted in activationdependent cell death, or apoptosis. Impressively, they were able to prime T cells for cell death with picomolar concentrations of gp120 and they offered these results as a potential mechanism for T cell depletion in AIDS. However, Liegler and Stites31 found no evidence for significant cell death by apoptosis in gp120 treated and TCR-stimulated PBMCs and they reported that, as in earlier studies,28 suppression of proliferation by gp120 could be reversed by addition of IL-2, thus suggesting that suppressed proliferation was not an apoptotic mechanism. Although based on the use of human T-cell clones, the studies ofAmendola et al.36 reported that preincubation of clones specific for influenza virus hemagglutinin with gp120 induced a significant inhibition of their antigen-stimulated proliferation which paralleled the induction of apoptosis. In these studies, however, antigen stimulation alone triggered apoptosis in a significant number of cells and gp120 merely potentiated the antigen effect. One potential mechanism for gp120-associated apoptosis was offered by the studies of Oyaizu et al..37 They showed that cross-linking of CD4 molecules (CD4XL) with HIV-1 envelope protein gp160 resulted in increased Fas expression as well as Fas mRNA in normal PBMCs and that upregulated Fas closely correlated with apoptotic cell death. The cytokines IFN and tumor necrosis factor-a (TNF-a) were implicated in CD4XL-mediated effects since antibodies to both cyto-kines blocked both Fas upregulation and apoptosis. This same group later showed38 that CD4XL by either anti-CD4 monoclonal antibody or HIV gp120 reduces the expression of the protooncogene Bcl-2 in CD4+ T cells, but not in CD8+ T cells, concurrent with the induction of apoptosis in CD4+ T cells. Addition of IL-2 to the cell cultures rescued CD4+ T cells from CD4XL-induced Bcl-2 downmodulation and apoptosis, a result consistent with earlier reports2831 that gp120-mediated suppression of proliferative responses of PBMCs to CMV or TCR stimulation could be reversed by IL-2.

Mechanisms other than apoptosis have been suggested to account for gp120-mediated immunosuppression. For instance, there are several reports of gp120 effects on monocytes which are important for the responses of PBMCs. Wahl et al.39 reported that gp120-treated monocytes were impaired in their ability to respond to chemotactic ligands due to receptor downregulation and that treated cells underwent differentiation, as evidenced by HLA-DR expression. Durrbaum-Landmann et al.40 treated cultured monocytes with HIV-1 rgpl20 and found that rgpl20 significantly reduced the accessory function of monocytes to stimulate autologous lymphocytes with anti-CD3. In addition, they found that Fc receptor-mediated chemiluminescence was reduced as was the expression of CD4 and Fc receptor 1/11, while CD14 expression and major histocompatibility complex classes I and II were unchanged. As suggested earlier, at least some of the anergy attributed to gp120 may be dependent on monocyte/macrophage-derived TNF-a. Kaneko et al.41 reported that gp120 inhibition of early T cell activation and mitogen-mediated IL-2 production was blocked in the presence of antibody to TNF-a. However, inhibition of the mixed lymphocyte reaction (MLR) in CD4+ T cells by gp120 was observed even in the absence of macrophage-derived TNF-a, suggesting that both TNF-a-depleted and TNF-a-independent events may play a role in T cell anergy associated with HIV. Zembala et al.42 used a recombinant gp120 fragment (rp120cd; amino acids 410-511), encompassing the CD4-binding region, which had been previously shown to induce TNF-a production in monocytes, to demonstrate inhibition of antigen (PPD) presentation by monocytes to autologous T lymphocytes. Another fragment of gp120 not containing the CD4-binding region was inactive. Anti-TNF-receptor antibody blocked the depression of antigen presentation by rpl20cd, suggesting a role for TNF and its receptor in impaired antigen presentation mediated by gp120.

A possible role for gp120-mediated effects on costimulatory molecules was suggested by the studies of Chirmule et al.43 Interactions between CD28/B7-1 and CD40 ligand (CD40L)/CD40 are essential for anti-CD3 monoclonal antibody (mAb)-induced T cell proliferation as evidenced by the upregulation of B7-1 and CD40L and the ability of mAbs to B7-1 and CD40L to block anti-CD3-stimulated proliferation. Pretreatment of CD4+ T cells with gp120 before CD3 ligation with anti-CD3 mAb inhibited upregulation of CD40 ligand (CD40L) on T cells and B7-1 on antigen-presenting cells (APC). Addition of anti-CD28 mAb overcame the inhibitory effect of gp120 on anti-CD3 mAb-induced T cell proliferation, supporting a role for gp120 in dysregulation of costimulatory molecules on both T cells and APC.

There are a number of reports of cytokine dysregulation by gp120. Capobianchi et al.44 reported that recombinant gp120 could induce IFN-a from human PBMCs and that this induction could be blocked by anti-CD4 antibody or soluble CD4. In a later study, Ferbas et al.45 reported that the major cell type responsible for the production of IFN-a in response to stimulation with HIV was the dendritic cell, as evidenced by their phenotype, large size, and veiled and ruffled morphology. Purified dendritic cells produced as much as 60-fold more IFN-a compared with HLA-DR+ CD 14+ monocytes and IFN-a was not produced by CD3+ T cells or CD56+ natural killer cells. The induction of IFN-a by HIV-1 could be blocked by anti-CD4 mAb or anti-gpl20 antiserum, suggesting that the gp120/CD4 interaction was required.

The potential for modulation of Th1 and Th2 cytokine profiles by HIV-1 gp160 was reported in a 1994 study by Hu et al.46 The authors pretre-ated cells (unfractionated PBMCs, CD4+ T cell lines, PBMCs depleted of CD8+ cells) with HIV-1 gp160 and demonstrated significant reduction of PHA-induced secretion of interferon-y (IFN-y) and IL2, but augmentation of IL-4 production. This effect was not observed when the PBMCs were depleted of either CD4+ or CD2+ cells or when the gp160 was pretreated with soluble CD4-Ig chimeric molecules, suggesting that gp120-CD4 interaction was required. Additional studies examining the balance between Th1 and Th2 cytokines have focused on IL-10. Ameglio et al.47 reported that recombinant HIV-1 gp120 was a potent inducer in normal human PBMCs of IL-10. In addition, they also reported induction of IFN-a, IFN-y, TNF-a, IL6, IL-10, and IL-1a, but not IL-2 or IL-4. Another study from the same institution also reported increased expression of IL-10. In those studies Borghi et al.48 showed that treatment of either 1-day monocytes or 7-day monocyte-derived macrophages with recombinant gp120 induced IL-10 mRNA expression and caused a marked increase in IL-10 secretion. This effect of gp120 was abrogated by mAb to gp120. In a study49 published a year earlier, these investigators had examined serum IL-10 levels in HIV-positive patients and correlated them with Centers for Disease Control (CDC) stages. Using a competition enzyme-linked immunosorbent assay (ELISA) for IL-10, they found that serum IL-10 levels were significantly higher in HIV-positive patients compared to HIV-negative controls. The IL-10 levels progressively increased in the subsequent CDC stages, without further changes from stage III to stage IV. Patients evaluated twice in CDC stage 11, with a time interval of at least 1 year, showed significant IL-10 increases and the increases were even more pronounced when the patients progressed from CDC stage II to CDC stage 111. In addition, a significant negative correlation was established between the patients' IL-10 levels and their CD4/CD8 ratios, suggesting that IL-10 might be involved in some of the immunological abnormalities associated with AIDS. More recently, Taoufik et al.50 reported that in human monocyte cultures, HIV gp120 induces a significant IL-10 synthesis and that this inhibits mRNA for IL-12 subunits and the subsequent expression of IL-12 p40 and p70 proteins in response to stimulation with Staphylococcus aureus strain cowan I.

Still another gp120-mediated mechanism of immunosuppression is the effect ofgp120 on hematopoietic stem cells. In 1991 and 1992 Zauli et al.51-53 reported that treatment of human CD34+ cell cultures with either HIV-1 or HIV-1 gp120 or HIV-1 gp160 caused a progressive and significant decrease in viability and a reduced percentage of committed progenitors. Both gp120 and gp160, at concentrations from 0.01 to 10 ^g/ml, decreased CD34+ cell viability as measured by trypan blue dye exclusion and tritiated thymidine incorporation in liquid cultures supplemented with human recombinant IL-3. In the absence of IL-3, no inhibition was seen at even the highest concentrations of gp120 or gp160. In virus-treated cells there were no signs of active virus replication and latent infection was ruled out by polymerase chain reaction (PCR). The cytotoxic activity of either HIV or gp120 could be abrogated by neutralizing antibody against gp120. Both gp120 and gp160 inhibited the in vitro growth of CFU-GM in a dosedependent fashion. In contrast to the results of Zauli et al,51-53 Sugiura et al.54 reported that culture of cord blood mononuclear cells with gp160 resulted in enhancement of the in vitro growth of myeloid hematopoietic progenitors. Using cultures of adherent cells, purified T cells, or CD34+ progenitors, the authors found that gp160 had no direct effect on highly purified hematopoietic progenitors, but acted indirectly via induction of a colony-stimulating factor(s) from T cells. The enhancing activity of gp160 could be blocked by soluble CD4 or polyclonal antisera to gp120.

Maciejewski et al.55 also observed suppressed hematopoiesis by gp120, but their studies suggested a role for TNF-a. They found that hematopoietic colony formation by CFU-E or CFU-GM was inhibited by both active and heat-inactivated HIV-1 virus as well as by HIV-1 gp120. Inhibition required the presence of macrophages and was not observed in cultures of highly enriched CD34+ cells. The addition of anti-TNF-a neutralizing antibodies to marrow cultures abrogated the inhibition by either gp120 or virus. Neutralizing antibodies to IL-4, IFN-a, or TGF-P had no effect on inhibition of colony formation. The authors demonstrated production of TNF-a from blood monocytes and marrow mononuclear cells exposed to gp120 and suggested that viral suppression of hematopoiesis did not require direct infection of progenitor cells, but might be mediated indirectly by TNF-a induced by virus or viral envelope protein. Zauli et al.56 reported data suggesting the involvement of TGF-P1, rather than TNF-a, in mediating gp120 effects on hematopoietic precursors. Highly purified CD34+ progenitor cells from the peripheral blood of 20 normal donors had impaired survival and clonogenic capacity after exposure to HIV-1 or cross-linked gp120. Cell cycle analysis suggested that HIV or gp120 cells were undergoing apoptosis. Blocking experiments with anti-TGF-P1 neutralizing serum suggested that HIV- or gp120-mediated suppression was almost entirely due to upregula-tion of endogenous TGF-P1. Increased levels of bioactive TGF-P1 could be detected in the supernatants of HIV-1- or HIV-1 gp120-treated CD34+ cells, and anti-TGF-1 neutralizing antiserum caused a significant increase in plating efficiency of CD34+ cells from the peripheral blood of HIV-1 -seropositive patients.

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