Peptide T from human immunodeficiency virus envelope does not interact with hepatic, intestinal and colonic vasoactive intestinal peptide (VIP) receptors

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is a considerable structural homology between peptide T and VIP, it has been proposed that the VIP receptor may be the naturally occurring protein which provides the corresponding cellular attachment site. In three different models (rat intestinal epithelial cell membranes, rat liver plasma membranes and human colonic cells), we document the lack of interaction between peptide T and the VIP receptor. These observations would also exclude any pathophysiologic effect caused by the crossreactivity of peptide T or its analogues and these VIP receptors.     

Interaction of vasoactive intestinal peptide with mouse lymphocytes: specific binding and the modulation of mitogen responses

Binding of radioiodinated vasoactive intestinal peptide (VIP) to mouse lymphocytes has been investigated. Specific cell binding of 125I-VIP was demonstrated with lymphocytes from mesenteric lymph nodes, subcutaneous lymph nodes, spleen, and Peyer's patches. The binding of VIP by these cells was accounted for by VIP binding sites upon T cells rather than non-T cells. In the presence of VIP, the in vitro response of lymphocytes to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) was inhibited in a dose-dependent fashion, whereas that to the B cell mitogen lipopolysaccharide (LPS) was not. There was a close correlation between the potency of VIP and some structurally related peptides for inhibition of 125I-VIP binding and the effect of those peptides on T cell mitogen responses. These observations demonstrate that mouse T lymphocytes have specific VIP receptors and that VIP can modulate the response of T cells to mitogenic stimulation. VIP may be an important immunoregulatory molecule, and may be implicated in the regulation of T cell function in mucosal tissues innervated by VIP-containing neurons.     

Peptide T does not affect induction of pineal N-acetyltransferase by vasoactive intestinal peptide

It has been suggested that the HIV virus binds to VIP recognition sites which can be blocked by the octapeptide, peptide T. Stimulation of VIP receptors on pinealocytes activates adenylate cyclase and increases the activity of the enzyme serotonin N-acetyltransferase (NAT). We examined whether peptide T or D-Ala peptide T amide affected this induction. We found no evidence for peptide T interference with NAT induction and conclude that if peptide T inhibits attachment of HIV virus to VIP receptors, it does so at regions other than that occupied by VIP in stimulating adenylate cyclase and NAT.     

In vivo selection of lymphocyte-tropic and macrophage-tropic variants of lymphocytic choriomeningitis virus during persistent infection.

This study demonstrates cell-specific selection of viral variants during persistent lymphocytic choriomeningitis virus infection in its natural host. We have analyzed viral isolates obtained from CD4+ T cells and macrophages of congenitally infected carrier mice and found that three types of variants are present in individual carrier mice: (i) macrophage-tropic, (ii) lymphotropic, and (iii) amphotropic. The majority of the isolates were amphotropic and exhibited enhanced growth in both lymphocytes and macrophages. However, some of the lymphocyte-derived isolates grew well in lymphocytes but poorly in macrophages, and a macrophage-derived isolate replicated well in macrophages but not in lymphocytes. In striking contrast, the original wild-type (wt) Armstrong strain of lymphocytic choriomeningitis virus that was used to initiate the chronic infection and from which the variants are derived grew poorly in both lymphocytes and macrophages. These three types of variants also differed from the parental virus in their ability to establish a chronic infection in immunocompetent hosts. Adult mice infected with the wt Armstrong strain cleared the infection within 2 weeks, whereas adult mice infected with the variants harbored virus for several months. These results suggest that the ability of the variants to persist in adult mice is due to enhanced replication in macrophages and/or lymphocytes. This conclusion is further strengthened by the finding that the variants and the parental wt virus grew equally well in mouse fibroblasts and that the observed growth differences were specific for cells of the immune system.     

Granulomas in murine schistosomiasis mansoni contain vasoactive intestinal peptide-responsive lymphocytes.

Granulomas develop around schistosome ova in murine Schistosoma mansoni. These granulomas have eosinophils that produce VIP. It is possible that VIP participates in immunoregulation. VIP-mediated effects usually operate through a cAMP-dependent mechanism. To identify VIP-responsive inflammatory cells in murine schistosomiasis, inflammatory cells were exposed to VIP and assessed for adenylate cyclase activation and VIP binding. VIP increased adenylate cyclase activity in splenic lymphocytes from both normal and infected mice. In each case, the half-maximal stimulation was at about 5 x 10(-8) M. [125I]VIP bound to splenic lymphocytes specifically, with a Kd of 10(-8) M. This suggested that maximal adenylate cyclase activation requires full receptor occupancy. The receptor was highly specific for VIP. Hormone analogs, that are VIP receptor antagonists in some tissues, were only weak agonists of the lymphocyte VIP receptor. Granuloma cells also bound VIP and responded with adenylate cyclase activation in a manner similar to that of spleen cells. Both splenic T and B lymphocytes responded to VIP. Deletion experiments, using anti-Thy 1.2, suggested that most of the responsive granuloma cells were T lymphocytes. Thus, VIP alters cAMP metabolism in granuloma T cells through a receptor-coupled mechanism similar to that observed for spleen cells. Binding studies on mouse intestinal epithelial cells suggested that their VIP receptor is functionally and possibly structurally different from the VIP receptor on mouse lymphocytes. Additional experiments suggested that VIP and other neuropeptides are unlikely to alter the granulomatous response through a primary interaction with the granuloma macrophages.     

Resistance to apoptosis in HIV-infected CD4+ T lymphocytes is mediated by macrophages: role for Nef and immune activation in viral persistence

Apoptosis or programmed cell death may play a critical role in AIDS pathogenesis through depletion of both CD4(+) and CD8(+) T lymphocytes. Using a reporter virus, a recombinant HIV infectious clone expressing the green fluorescent protein (GFP), apoptosis was measured in productively infected CD4(+) T lymphocytes, in the presence and absence of autologous macrophages. The presence of macrophages in the culture increased the frequency of nonapoptotic GFP-positive productively infected CD4(+) T lymphocytes. The appearance of nonapoptotic productively infected CD4(+) T lymphocytes in the culture required intercellular contacts between macrophages and PBLs and the expression of the HIV Nef protein. The presence of macrophages did not reduce apoptosis when CD4(+) T lymphocytes were infected with a GFP-tagged virus deleted for the nef gene. TNF-alpha (TNF) expressed on the surface of macrophages prevented apoptosis in nef-expressing, productively infected CD4(+) T lymphocytes. Similarly, following TNF stimulation, apoptosis was diminished in Jurkat T cells transfected with a nef-expressing plasmid. TNF stimulation of nef-expressing Jurkat T cells resulted in NF-kappaB hyperactivation, which has been shown to deliver anti-apoptotic signals. Our results indicate that intercellular contacts with macrophages increase the rate of productively infected nonapoptotic CD4(+) T lymphocytes. The survival of productively infected CD4(+) T lymphocytes requires Nef expression as well as activation by TNF expressed on the surface of macrophages and might participate in the formation and maintenance of viral reservoirs in HIV-infected persons.     

In vivo CD40-CD154 (CD40 ligand) interaction induces integrated HIV expression by APC in an HIV-1-transgenic mouse model

Because of their relative resistance to viral cytopathic effects, APC can provide an alternative reservoir for latently integrated HIV. We used an HIV-transgenic mouse model in which APC serve as the major source of inducible HIV expression to study mechanisms by which integrated virus can be activated in these cells. When admixed with transgenic APC, activated T lymphocytes provided a major contact-dependent stimulus for viral protein expression in vitro. Using blocking anti-CD154 mAb as well as CD154-deficient T cells, the HIV response induced by activated T lymphocytes was demonstrated to require CD40-CD154 interaction. The role of this pathway in the induction of HIV expression from APC in vivo was further studied in an experimental model involving infection of the HIV-transgenic mice with PLASMODIUM: chabaudi parasites. Enhanced viral production by dendritic cells and macrophages in infected mice was associated with up-regulated CD40 expression. More importantly, in vivo treatment with blocking anti-CD154 mAb markedly reduced viral expression in P. chabaudi-infected animals. Together, these findings indicate that immune activation of integrated HIV can be driven by the costimulatory interaction of activated T cells with APC. Because chronic T cell activation driven by coinfections as well as HIV-1 itself is a characteristic of HIV disease, this pathway may be important in sustaining viral expression from APC reservoirs.     

CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis. Relationship to vasoactive intestinal polypeptide

The octapeptide Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr (peptide T) and two structural analogs are potent agonists of human monocyte chemotaxis, evincing identical rank potency orders as was previously shown for their inhibition of human immunodeficiency virus (HIV) envelope binding and T cell infectivity. Chemotactic activity could be inhibited by anti-CD4 monoclonal antibodies (Mabs), but not other mononuclear cell Mabs. The core peptide required for chemotactic activity is a pentapeptide related to the sequence Thr-Thr-Asn-Tyr-Thr. Homologous pentapeptides, identified by computer search, were detected in several other non-HIV-related viruses as well as the neuropeptide vasoactive intestinal polypeptide (VIP). The CD4 molecule, therefore, appears to be a recognition molecule for a small signal peptide ligand whose active sequence is a homolog of peptide T and which may be the neuropeptide VIP.     

Upregulation of Fas ligand expression by human immunodeficiency virus in human macrophages mediates apoptosis of uninfected T lymphocytes.

Apoptosis has been proposed to mediate CD4+ T-cell depletion in human immunodeficiency virus (HIV)-infected individuals. Interaction of Fas ligand (FasL) with Fas (CD95) results in lymphocyte apoptosis, and increased susceptibility to Fas-mediated apoptosis has been demonstrated in lymphocytes from HIV-infected individuals. Cells undergoing apoptosis in lymph nodes from HIV-infected individuals do not harbor virus, and therefore a bystander effect has been postulated to mediate apoptosis of uninfected cells. These data raise the possibility that antigen-presenting cells are a source of FasL and that HIV infection of cells such as macrophages may induce or increase FasL expression. In this report, we demonstrate that HIV infection of monocytic cells not only increases the surface expression of Fas but also results in the de novo expression of FasL. Interference with the FasL-Fas interaction by anti-Fas blocking antibodies abrogates HIV-induced apoptosis of monocytic cells. Human monocyte-derived macrophages from healthy donors contain detectable FasL mRNA, which is further upregulated following HIV infection with monocytotropic strains. HIV-infected human macrophages result in the apoptotic death of Jurkat T cells and peripheral blood T lymphocytes. Interruption of the FasL-Fas interaction abrogates the HIV-infected macrophage-dependent death of T lymphocytes. These results provide evidence that human macrophages can provide a source of FasL, especially following HIV infection, and can thus participate in lymphocyte depletion in HIV-infected individuals.     

Carrageenan formulation prevents macrophage trafficking from vagina: implications for microbicide development

Considerable evidence suggests that human immunodeficiency virus (HIV)-infected macrophages and/or lymphocytes may mediate sexual transmission of HIV. We and others have previously demonstrated that when vitally stained donor mouse lymphocytes or macrophages are placed in the vaginas of mice, some of the stained cells can later be found in the iliac lymph nodes. The aim of this study was to assess the extent of mononuclear cell trafficking from the vagina and to test the possibility that carrageenan formulation, a sulfated polysaccharide formulation containing 3% PDR98-15 carrageenan (PC-515; FMC Biopolymer, Rockland, ME), a vaginal microbicide, would prevent vaginal transmigration of macrophages. When supravitally stained mouse macrophages and T cells were inoculated into the vagina of recipient mice, discrete numbers of donor cells migrated to the recipient iliac and inguinal lymph nodes and spleen. When recipient mice were preinoculated with the carrageenan formulation, the number of macrophages in lymph nodes and spleen was reduced by >90%. In contrast, a methylcellulose formulation, which is believed to be inactive, did not significantly reduce migration to the lymphoid organs. Our findings suggest that the carrageenan formulation blocks cell trafficking of macrophages from vagina and that blocking does not result from cytotoxicity. Blocking cell trafficking may help to prevent sexual transmission of HIV.     

Impact of VIP and cAMP on the regulation of TNF-alpha and IL-10 production: implications for rheumatoid arthritis

Vasoactive intestinal peptide (VIP) is an anti-inflammatory immunomodulatory neuropeptide with therapeutic potential demonstrated for collagen-induced arthritis. The aim of this study was to characterise its potential anti-arthritic effect on human monocytes, macrophages, T cells, and rheumatoid arthritis synovial membrane cells. Monocytes, macrophages, and T cells derived from human peripheral blood were treated with VIP and compared with other cAMP-elevating drugs for a range of activating stimuli. Cytokine production was assessed for cell cultures and, in addition, the ability of VIPs to activate cAMP response element binding protein. VIP partially suppressed monocyte- and macrophage-derived tumour necrosis factor alpha (TNF-alpha) with no effect on IL-10, whereas VIP fails to regulate IL-10 and TNF-alpha production by T lymphocytes. No such modulation of cytokine profile was observed for rheumatoid arthritis synovial membrane cells. Elevation of intracellular cAMP, on the other hand, potently suppressed macrophage TNF-alpha production and modulated T-cell response by inhibiting TNF-alpha and IFN-gamma. VIP's lack of effect on IL-10 and its slight effect on TNF-alpha results from cAMP being rapidly degraded as the phosphodiesterase IV inhibitor, rolipram, rescues cAMP-dependent activation of cAMP response element binding protein. Interestingly, macrophages stimulated with phorbol 12-myristate 13-acetate/ionomycin displayed an augmented IL-10 response upon addition of dibutyryl cAMP, with corresponding downregulation in TNF-alpha, suggesting a complex interaction between protein kinase C and protein kinase A in cytokine regulation. In conclusion, VIP may represent an efficaceous anti-arthritic treatment modulating macrophage and T-cell cytokine profiles when used alongside a phosphodiesterase inhibitor.     

Differential transmission of HIV traversing fetal oral/intestinal epithelia and adult oral epithelia

While human immunodeficiency virus (HIV) transmission through the adult oral route is rare, mother-to-child transmission (MTCT) through the neonatal/infant oral and/or gastrointestinal route is common. To study the mechanisms of cell-free and cell-associated HIV transmission across adult oral and neonatal/infant oral/intestinal epithelia, we established ex vivo organ tissue model systems of adult and fetal origin. Given the similarity of neonatal and fetal oral epithelia with respect to epithelial stratification and density of HIV-susceptible immune cells, we used fetal oral the epithelium as a model for neonatal/infant oral epithelium. We found that cell-free HIV traversed fetal oral and intestinal epithelia and infected HIV-susceptible CD4(+) T lymphocytes, Langerhans/dendritic cells, and macrophages. To study the penetration of cell-associated virus into fetal oral and intestinal epithelia, HIV-infected macrophages and lymphocytes were added to the surfaces of fetal oral and intestinal epithelia. HIV-infected macrophages, but not lymphocytes, transmigrated across fetal oral epithelia. HIV-infected macrophages and, to a lesser extent, lymphocytes transmigrated across fetal intestinal epithelia. In contrast to the fetal oral/intestinal epithelia, cell-free HIV transmigration through adult oral epithelia was inefficient and virions did not infect intraepithelial and subepithelial HIV-susceptible cells. In addition, HIV-infected macrophages and lymphocytes did not transmigrate through intact adult oral epithelia. Transmigration of cell-free and cell-associated HIV across the fetal oral/intestinal mucosal epithelium may serve as an initial mechanism for HIV MTCT.     

The vasoactive intestinal peptide receptor-effector system is developmentally regulated in rat jejuno-ileal epithelial cells

The specific binding of vasoactive intestinal peptide (VIP) to its specific receptors as well as the stimulatory effect of the neuropeptide on cyclic AMP accumulation were studied in jejuno-ileal epithelial cells from 14-, 20- and 60-day-old rats. The potency and specificity of the VIP receptor-effector system did not vary during development. However, the concentration of VIP receptors and the efficiency of VIP stimulation of cyclic AMP generation increased from suckling to adult conditions, and VIP levels in jejuno-ileal tissue followed a parallel course.     

Complementary antiviral efficacy of hydroxyurea and protease inhibitors in human immunodeficiency virus-infected dendritic cells and lymphocytes

Dendritic cells are susceptible to human immunodeficiency virus (HIV) infection and may transmit the virus to T cells in vivo. Scarce information is available about drug efficacy in dendritic cells because preclinical testing of antiretroviral drugs has been limited predominantly to T cells and macrophages. We compared the antiviral activities of hydroxyurea and two protease inhibitors (indinavir and ritonavir) in monocyte-derived dendritic cells and in lymphocytes. At therapeutic concentrations (50 to 100 microM), hydroxyurea inhibited supernatant virus production from monocyte-derived dendritic cells in vitro but the drug was ineffective in activated lymphocytes. Concentrations of hydroxyurea insufficient to be effective in activated lymphocytes cultured alone strongly inhibited supernatant virus production from cocultures of uninfected, activated lymphocytes with previously infected monocyte-derived dendritic cells in vitro. In contrast, protease inhibitors were up to 30-fold less efficient in dendritic cells than in activated lymphocytes. Our data support the rationale for testing of the combination of hydroxyurea and protease inhibitors, since these drugs may have complementary antiviral efficacies in different cell compartments. A new criterion for combining drugs for the treatment of HIV infection could be to include at least one drug that selectively targets HIV in viral reservoirs.     

Cutting edge: is vasoactive intestinal peptide a type 2 cytokine?

A component of the chemical language shared by the immune and nervous system is the expression of neuropeptides by immune cells. Vasoactive intestinal peptide (VIP) was shown to be produced by T lymphocytes. Here we investigate whether T cell subsets differentially express VIP. Our studies indicate that, upon specific Ag stimulation, Th2 and T2 cells, but not Th1 and T1 cells derived from TCR transgenic (Tg) mice, express VIP mRNA and protein, and secrete VIP. Following immunization with the specific Ag, significant levels of VIP are present in the serum of syngeneic, non-Tg hosts that receive Th2, but not Th1 Tg cells. Th2 Tg cells recovered from the non-Tg hosts immunized with the specific Ag, but not with an irrelevant Ag, express intracellular VIP. Because VIP is produced by Ag-stimulated type 2 T cells, and differentially affects Th1 and Th2 cells, could VIP be viewed as a type 2 cytokine?     

Human immunodeficiency virus (HIV)-positive sera obtained shortly after seroconversion neutralize autologous HIV type 1 isolates on primary macrophages but not on lymphocytes

The aim of this study was to analyze the role of humoral immunity in early human immunodeficiency virus (HIV) infection. As neutralizing activities in HIV-positive sera are rarely detectable earlier than 9 to 12 months after infection using primary lymphocytes as target cells in neutralization assays, humoral immunity is generally thought not to contribute significantly to early virus control in the patients. Besides lymphocytes, cells of the monocyte/macrophage lineage are known to be important target cells for HIV in vivo during the establishment of the infection. Therefore, we studied the neutralization of early primary HIV isolates by autologous serum samples using primary macrophages as target cells in the neutralization assays. We analyzed neutralizing activities against the autologous HIV-1 isolates in 10 patients' sera taken shortly after seroconversion, both on primary macrophages and, for comparison, on lymphocytes. Viruses were isolated and expanded in primary mixed cultures containing macrophages and lymphocytes in order to avoid selection for one particular cell type. All viruses replicated to different degrees in macrophages and lymphocytes; nine had a nonsyncytium-inducing phenotype, and one was syncytium inducing. The detection of neutralizing antibodies in acute primary HIV infection depended on the target cells used. Confirming previous studies, we did not find neutralizing activities on lymphocytes at this early time point. In contrast, neutralizing activities were detectable in the same sera if primary macrophages were used as target cells. Differences in neutralizing activities on macrophages and lymphocytes were not due to different virus variants being present in the different cell systems, as gp120 sequences derived from both cell types were homogeneous. Neutralization activities on macrophages did not correlate with the amount of beta-chemokines in the sera. As affinity-purified immunoglobulin G preparations from an early patient serum also exhibited neutralization of the autologous virus isolate on primary macrophages, but not on lymphocytes, neutralization is very likely due to antibodies against viral epitopes necessary for infection of macrophages but not for infection of lymphocytes. Our data suggest that, along with cell-mediated immunity, humoral immunity may contribute to the reduction of primary viremia in the patient. This was further supported by a certain association between neutralizing antibody titers on macrophages and viral load in the patients.     

Rodent cells support key functions of the human immunodeficiency virus type 1 pathogenicity factor Nef

After infection with human immunodeficiency virus (HIV), progression toward immunodeficiency is governed by a complex interplay of viral and host determinants. The viral accessory protein Nef is a key factor for the development of AIDS. Strains of HIV and simian immunodeficiency virus that lack functional nef genes either do not induce AIDS or do so only after a significant delay. The validity of a transgenic-small-animal model for de novo infection by HIV will depend on its ability to recapitulate the actions of critical factors of viral pathogenicity, such as Nef. We assessed the ability of rat, mouse, and hamster cells to support key effector functions of Nef. In cell lines from rodents, the subcellular distribution of wild-type HIV type 1 strain SF2 Nef and mutants was comparable to that in human cells. Nef downregulated human CD4 from the cell surface, was associated with p21-activated kinase activity, and enhanced the infectivity of HIV-1 virions. Importantly, these Nef-induced effects, as well as the downregulation of rat CD4 and major histocompatibility complex class I molecules, could also be demonstrated in primary T lymphocytes and macrophages from human CD4-transgenic rats. Thus, HIV-1 Nef exerts key functions in rodent cells. In line with our ongoing efforts to establish a transgenic-rat model of HIV disease, these results indicate that important aspects of viral pathogenesis could be addressed in a transgenic-rodent model permissive for de novo infection and that such a model would be valuable for evaluating the function of Nef in vivo.     

Macrophage colony-stimulating factor antagonists inhibit replication of HIV-1 in human macrophages

Macrophages infected with HIV-1 produce high levels of M-CSF and macrophage-inflammatory protein-1alpha (MIP-1alpha). M-CSF facilitates the growth and differentiation of macrophages, while the chemotactic properties of MIP-1alpha attract both T lymphocytes and macrophages to the site of HIV infection. Studies described in this work indicate M-CSF may function in an autocrine/paracrine manner to sustain HIV replication, and data suggest possible therapeutic strategies for decreasing viral load following HIV infection. We show that macrophage infection with measles virus or respiratory syncytial virus, in contrast to HIV-1, results in production of MIP-1alpha, but not M-CSF. Thus, M-CSF appears to be specifically produced upon infection of macrophages with HIV-1. Furthermore, addition of M-CSF antagonists to HIV-1-infected macrophages, including anti-M-CSF monoclonal or polyclonal Abs or soluble M-CSF receptors, dramatically inhibited HIV-1 replication and reduced production of MIP-1alpha. Our results suggest that biologic antagonists for M-CSF may represent novel strategies for inhibiting the spread of HIV-1 by 1) blocking virus replication in macrophages, 2) reducing recruitment of HIV-susceptible T cells and macrophages by MIP-1alpha, and 3) preventing the establishment and maintenance of infected macrophages as a reservoir for HIV.     

Macrophage-derived simian immunodeficiency virus exhibits enhanced infectivity by comparison with T-cell-derived virus

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infect and productively replicate in macrophages and T lymphocytes. Here, we show that SIV virions derived from macrophages have higher levels of infectivity than those derived from T cells. The lower infectivity of T-cell-derived viruses is influenced by the quantity or type of mannose residues on the virion. Our results demonstrate that the cellular origin of a virus is a major factor in viral infectivity. Cell-type-specific factors in viral infectivity, and organ-specific or disease stage-specific differences in cellular derivation of virions, can be critical in the pathogenesis of HIV and AIDS.