β-Chemokine Production Research Articles

Changes in CCR5 and CXCR4 Expression and β-Chemokine Production in HIV-1–Infected Patients Treated With Highly Active Antiretroviral Therapy

Changes in CCR5 and CXCR4 Expression and β-Chemokine Production in HIV-1–Infected Patients Treated With Highly Active Antiretroviral Therapy

CCR5 and CXCR4 chemokine receptor expression and β-chemokine production during early T cell repopulation induced by highly active anti-retroviral therapy

Expression of chemokine receptors and beta-chemokine production by peripheral blood mononuclear cells (PBMC) were determined in HIV-1-infected individuals before and after highly active anti-retroviral therapy (HAART) and their relationship to viral load, T cell phenotype and the expression of immunological activation markers was examined. We found that the expression of CCR5 is up-regulated in HIV-1-infected individuals while CXCR4 appears down-regulated on both CD4 and CD8 T cells compared with normal controls. These alterations are associated with the high levels of viral load. In addition, a relationship was observed between the degree of immune activation and chemokine receptor expression on T cells. However, after 3 months of combined anti-retroviral regimen, expression of CXCR4 significantly increased while CCR5 decreased when compared with pretherapy determinations. This was seen in strict association with a dramatic decrease of viral load and an increase of both CD45RA+/CD62L+ (naive) and CD45RA-/CD62L+ or CD45RA+/CD62L- (memory) T cells accompanied by a significant decrease of the expression of immune activation markers such as HLA-DR and CD38. At enrolment, both spontaneous and lectin-induced RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta production by PBMC were higher in HIV-1-infected individuals compared with normal controls, although differences for MIP-1beta were not statistically significant. However, RANTES and MIP-1alpha production decreased during HAART at levels closer to that determined with normal controls, while MIP-1beta production was less consistently modified. These data indicate that the expression of chemokine receptors CCR5 and CXCR4 and the production of beta-chemokines are altered in HIV-infected individuals, and suggest that their early modifications during HAART reflect both the peripheral redistribution of naive/memory T cell compartments and the decrease in levels of T cell activation. Such modifications in the expression of host determinants of viral tropism and the production of anti-viral molecules may play a role in the emergence of virus variants when a failure of HAART occurs.

Regulation of human immunodeficiency virus type 1 infection, beta-chemokine production, and CCR5 expression in CD40L-stimulated macrophages: immune control of viral entry.

Mononuclear phagocytes (MP) and T lymphocytes play a pivotal role in the host immune response to human immunodeficiency virus type 1 (HIV-1) infection. Regulation of such immune responses can be mediated, in part, through the interaction of the T-lymphocyte-expressed molecule CD40 ligand (CD40L) with its receptor on MP, CD40. Upregulation of CD40L on CD4+ peripheral blood mononuclear cells during advanced HIV-1 disease has previously been reported. Based on this observation, we studied the influence of CD40L-CD40 interactions on MP effector function and viral regulation in vitro. We monitored productive viral infection, cytokine and beta-chemokine production, and beta-chemokine receptor expression in monocyte-derived macrophages (MDM) after treatment with soluble CD40L. Beginning 1 day after infection and continuing at 3-day intervals, treatment with CD40L inhibited productive HIV-1 infection in MDM in a dose-dependent manner. A concomitant and marked upregulation of beta-chemokines (macrophage inhibitory proteins 1alpha and 1beta and RANTES [regulated upon activation normal T-cell expressed and secreted]) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) was observed in HIV-1-infected and CD40L-treated MDM relative to either infected or activated MDM alone. The addition of antibodies to RANTES or TNF-alpha led to a partial reversal of the CD40L-mediated inhibition of HIV-1 infection. Surface expression of CD4 and the beta-chemokine receptor CCR5 was reduced on MDM in response to treatment with CD40L. In addition, treatment of CCR5- and CD4-transfected 293T cells with secretory products from CD40L-stimulated MDM prior to infection with a CCR5-tropic HIV-1 reporter virus led to inhibition of viral entry. In conclusion, we demonstrate that CD40L-mediated inhibition of viral entry coincides with a broad range of MDM immune effector responses and the down-modulation of CCR5 and CD4 expression.

LFA-3 Plasmid DNA Enhances Ag-Specific Humoral- and Cellular-Mediated Protective Immunity against Herpes Simplex Virus-2 in Vivo: Involvement of CD4+ T Cells in Protection

Adhesion molecules are important for cell trafficking and delivery of secondary signals for stimulation of T cells and antigen-presenting cells (APCs) in a variety of immune and inflammatory responses. Adhesion molecules lymphocyte function-associated antigen (LFA)-1 and CD2 on T cells recognize intercellular adhesion molecule (ICAM)-1 and LFA-3 on APCs, respectively. Recent studies have suggested that these molecules might play a regulatory role in antigen-specific immune responses. To investigate specific roles of adhesion molecules in immune induction we coimmunized LFA-3 and ICAM-1 cDNAs with a gD plasmid vaccine and then analyzed immune modulatory effects and protection against lethal herpes simplex virus (HSV)-2 challenge. We observed that gD-specific IgG production was enhanced by LFA-3 coinjection. However, little change in IgG production was observed by ICAM-1 coinjection. Furthermore, both Th1 and Th2 IgG isotype production was driven by LFA-3. LFA-3 also enhanced Th cell proliferative responses and production of interleukin (IL)-2, interferon-γ, IL-4, and IL-10 from splenocytes. In contrast, ICAM-1 showed slightly increasing effects on T-cell proliferation responses and cytokine production. β-Chemokine production (RANTES, MIP-1α, and MCP-1) was also influenced by LFA-3 or ICAM-1. When animals were challenged with a lethal dose of HSV-2, LFA-3-coimmunized animals exhibited an enhanced survival rate, as compared to animals given ICAM-1 or gD DNA vaccine alone. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vitro and in vivo T-cell subset deletion. These studies demonstrate that adhesion molecule LFA-3 can play an important role in generating protective antigen-specific immunity in the HSV model system through increased induction of CD4+ Th1 T-cell subset.

Impact of alcohol on the ability of Kupffer cells to produce chemokines and its role in alcoholic liver disease.

Chemokines are implicated in the pathogenesis of alcoholic liver disease in humans and in experimental models of alcohol intoxication. The major sources of these chemokines are Kupffer cells which represent more than 80% of tissue macrophages in the body. Kupffer cells are highly responsive to the effects of ethanol, endotoxin and human immunodeficiency virus (HIV)-1 glycoprotein120. These agents, either independently or in combination, may exacerbate the production of chemokines. Chemokines are agents that are highly chemotactic to mononuclear cells and granulocytes. The levels of these chemokines in sera and tissue are elevated in patients with alcoholic hepatitis, alcoholic cirrhosis, diseased livers, viral hepatitis, and in experimental models of chronic alcohol intoxication. Alcohol-induced influx of endotoxin from the gut into the portal circulation is suggested to play an important role in the activation of Kupffer cells which leads to enhanced chemokine release. The up-regulation of chemokines during alcohol consumption is selective. During the early phase of alcoholic liver disease, C-X-C or alpha-chemokines predominate. This is also associated with neutrophilic infiltration of the liver. In the later stage, up-regulation of C-C or beta-chemokine production and migration of mononuclear cells into the liver are observed, and this may lead to liver cirrhosis. Selective up-regulation of chemokine synthesis and release may involve differential modulation of the transcription factors required for chemokine gene expression. Increased cytokine release following alcohol consumption may also regulate chemokine secretion in Kupffer cells via paracrine and autocrine mechanisms and vice versa. In addition, infection with HIV-1 may further compromise the liver to more damage. During HIV-1 infection, a pre-existing liver disease superimposed on chronic alcohol consumption may also exacerbate HIV-1 replication and lymphocytic infiltration in the liver, because of the ability of HIV-1 gp120 to stimulate chemokine production by Kupffer cells and stimulate migration of inflammatory leucocytes in the liver.

Susceptibility of in Vitro Stimulated PBMC to Infection with NSI HIV-1 Is Associated with Levels of CCR5 Expression and β-Chemokine Production

Susceptibility of PHA/rIL-2-stimulated PBMC from 14 healthy blood donors for NSI HIV-1 infection was analyzed in relation to CCR5 expression and β-chemokine production. After 1 week of culture in the presence of rIL-2, but not at the moment of inoculation, CCR5 surface expression was positively and β-chemokine production was inversely associated with susceptibility to NSI HIV-1 infection. Surprisingly, no association was observed between CCR5 genotype and in vitro NSI HIV-1 susceptibility, which was in agreement with similar levels of CCR5 surface expression and β-chemokine production in CCR5Δ32/+ and CCR5 +/+ PBMC after PHA/rIL-2 stimulation. In contrast to what was observed in vitro, CCR5 genotype did associate with CCR5 surface expression levels in vivo in resting as well as in activated CD4+ T cell populations that were identified by the expression of CD45RO, CD27, HLA-DR, and CD69. The association between CCR5 expression and susceptibility to infection by NSI HIV-1 observed in vitro might offer an explanation for the in vivo observed protective effect of CCR5 polymorphisms that influence CCR5 expression on disease progression.

Differential induction of chemokines in human microglia by type i and ii interferonsThe first two authors contributed equally to this work.

Chemokines are secreted proteins that function as chemoattractants, mediating the recruitment of specific subsets of leukocytes to sites of tissue damage and immunological reactions. Chemokines may also function as antiviral agents, since viruses such as human immunodeficiency virus type 1 (HIV-1) use chemokine receptors as co-receptors for viral entry. This study examines whether virus-induced interferon, IFNβ, or immune-related interferon, IFNγ, affects the production of β-chemokines by CNS microglia and peripheral monocytes. When IFNβ was used as the stimulus, induction of MIP-1α, MIP-1β, MCP-1, and RANTES mRNA and protein was observed within 12 h of stimulation in microglia. By contrast, when IFNγ was used as the stimulus, only MCP-1 was induced. IFNβ stimulation of blood monocytes resulted in upregulation of MIP-1α, MIP-1β, and MCP-1. Thus, type I and II interferons differentially regulate β-chemokines in human fetal microglia and peripheral blood monocytes. These observations may have relevance for the therapeutic activity of IFNβ in multiple sclerosis and for the antiviral effects of IFNβ for HIV-1 infection of monocytes and microglia. GLIA 29:273–280, 2000. © 2000 Wiley-Liss, Inc.

Heat shock proteins generate β-chemokines which function as innate adjuvants enhancing adaptive immunity

Heat shock proteins (HSP) are widely distributed and highly immunogenic molecules. A novel property reported here is that stimulation with HSP70 of CD8-enriched T cells derived from naive non-human primates caused a dose-dependent increase in concentrations of the β-chemokines RANTES, macrophage inflammatory protein (MIP)-1α or MIP-1β. However, the concentrations of these β-chemokines were greatly increased when the CD8 T cells derived from HSP70-immunized non-human primates were stimulated with HSP70. HSP linked to peptides or proteins combined generation of β-chemokines with an adjuvant function by enhancing specific T cell proliferative responses and IgG and IgA antibodies. The β-chemokine and adjuvant functions were also elicited by topical mucosal administration of HSP linked to an antigen. We postulate that microbial HSP can stimulate β-chemokine production which may be responsible for innate adjuvanticity, as was found in cells eluted from normal rectal mucosal tissue, and constitutes a significant component of the mucosal-associated lymphoid system. Furthermore, stimulation of innate immunity may drive adaptive immunity and account for the protective effects of HSP against tumors and viruses.

Inhibition of CCR5 Expression by IL-12 Through Induction of β-Chemokines in Human T Lymphocytes

AbstractIL-12 induces initiation of the differentiation of naive CD4+ T lymphocytes into Th1 cells and is important for the control of cell-mediated immunity. β-Chemokines serve to attract various types of blood leukocytes to sites of infection and inflammation. The specific receptor for the β-chemokines (macrophage-inflammatory protein (MIP)-1α, MIP-1β, and RANTES), CCR5, also functions as the primary coreceptor for macrophage-tropic isolates of HIV-1. IL-12, but not IL-4, IL-10, or IL-13, now has been shown to down-modulate the surface expression of CCR5 induced by IL-2 on both CD4+ and CD8+ T lymphocytes. Decreased CCR5 surface expression was not secondary to transcriptional inhibition, given that CCR5 mRNA was enhanced in cells cultured in IL-12/IL-2 compared with those cultured in IL-2 only. The effect of IL-12 in down-modulation of CCR5 surface expression was shown to be mediated by soluble factors secreted from the T cells. Rapid and transient intracellular Ca2+ mobilization was induced in monocytes by IL-12-induced supernatants, which desensitized the response of monocytes to MIP-1α, but not their response to stromal cell-derived factor-1α. Neutralization with specific Abs identified these factors as MIP-1α and MIP-1β from most donors. IL-4, IL-10, IFN-γ, and IL-18 primarily inhibited MIP-1β secretion and also weakly suppressed MIP-1α secretion. HIV-1 replication was inhibited in IL-2/IL-12-containing cultures that correlated with chemokine and chemokine-receptor levels. These data suggest that the effects of IL-12 on β-chemokine production and chemokine-receptor expression may contribute to the immunomodulatory activities of IL-12 and may have potential therapeutic relevance in controlling HIV-1 replication.

Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and beta-chemokine production.

Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4(+) T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-gamma, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Deltanef, up to 2% of all CD4(+)T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.

The mode and duration of anti-CD28 costimulation determine resistance to infection by macrophage-tropic strains of human immunodeficiency virus type 1 in vitro.

We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) in vitro. Our results confirm the observations of Levine et al. (15) that stimulation of CD4 T cells with anti-CD3/anti-CD28 antibodies coimmobilized on magnetic beads renders the cells resistant to infection by M-tropic strains of HIV-1. The resistance was strongest when the beads were left in the cultures throughout the experiment. In contrast, stimulation of CD4 T cells with the same antibodies immobilized on the surface of plastic culture dishes failed to induce resistance and resulted in high levels of p24 production. This was true even if the cells were passaged continuously on freshly coated plates. If the beads were removed after initial stimulation, p24 production increased over time and produced a result intermediate to the other forms of stimulation. For beads-in, beads-out, and one-time plate stimulated cultures, resistance to infection correlated with down-regulation of CCR5 expression at the cell surface and with increased production of beta-chemokines. However, cultures of CD4 T cells continuously passaged on anti-CD3/anti-CD28-coated plates produced large amounts of p24 despite decreased levels of CCR5 expression and increasing production of beta-chemokines. Expression of the T-cell activation markers CD25 and CD69 and production of gamma interferon further supported the differences in plate versus bead stimulation. Our results explain the apparent contradiction between the ability of anti-CD28 antibody costimulation to induce resistance to HIV infection when presented on magnetic beads and the increased ability to recover virus from the cells of HIV-positive donors who are on highly active antiretroviral therapy when cells are stimulated by anti-CD3/anti-CD28 immobilized on plastic dishes.

HIV-1 DNA vaccines and chemokines

DNA vaccines have a demonstrated ability to induce humoral and cellular immune responses in animal models and humans. The technology, although it dates back to the 1950's, has had an insurgence of interest within the past few years following concurrent research papers. The basic technology is being applied broadly to viral, bacterial and parasitic infections. It has also been demonstrated that genes delivered via plasmid expression vectors result in expression of functional proteins in the inoculated host. Further, injection of plasmids encoding cytokine, chemokine or co-stimulatory molecules, also referred to as immunomodulatory plasmids can lead to the further expansion of this technology to include directed immunology. We have been developing DNA technology specifically with a focus as a vaccine against HIV-1 infection. We report that such vaccines can stimulate immune responses in a variety of relevant animal systems including humoral and cellular responses as well as the production of β-chemokines. We describe that the β-chemokines can both modulate the immune response induced by DNA vaccines and be modulated by the DNA vaccines in the murine and chimpanzee models as well as in humans.

Normal Immune Function of Monocyte-Derived Dendritic Cells from HIV-Infected Individuals: Implications for Immunotherapy

Dendritic cells (DC) are the most potent cells involved in the generation of primary and secondary immune responses. To assess the feasibility of using autologous DC as immunotherapy for HIV disease, we analyzed a variety of immune parameters using DC isolated from HIV-infected (HIV+) individuals, as well as DC obtained from HIV-uninfected (HIV-) individuals infected in vitro with HIV. After stimulation with recombinant CD40 ligand (CD40LT), cytokine and beta-chemokine production were similar by DC from HIV- donors infected in vitro with the CCR5-using HIV Ba-L strain (n = 8) compared with uninfected DC from the same donors. Production of beta-chemokines, but not of cytokines, was increased by a CXCR4-using IIIB strain-infected DC (n = 7). Stimulation of HIV-infected DC with CD40LT decreased infection in Ba-L-infected DC, but had no effect on IIIB-infected DC. Consistent with this finding, CD40LT down-regulated CCR5 and up-regulated CXCR4 expression on DC. Monocyte-derived DC were also propagated from 15 HIV+ and 13 HIV- donors. They exhibited similar expression of costimulatory molecules and produced similar amounts of IL-12, IL-10, and beta-chemokines, following stimulation. By contrast, stimulated PBMC from HIV+ patients exhibited decreased IL-12 and increased IL-10 production. In summary, phenotype, cytokine secretion, and beta-chemokine production by DC from HIV+ individuals were normal. These cells may prove useful in boosting cellular immune responses in HIV+ individuals.

Protective Role of β-Chemokines Associated with HIV-Specific Th Responses Against Perinatal HIV Transmission

AbstractTo examine the protective role of cellular immunity in the vertical transmission of HIV, we analyzed HIV-specific IL-2 and CTL responses, as well as β-chemokine expression in HIV-infected and uninfected infants of HIV+ mothers. Our results showed that HIV envelope (env) peptide-specific IL-2 responses associated with β-chemokine production were detectable at birth in the majority of uninfected infants of HIV+ mothers. The responses falling to background before the infants were 1 yr old were rarely associated with HIV-specific CTL activity. Conversely, HIV-specific Th and CTL cellular responses were absent at birth in HIV-infected infants. Infants with AIDS-related symptoms exhibited undetectable or very low levels of HIV-specific cellular immunity during the first year of life, whereas those with a slowly progressive disease showed evidence of such immunity between their second and ninth month. The latter group of infected infants tested negative for plasma HIV RNA levels shortly after birth, suggesting lack of intrauterine exposure to HIV. The presence of HIV-specific Th responses at birth in uninfected newborns of HIV+ mothers, but absence of such activities in HIV-infected infants without evidence of intrauterine HIV infection, suggests that in utero development of HIV-specific Th responses associated with β-chemokines could mediate nonlytic inhibition of infection during vertical transmission of HIV.

Differential effects of human immunodeficiency virus isolates on beta-chemokine and gamma interferon production and on cell proliferation.

All human immunodeficiency virus (HIV) isolates can grow readily in primary CD4(+) T cells, but they can be distinguished by their ability to replicate in macrophages and established T-cell lines. The macrophage-tropic viruses are generally non-syncytium inducing (NSI), whereas the T-cell-line-tropic viruses are syncytium inducing (SI) in cultured cells. We now demonstrate that infection of CD4(+) T cells by NSI and SI viruses shows a differential effect on production of beta-chemokines and gamma interferon. Infection by NSI viruses increased production of MIP-1alpha, MIP-1beta, and gamma interferon, whereas infection by SI viruses had no effect or decreased production of these cytokines. Production of RANTES was slightly increased during infection by both virus phenotypes. This differential effect of NSI and SI viruses was observed at the level of beta-chemokine mRNA as well as at the level of protein expression. Infection by NSI viruses also increased CD4(+) cell proliferation. These results may have relevance for a differential role of HIV strains in AIDS pathogenesis.

Differential Development of Type 1 and Type 2 Cytokines and β-Chemokines in the Ontogeny of Healthy Newborns

Interleukin (IL)-2, interferon gamma (IFN-γ; type 1 cytokines), IL-4, and IL-10 (type 2 cytokines), and β-chemokines (MIP-1α and RANTES) production by cord blood lymphocytes (CBL) and peripheral blood lymphocytes (PBL) of newborns was analyzed in a cross-sectional study to examine the maturation of these components of the immune response. Immunophenotyping was performed on the same specimens. Results showed that the CD4/CD8 ratio remains stable, the percentage of natural killer cells decreases, and the number and percentage of B cells increase after birth. Analysis of cytokine production suggested that the production of all cytokines increases gradually and steadily after birth, and that IFN-γ and IL-10 production is reduced at birth whereas IL-2 and IL-4 production is not. Finally, mitogen-stimulated β-chemokine production was present at birth and increased slightly but significantly with age. These data indicate that a differential functional maturation of immune response after birth favoring a more precocious development of IL-2 (a type 1 cytokine) is present and should help to analyze the ontogeny of the immune system.

Differences in HIV replication in CD4+ lymphocytes are not related to beta-chemokine production.

CD4+ T lymphocytes from different donors vary in their ability to replicate different isolates of HIV. Beta-chemokines have been shown to reduce the rate of HIV replication in cultured cells. We now demonstrate, using CD4+ cells from 19 different donors, that the variations in viral replication observed in CD4+ lymphocytes are not due to endogenous production of beta-chemokines by the cells. Instead of finding a correlation of high-level beta-chemokine production with low-level replication of virus, we found either no consistent relationship between these two parameters or a correlation between high-level beta-chemokine production and high-level virus replication. This observation was made with both chemokine-sensitive and chemokine-resistant HIV isolates. Thus, other mechanisms appear to be involved in the variability in HIV replication in cultured CD4+ cells.

Binding of HIV-2 envelope glycoprotein to CD8 molecules and related chemokine production.

We recently found that human immunodeficiency virus (HIV)-2 envelope glycoprotein, but not that of HIV-1, could bind to CD4 and CD8 molecules on T cells, and that the binding site of HIV-2 envelope glycoprotein was located on the alpha-chain (but not the beta-chain) of CD8. This study showed that the binding of HIV-2 envelope glycoprotein could induce phosphorylation of protein tyrosine kinase p56lck in CD8+ T cells. We also found that production of beta-chemokines in response to HIV-2 envelope glycoprotein was significantly higher than that in response to HIV-1 envelope glycoprotein, and that CD8+ T cells were the main source of beta-chemokines production among the T-cell population. These findings indicate the possibility that the binding of envelope glycoprotein to CD8 molecules are related to signal transduction into CD8+ T cells and the resultant beta-chemokine production in HIV-2 infection. Our results may help to explain the differences in disease manifestations between HIV-1 and HIV-2, including the lower virulence of HIV-2 and the longer survival of HIV-2-infected individuals.

Naı̈ve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis

In vitro evidence suggests that memory CD4(+) cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA+ CD4(+) (naïve) and CD45RO+ CD4(+) (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4(+) cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4(+) subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta upon stimulation. Neutralization of these beta-chemokines rendered memory CD4(+) cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and beta-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4(+) T cells serve as the targets for early rounds of HIV-1 replication.

CD40 ligand (CD154) stimulation of macrophages to produce HIV-1-suppressive beta-chemokines.

beta-chemokines play an important role in the development of immunologic reactions. Macrophages are major beta-chemokine-producing cells during T-cell directed, delayed-type hypersensitivity reactions in tissues, and have been reported to be important producers of beta-chemokines in the lymph nodes of HIV-1-infected individuals. However, the physiological signals responsible for inducing macrophages to produce beta-chemokines have not been established. Two soluble T cell products, interferon-gamma and granulocyte-macrophage colony stimulating factor, were added to cultured macrophages, but failed to stimulate the production of macrophage inflammatory protein-1alpha and -1beta; regulated upon activation, normal T cell expressed and secreted (RANTES); or monocyte chemoattractant protein-1. Instead, direct cell-cell contact between macrophages and cells engineered to express CD40L (also known as CD154) resulted in the production of large amounts of macrophage inflammatory protein-1alpha and -1beta, and RANTES (all ligands for CCR5), and monocyte chemoattractant protein-1 (a ligand for CCR2). Supernatants from CD40L-stimulated macrophages protected CD4(+) T cells from infection by a nonsyncytium-inducing strain of HIV-1 (which uses CCR5 as a coreceptor). These results have implications for granulomatous diseases, and conditions such as atherosclerosis and multiple sclerosis, where CD40L-bearing cells have been found in the macrophage-rich lesions where beta-chemokines are being produced. Overall, these findings define a pathway linking the specific recognition of antigen by T cells to the production of beta-chemokines by macrophages. This pathway may play a role in anti-HIV-1 immunity and the development of immunologic reactions or lesions.