Exclusion of HIV coreceptors CXCR4, CCR5, and CCR3 from the HIV envelope.

Both HIV-1 primary isolates and laboratory strains incorporate cell-derived molecules into their envelopes depending on the host cell in which they are grown. This incorporation is not random and, specifically, HIV-1 has been shown to select against the incorporation into its surface of CD4, its main receptor. In this study, we have looked at the incorporation of HIV coreceptors CXCR4, CCR5, and CCR3 into the HIV envelope. For this purpose, we grew HIV-1 primary isolate BZ167 in several cell lines and PBMCs, and the envelope profiles of the resulting viruses were determined with a virus-binding ELISA. While the virus particle gained several molecules when passed through the different cell lines (e.g., ICAM-3, LFA-1, ICAM-1, or MHC class II), BZ167 never incorporated significant levels of CXCR4, CCR5, or CCR3 into its envelope even though some or all of the cell lines in which it was grown expressed them. These results show that HIV-1 selects against the incorporation of these chemokine receptors into its envelope molecule, as it does against the incorporation of CD4.     

Mycobacterium tuberculosis upregulates coreceptors CCR5 and CXCR4 while HIV modulates CD14 favoring concurrent infection

Tuberculosis is the most frequent coinfection in humans infected with HIV-1, but little is known about mechanisms that favors coinfection. The aim of this work is to understand tuberculosis and HIV infections. We determined the pattern of expression of CD11c, CD14, CD40, CCR5, and CXCR4 and quantified IL-1beta, IL-6, IL-8, TNF-alpha, and RANTES in tuberculosis patients and HIV patients. Monocytes from healthy PPD+ volunteers (HP(+)V) stimulated with intracellular proteins (IP), lipids, and polysaccharides (PLS) from Mycobacterium tuberculosis down regulate CD11c expression (p < 0.05). On the contrary, CD14 expression was elevated in tuberculosis patients (p < 0.05) and HIV-infected patients (p > 0.05). CD14 expression was elevated on monocytes from HP(+)V stimulated with PLS and lipids (p < 0.05). CD40 low expression was found in tuberculosis patients and on monocytes from HP(+)V stimulated with lipids, but it was elevated in HIV-infected patients (p < 0.05). CXCR4 and CCR5 expression was high in pulmonary tuberculosis patients and low in HIV-infected patients (p < 0.05). Finally, CCR5+ monocytes from HP(+)V after stimulation with PLS and CXCR4+ lymphocytes were elevated after stimulation with IP (p < 0.05). In general, high levels of IL-1beta, IL-6, and TNF-alpha were found in all groups, but low levels of RANTES were found in pulmonary tuberculosis patients. In conclusion, the pulmonary tuberculosis patients have a microenvironment that facilitates the HIV infection through three possible mechanisms: (1) increasing the coreceptor for HIV entrance, (2) increasing proinflammatory cytokines, and (3) down-regulating RANTES. At the same time, HIV patients have a microenvironment that facilitates entry of M. tuberculosis into macrophages through CD14.     

Patients with active tuberculosis have increased expression of HIV coreceptors CXCR4 and CCR5 on CD4(+) T cells.

Expression of human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5 was found to be elevated on CD4(+) T cells (1) in blood samples obtained from patients with tuberculosis and (2) in blood samples obtained from healthy subjects and stimulated with mycobacterial lipoarabinomannan in vitro. These data suggest that the increase in HIV viremia that occurs in association with tuberculosis may result from up-regulation of CXCR4 and CCR5 on CD4(+) T cells, thereby causing acceleration of HIV infection.     

gp120 envelope glycoproteins of human immunodeficiency viruses competitively antagonize signaling by coreceptors CXCR4 and CCR5

Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K⁺ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1α for CXCR4 and MIP-1α; MIP-1β and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5–7 min and recovery time constants of 12–19 min. CXCR4 and CCR5 exhibited heterologous desensitization in this oocyte system, suggesting possible inhibition of a common downstream step in their signaling pathways. In contrast to chemokines, perfusion with monomeric or oligomeric preparations of the glycoprotein of M_r 120,000 (gp120) derived from several isolates of HIV-1 did not activate signaling by CXCR4 or CCR5 regardless of CD4 coexpression. However, adsorption of the gp120 from a T-cell-tropic virus resulted in CD4-dependent antagonism of CXCR4 response to SDF-1α, whereas gp120 from macrophage-tropic viruses caused CD4-dependent antagonism of CCR5 response to MIP-1α. These antagonisms could be partially overcome by high concentrations of chemokines and were specific for coreceptors of the corresponding HIV-1 isolates, suggesting that they resulted from direct interactions of gp120–CD4 complexes with coreceptors and that they did not involve the desensitization pathway. These results indicate that monomeric or oligomeric gp120s specifically antagonize CXCR4 and CCR5 signaling in response to chemokines, but they do not exclude the possibility that gp120s might also function as weak agonists in some cells. The gp120-mediated disruption of CXCR4 and CCR5 signaling may contribute to AIDS pathogenesis.     

Thalidomide suppresses Up-regulation of human immunodeficiency virus coreceptors CXCR4 and CCR5 on CD4+ T cells in humans

Concurrent infection in patients with human immunodeficiency virus (HIV) infection increases the expression of HIV coreceptors CXCR4 and CCR5. Thalidomide has beneficial effects in a number of HIV-associated diseases. The effect of thalidomide on CXCR4 and CCR5 expression on CD4+ T cells was determined. Thalidomide produced a dose-dependent inhibition of lipopolysaccharide (LPS)-induced up-regulation of CXCR4 and CCR5 in vitro. Antibody to tumor necrosis factor-alpha (TNF-alpha) also attenuated the LPS-induced HIV coreceptor up-regulation, which was not further reduced by thalidomide. Thalidomide (400 mg) was orally administered to 6 men, and their blood was stimulated ex vivo with LPS, staphylococcal or mycobacterial antigens, or antibody to CD3 or CD28 cells. All stimuli induced up-regulation of HIV coreceptors, which was reduced after ingestion of thalidomide. Thalidomide may be beneficial in the treatment of intercurrent infections during HIV infection by reducing the up-regulation of CXCR4 and CCR5 expression on CD4+ T cells induced by bacterial and mycobacterial antigens, by a mechanism that involves inhibition of TNF-alpha.     

Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection

HIV entry into human cells is mediated by CD4 acting in concert with one of several members of the chemokine receptor superfamily. The resistance to HIV infection observed in individuals with defective CCR5 alleles indicated that this particular chemokine receptor plays a crucial role in the initiation of in vivo HIV infection. Expression of human CD4 transgene does not render mice susceptible to HIV infection because of structural differences between human and mouse CCR5. To ascertain whether expression of human CD4 and CCR5 is sufficient to make murine T lymphocytes susceptible to HIV infection, the lck promoter was used to direct the T cell-specific expression of human CD4 and CCR5 in transgenic mice. Peripheral blood mononuclear cells and splenocytes isolated from these mice expressed human CD4 and CCR5 and were infectible with selected M-tropic HIV isolates. After in vivo inoculation, HIV-infected cells were detected by DNA PCR in the spleen and lymph nodes of these transgenic mice, but HIV could not be cultured from these cells. This indicated that although transgenic expression of human CD4 and CCR5 permitted entry of HIV into the mouse cells, significant HIV infection was prevented by other blocks to HIV replication present in mouse cells. In addition to providing in vivo verification for the important role of CCR5 in T lymphocyte HIV infection, these transgenic mice represent a new in vivo model for understanding HIV pathogenesis by delineating species-specific cellular factors required for productive in vivo HIV infection. These mice should also prove useful for the assessment of potential therapeutic and preventative modalities, particularly vaccines.     

Evolution of HIV-1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains

The chemokine receptor CXCR4 functions as a fusion coreceptor for T cell tropic and dual-tropic HIV-1 strains. To identify regions of CXCR4 that are important for coreceptor function, CXCR4–CXCR2 receptor chimeras were tested for the ability to support HIV-1 envelope (env) protein-mediated membrane fusion. Receptor chimeras containing the first and second extracellular loops of CXCR4 supported fusion by T tropic and dual-tropic HIV-1 and HIV-2 strains and binding of a monoclonal antibody to CXCR4, 12G5, that blocks CXCR4-dependent infection by some virus strains. The second extracellular loop of CXCR4 was sufficient to confer coreceptor function to CXCR2 for most virus strains tested but did not support binding of 12G5. Truncation of the CXCR4 cytoplasmic tail or mutation of a conserved DRY motif in the second intracellular loop did not affect coreceptor function, indicating that phosphorylation of the cytoplasmic tail and the DRY motif are not required for coreceptor function. The results implicate the involvement of multiple CXCR4 domains in HIV-1 coreceptor function, especially the second extracellular loop, though the structural requirements for coreceptor function were somewhat variable for different env proteins. Finally, a hybrid receptor in which the amino terminus of CXCR4 was replaced by that of CCR5 was active as a coreceptor for M tropic, T tropic, and dual-tropic env proteins. We propose that dual tropism may evolve in CCR5-restricted HIV-1 strains through acquisition of the ability to utilize the first and second extracellular loops of CXCR4 while retaining the ability to interact with the CCR5 amino-terminal domain.     

Distribution of the human immunodeficiency virus coreceptors CXCR4 and CCR5 in fetal lymphoid organs: implications for pathogenesis in utero

HIV entry into a cell requires the coordinate cell surface expression of CD4 and one of several chemokine coreceptors. Here we have examined the expression of the two most widely utilized HIV coreceptors, CXCR4 and CCR5, on various lymphoid tissues derived from the fetus. CXCR4 and CCR5 are differentially expressed on lymphocytes in different lymphoid organs, which may reflect the maturational and functional status of cells within the organ. The different levels of coreceptor expression on CD4+ cells in the various organs may directly affect the targeting of CXCR4- and CCR5-tropic strains of HIV toward different fetal lymphoid tissues during in utero infection.     

Up-regulation of HIV coreceptors CXCR4 and CCR5 on CD4(+) T cells during human endotoxemia and after stimulation with (myco)bacterial antigens: the role of cytokines

Concurrent infections in patients with human immunodeficiency virus (HIV) infection stimulate HIV replication. Chemokine receptors CXCR4 and CCR5 can act as HIV coreceptors. The authors hypothesized that concurrent infection increases the HIV load through up-regulation of CXCR4 and CCR5. Using experimental endotoxemia as a model of infection, changes in HIV coreceptor expression were assessed in 8 subjects injected with lipopolysaccharide (LPS, 4 ng/kg). The expression of CXCR4 and CCR5 on CD4(+) T cells was increased 2- to 4-fold, 4 to 6 hours after LPS injection. In whole blood in vitro, LPS induced a time- and dose-dependent increase in the expression of CXCR4 and CCR5 on CD4(+) T cells. Similar changes were observed after stimulation with cell wall components of Mycobacterium tuberculosis (lipoarabinnomannan) or Staphylococcus aureus (lipoteichoic acid), or with staphylococcal enterotoxin B. LPS increased viral infectivity of CD4-enriched peripheral blood mononuclear cells (PBMCs) with a T-tropic HIV strain. In contrast, M-tropic virus infectivity was reduced, possibly because of elevated levels of the CCR5 ligand cytokines RANTES and MIP-1beta. LPS-stimulated up-regulation of CXCR4 and CCR5 in vitro was inhibited by anti-TNF and anti-IFN gamma. Incubation with recombinant TNF or IFN gamma mimicked the LPS effect. Anti-interleukin 10 (anti-IL-10) reduced CCR5 expression, without influencing CXCR4. In accordance, rIL-10 induced up-regulation of CCR5, but not of CXCR4. Intercurrent infections during HIV infection may up-regulate CXCR4 and CCR5 on CD4(+) T cells, at least in part via the action of cytokines. Such infections may favor selectivity of HIV for CD4(+) T cells expressing CXCR4. (Blood. 2000;96:2649-2654)     

CC chemokines and the receptors CCR3 and CCR5 are differentially expressed in the nonneoplastic leukocytic infiltrates of Hodgkin disease

Lymph nodes with Hodgkin disease (HD) harbor few neoplastic cells in a marked leukocytic infiltrate. Since chemokines are likely to be involved in the recruitment of these leukocytes, the expression of potentially relevant chemokines and chemokine receptors were studied in lymph nodes from 24 patients with HD and in 5 control lymph nodes. The expression of regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta was analyzed by in situ hybridization and that of CCR3 and CCR5 by immunohistochemistry and flow cytometry. It was found that, overall, the expression of all 4 chemokines was markedly enhanced, but the cellular source was different. RANTES was expressed almost exclusively by T cells whereas the expression of MCP-1, MIP-1alpha, and MIP-1beta was confined largely to macrophages. In control lymph nodes, chemokine expression was low, with the exception of MIP-1alpha in macrophages. CCR3 and CCR5 were highly expressed in T cells of HD involved but not of control lymph nodes. CCR3 was equally distributed in CD4+ and CD8+ cells, but CCR5 was associated largely with CD4+ cells. In HD lymph nodes, CCR3 and CCR5 were also expressed in B cells, which normally do not express these receptors. All these chemokines and receptors studied, by contrast, were absent in the neoplastic cells. It was concluded that chemokines are involved in the formation of the HD nonneoplastic leukocytic infiltrate. Expression of CCR3 and CCR5 appears to be characteristic of HD, but the roles of these receptors' up-regulation for the disease process remain unclear.     

R5 HIV productively infects Langerhans cells,and infection levels are regulated by compound CCR5 polymorphisms

Langerhans cells (LCs) are suspected to be initial targets for HIV after sexual exposure (by becoming infected or by capturing virus). Here, productive R5 HIV infection of LC ex vivo and LC-mediated transmission of virus to CD4+ T cells were both found to depend on CCR5. By contrast, infection of monocyte-derived dendritic cells and transfer of infection from monocyte-derived dendritic cells to CD4+ T cells were mediated by CCR5-dependent as well as DC-specific ICAM-3-grabbing nonintegrin-dependent pathways. Furthermore, in 62 healthy individuals, R5 HIV infection levels in LCs ex vivo were associated with CCR5 genotype. Specifically, genotyping for ORF Delta 32 revealed that LCs isolated from ORF Delta 32/wt individuals were significantly less susceptible to HIV when compared with LCs isolated from ORFwt/wt individuals (P = 0.016). Strikingly, further genetic analyses of the A-2459G CCR5 promoter polymorphism in ORF Delta 32/wt heterozygous individuals revealed that LCs isolated from -2459A/G + ORF Delta 32/wt individuals were markedly less susceptible to HIV than were LCs from -2459A/A + ORF Delta 32/wt individuals (P = 0.012). Interestingly, these genetic susceptibility data in LCs parallel those of genetic susceptibility studies performed in cohorts of HIV-infected individuals. Thus, we suggest that CCR5-mediated infection of LCs, and not capture of virus by LCs, provides a biologic basis for understanding certain aspects of host genetic susceptibility to initial HIV infection.     

Interclade neutralization and enhancement of human immunodeficiency virus type 1 identified by an assay using HeLa cells expressing both CD4 receptor and CXCR4/CCR5 coreceptors

We report on the development and evaluation of a human immunodeficiency virus (HIV) neutralization assay that uses P4P cells, which are CD4+CXCR4+CCR5+ HeLa cells that carry the lacZ gene under the control of the HIV-1 long terminal repeat. The results of the present study suggest that the P4P assay can be used for the extensive study of both neutralizing and enhancing activity in serum samples from HIV-1-infected patients and from vaccinated individuals.     

Kinetics of CXCR4 and CCR5 up-regulation and human immunodeficiency virus expansion after antigenic stimulation of primary CD4(+) T lymphocytes

The chemokine receptors CCR5 and CXCR4 are coreceptors for the human immunodeficiency virus (HIV) and determine the cell tropism of different HIV strains. Previous studies on their regulation were performed under conditions of unspecific T-lymphocyte stimulation and provided conflicting results. To mimic physiologic conditions, highly purified primary Staphylococcus enterotoxin B (SEB)-reactive CD4 T lymphocytes were stimulated in the presence of autologous antigen-presenting cells and the kinetics of CCR5 and CXCR4 surface expression and HIV replication were studied. Both chemokine receptors were transiently up-regulated with maximal expression at day 3 after stimulation. The stimulated T cells were equally susceptible to productive infection with R5-and X4-tropic virus strains. Thus, antigenic stimulation of T cells promotes efficient replication of both, T cell-tropic and macrophage-tropic HIV. (Blood. 2000;96:1853-1856)     

A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes

Preferential homing of naive lymphocytes to secondary lymphoid organs is thought to involve the action of chemokines, yet no chemokine has been shown to have either the expression pattern or the activities required to mediate this process. Here we show that a chemokine represented in the EST database, secondary lymphoid-tissue chemokine (SLC), is expressed in the high endothelial venules of lymph nodes and Peyer’s patches, in the T cell areas of spleen, lymph nodes, and Peyer’s patches, and in the lymphatic endothelium of multiple organs. SLC is a highly efficacious chemoattractant for lymphocytes with preferential activity toward naive T cells. Moreover, SLC induces firm adhesion of naive T lymphocytes via β2 integrin binding to the counter receptor, intercellular adhesion molecule-1, a necessary step for lymphocyte recruitment. SLC is the first chemokine demonstrated to have the characteristics required to mediate homing of lymphocytes to secondary lymphoid organs. In addition, the expression of SLC in lymphatic endothelium suggests that the migration of lymphocytes from tissues into efferent lymphatics may be an active process mediated by this molecule.     

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.     

Lamina propria lymphocytes, not macrophages, express CCR5 and CXCR4 and are the likely target cell for human immunodeficiency virus type 1 in the intestinal mucosa

Most human immunodeficiency virus type 1 (HIV-1) infections are acquired via mucosal surfaces, and transmitted viruses are nearly always macrophage-tropic, suggesting that mucosal macrophages participate in early HIV-1 infection. Mucosal lymphocytes isolated from normal human intestine expressed CD4 (14,530+/-7970 antibody-binding sites [ABSs]/cell), CCR5 (2730+/-1524 ABSs/cell), and CXCR4 (2507+/-1840 ABSs/cell), but intestinal macrophages, which also expressed CD4 (2959+/-2695 ABSs/cell), displayed no detectable CCR5 or CXCR4 ABS. The absence of CCR5 on intestinal macrophages was not due to expression of the Delta32 deletion allele because matched-blood monocytes expressed CCR5. CCR5(+)CXCR4(+) intestinal lymphocytes supported both R5 (BaL) and X4 (IIIB) HIV-1 replication, whereas the CCR5(-)CXCR4(-) macrophages were not permissive to either isolate or other laboratory isolates (ADA and DJV) and primary isolates (MDR 24 and JOEL). In the intestinal mucosa, lymphocytes, not macrophages, are the likely target cell for R5 (and X4) HIV-1 and are the major source of HIV-1 production during early infection.     

Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages

CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2- to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4+/CD45RO+/CD62L-true memory cells compared with CD4+/CD45RO+/CD62L+ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitro maturation of PBDC resulted in median 3- and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from approximately 5,000 to approximately 50, 000 ABS) whereas granulocyte-macrophage colony-stimulating factor caused a marked decrease of CXCR4 (from approximately 5,000 ABS to <500) while up-regulating CCR5 expression (from approximately 5,000 to approximately 20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms.