Reciprocal desensitization of CCR5 and CD4 is mediated by IL-16 and macrophage-inflammatory protein-1 beta, respectively.

The ability of HIV-1 gp120 to inhibit chemokine signaling prompted us to determine whether signaling through CD4 by a natural ligand, IL-16, could alter cellular responsiveness to chemokine stimulation. These studies demonstrate that IL-16/CD4 signaling in T lymphocytes results in a selective loss of macrophage-inflammatory protein (MIP)-1 beta/CCR5-induced chemotaxis. There was no effect on monocyte chemoattractant protein-2/CCR1, -2, or -3-induced chemotaxis. Desensitization of CCR5 by IL-16 required at least 10 min of pretreatment; no modulation of CCR5 expression was observed, nor was MIP-1 beta binding to CCR5 altered. Using murine T cell hybridomas transfected to express native or mutated forms of CD4, it was determined that IL-16/CD4 induces a p56lck-dependent signal that results in desensitization of CCR5. The desensitization process is reciprocal and again selective, as prior CCR5 stimulation, but not CCR1, -2, or -3 stimulation, completely inhibits IL-16/CD4-induced T cell migration. Of interest, while p56lck enzymatic activity is not required for IL-16-induced migration, it was required for desensitization of CCR5. These studies indicate the existence of reciprocal receptor cross-desensitization between CD4 and CCR5 induced by two proinflammatory cytokines and suggest a selective relationship between the two receptors.     

Chemokine receptor CCR5 functionally couples to inhibitory G proteins and undergoes desensitization

Chemokine receptor CCR5 is not only essential for chemotaxis of leukocytes but also has been shown to be a key coreceptor for HIV-1 infection. In the present study, hemagglutinin epitope-tagged human CCR5 receptor was stably expressed in Chinese hamster ovary cells or transiently expressed in NG108–15 cells to investigate CCR5-mediated signaling events. The surface expression of CCR5 was confirmed by flow cytometry analysis. The CCR5 agonist RANTES stimulated [³⁵S]GTPγS binding to the cell membranes and induced inhibition on adenylyl cyclase activity in cells expressing CCR5. The effects of RANTES were CCR5 dependent and could be blocked by pertussis toxin. Furthermore, overexpression of Giα2 strongly increased both RANTES-dependent G-protein activation and inhibition on adenylyl cyclase in cells cotransfected with CCR5. These data demonstrated directly that activation of CCR5 stimulated membrane-associated inhibitory G proteins and indicated that CCR5 could functionally couple to G-protein subtype Giα2. The abilities of CCR5 to activate G protein and to inhibit cellular cAMP accumulation were significantly diminished after a brief prechallenge with RANTES, showing rapid desensitization of the receptor-mediated responsiveness. Prolonged exposure of the cells to RANTES caused significant reduction of surface CCR5 as measured by flow cytometry, indicative of agonist-dependent receptor internalization. Our data thus demonstrated that CCR5 functionally couples to membrane-associated inhibitory G proteins and undergoes agonist-dependent desensitization and internalization. J. Cell. Biochem. 71:36–45, 1998. © 1998 Wiley-Liss, Inc.     

Intrinsic AHR in IL-5 transgenic mice is dependent on CD4(+) cells and CD49d-mediated signaling

Overexpression of interleukin (IL)-5 by the airway epithelium in mice using the rat CC10 promoter (NJ.1726 line) leads to several histopathologies characteristic of human asthma, including airway hyperreactivity (AHR). We investigated the contribution of B and T cells, as well as CD4 expression, to the development of AHR in IL-5 transgenic mice. NJ.1726 mice on a T cell or CD4 knockout background, but not on a B cell knockout background, lost intrinsic AHR. These effects occurred without decreases in IL-5 or eosinophils. We further investigated the contribution of alpha(4)-integrin signaling to the development of AHR in IL-5 transgenic mice through the administration of anti-CD49d (alpha(4)-integrin) antibody (PS/2). Administration of PS/2 resulted in immediate (16-h) inhibition of AHR. The inhibition of AHR was not associated with a decrease in airway eosinophils. These studies demonstrate that, despite the presence of increased levels of IL-5 and eosinophils in the lungs of NJ.1726 mice, CD4(+) cells and alpha(4)-integrin signaling are necessary for the intrinsic AHR that develops in IL-5 transgenic mice.     

Evidence for CD4-enchanced signaling through the chemokine receptor CCR5

The chemokine receptor CCR5 is constitutively associated with the T cell co-receptor CD4 in plasma cell membranes, but the physiological role of this interaction has not been elucidated. Here we show that detergent-solubilized, purified CCR5 can directly associate with purified soluble fragments of the extracellular portion of CD4. We further demonstrate that the physical association of CCR5 and CD4 in membrane vesicles results in the formation of a receptor complex that exhibits macrophage inflammatory protein 1beta (MIP-1beta) binding properties that are distinct from CCR5. The affinity of the CD4-CCR5 complex for MIP-1beta was 3.5-fold lower than for CCR5, but the interaction of CD4 and CCR5 resulted in a receptor complex that exhibited enhanced G-protein signaling as compared with CCR5 alone. MIP-1beta-induced G-protein activation was further increased by simultaneous stimulation of CD4 with its natural agonist, interleukin-16. Thus, the physical association of CD4 and CCR5 results in receptor cross-talk with allosteric CD4-dependent regulation of the binding and signaling properties of CCR5. Although the precise physiological role of the CD4 effects on CCR5-mediated signaling remains unknown, one can speculate that the cross-talk is a component of mechanisms involved in the fine tuning of immune system cell responses.     

IL-16 promotes leukotriene C(4) and IL-4 release from human eosinophils via CD4- and autocrine CCR3-chemokine-mediated signaling

Human eosinophils are potential sources of inflammatory and immunomodulatory mediators, including cysteinyl leukotrienes, chemokines, and cytokines, which are pertinent to allergic inflammation. We evaluated the means by which IL-16, a recognized eosinophil chemoattractant, might act on eosinophils to affect their capacity to release leukotriene C(4) (LTC(4)) or their preformed stores of chemokines (eotaxin, RANTES) or Th1 (IL-12) or Th2 (IL-4) cytokines. IL-16 dose dependently (0.01-100 nM) elicited new lipid body formation, intracellular LTC(4) formation at lipid bodies, and priming for enhanced calcium ionophore-activated LTC(4) release. IL-16 also elicited brefeldin A-inhibitable, vesicular transport-mediated release of preformed IL-4, but not IL-12, from eosinophils. CD4 is a recognized IL-16R, and accordingly anti-CD4 Fab, soluble CD4, and a CD4 domain 4-based IL-16 blocking peptide inhibited the actions of IL-16 on eosinophils. Although CD4 is not G-protein coupled, pertussis toxin inhibited IL-16-induced eosinophil activation. IL-16 actions were found to be mediated by the autocrine activity, not of platelet-activating factor, but rather of endogenous CCR3-acting chemokines. IL-16 induced the rapid vesicular transport-mediated release of RANTES. The effects of IL-16 were blocked by CCR3 inhibitors (met-RANTES, anti-CCR3 mAb) and by neutralizing anti-eotaxin and anti-RANTES mAbs, but not by platelet-activating factor receptor antagonists (CV6209, BN52021). RANTES and eotaxin each enhanced LTC(4) and IL-4 (but not IL-12) release. Therefore, IL-16 activation of eosinophils is CD4-mediated to elicit the extracellular release of preformed RANTES and eotaxin, which then in an autocrine fashion act on plasma membrane CCR3 receptors to stimulate both enhanced LTC(4) production and the preferential release of IL-4, but not IL-12, from within eosinophils.     

Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4

This study was undertaken to demonstrate the unique specificity of the chemokine receptor CXCR4 antagonist AMD3100. Calcium flux assays with selected chemokine/cell combinations, affording distinct chemokine receptor specificities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in different cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not affect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium fluxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunodeficiency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis).     

High-Level Production,Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1

Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis.     

Cutting edge: IL-16/CD4 preferentially induces Th1 cell migration: requirement of CCR5

IL-16 binds to CD4 and induces a migratory response in CD4(+) T cells. Although it has been assumed that CD4 is the sole receptor and that IL-16 induces a comparable migratory response in all CD4(+) T cells, this has not been investigated. In this study, we determined that IL-16 preferentially induces a migratory response in Th1 cells. Because chemokine receptor CCR5 is expressed predominantly in Th1 cells and is physically associated with CD4, we investigated whether IL-16/CD4 stimulation was enhanced in the presence of CCR5. Using T cells from CCR5(null) mice, we determined that IL-16-induced migration was significantly greater in the presence of CCR5. The presence of CCR5 significantly increased IL-16 binding vs CD4 alone; however, IL-16 could not bind to CCR5 alone. Because CD4(+)CCR5(+) cells are prevalent at sites of inflammation, this intimate functional relationship likely plays a pivotal role for the recruitment and activation of Th1 cells.     

IL-16 activates plasminogen-plasmin system and promotes human eosinophil migration into extracellular matrix via CCR3-chemokine-mediated signaling and by modulating CD4 eosinophil expression

Increased eosinophil counts are a major feature of asthmatic airways. Eosinophil recruitment requires migration through epithelium and tissue extracellular matrix by activation of proteases. We assessed the capacity of IL-16, a CD4(+) cell chemotactic factor, to induce migration of eosinophils through a reconstituted basement membrane and evaluated the proteases, mediators, and receptors involved in this migration. IL-16 added to lower chambers of Invasion Chambers elicited eosinophil migration through Matrigel. This effect was decreased by inhibition of the plasminogen-plasmin system (Abs against urokinase plasminogen activator receptor or plasminogen depletion), but not by anti-matrix metalloproteinase-9 Abs. Abs against CD4 also inhibited IL-16-induced eosinophil migration. At the baseline level, few eosinophils (4.6% positive cells with a mean fluorescence of 0.9) expressed surface membrane CD4, while most permeabilized eosinophils (68% positive cells with a mean fluorescence of 18) express the CD4 Ag. TNF-pretreatment increased surface membrane CD4(+) expression by 6-fold as previously described, and increased IL-16-induced cell migration by 2.2-fold. Incubation of eosinophils with IL-16 also increased surface membrane CD4 expression by 5.4-fold, supporting the role of CD4 as receptor for IL-16. Abs against CCR3, eotaxin, or RANTES blocked IL-16-induced migration. In conclusion, IL-16 promotes eosinophil migration in vitro, by activating the plasminogen-plasmin system and increasing the membrane expression of its receptor. This effect is initiated via CD4 and mediated via the release of CCR3 ligand chemokines. Interestingly, most eosinophils express intracellular CD4. Hence, IL-16 may play an important role in the recruitment of blood eosinophils to the bronchial mucosa of asthmatics.     

FTY720-enhanced T cell homing is dependent on CCR2, CCR5, CCR7, and CXCR4: evidence for distinct chemokine compartments

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.     

HIV-1 entry into T-cells is not dependent on CD4 and CCR5 localization to sphingolipid-enriched,detergent-resistant,raft membrane domains

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56(lck). Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.     

Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12

Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12–CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of “selective blockade” of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.     

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor

The identification of chemokine receptors as HIV-1 coreceptors has focused research on developing strategies to prevent HIV-1 infection. We generated CCR2-01, a CCR2 receptor-specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T-tropic (X4) HIV-1 strains. This effect is explained by the ability of CCR2-01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV-1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells.     

HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children

Background

HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5^broad viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression.

Methodology/Principal Findings

Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5^narrow phenotype (n = 20), but R5^broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5^broad and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5^broad phenotype, however, the presence of the R5^broad virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5^broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5^broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn''s viral variant.

Conclusions/Significance

Our results show that R5^broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5^narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.     

CXCR4/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway

Chemokines and their receptors play a critical role in host immune surveillance and are important mediators of human immunodeficiency virus (HIV) pathogenesis and inflammatory response. The chemokine receptors CCR5 and CXCR4, which act as co-receptors along with CD4 for HIV docking and entry, are down-modulated by their respective ligands, MIP-1beta/SDF-1alpha or by the HIV envelope protein, gp120. We have studied the role of the proteasome pathway in the down-regulation of these receptors. Using the yeast and mammalian two-hybrid systems, we observed that the CCR5 receptor is constitutively associated with the zeta subunit of proteasome. Immunoprecipitation studies in CCR5 L1.2 cells revealed that this association was increased with MIP-1beta stimulation. The proteasome inhibitors, lactacystin and epoxomicin, attenuated MIP-1beta induced CCR5 down-modulation as detected by fluorescence-activated cell sorter analysis and confocal microscopy. The proteasome inhibitors also inhibited the SDF-1alpha and gp120 protein-induced down-modulation of the CXCR4 receptor in Jurkat cells. However, the inhibitors had no significant effect on the gp120-induced internalization of the CD4 receptor. These inhibitors also blocked cognate ligand-mediated chemotaxis but had no effect on SDF-1alpha-induced p44/42 MAP kinase or MIP-1beta-induced p38 kinase activities, thus indicating differential effects of the inhibitors on signaling mediated by these receptors. These results indicate that the CCR5 and CXCR4 receptor down-modulation mechanism and chemotaxis mediated by these receptors are dependent upon proteasome activity.     

Cutting Edge: Chemokine receptor CCR4 is necessary for antigen-driven cutaneous accumulation of CD4 T cells under physiological conditions

Dual expression of chemokine receptor CCR4 and E-selectin ligand is characteristic of skin-tropic CD4 T cells from blood, lymphoid organs, and the skin itself. A strong and specific correlation exists among CCR4, its ligand CCL17/TARC, and the cutaneous lymphocyte-homing process. Nevertheless, whether CCR4 function is required for skin-specific trafficking remains an open question, which we address in this study. We developed an Ag-specific, TCR-transgenic, murine CD4 T cell adoptive transfer model that induces a mixed Th1 and Th17 cutaneous response. Within the hosts, both CCR4(+/+) and CCR4(-/-) donor CD4 T cells contribute equally well to the circulating E-selectin ligand(+) pool in response to Ag. However, only CCR4(+/+) donor cells accumulate efficiently within the skin. CCR4(-/-) cells home normally to the peritoneum, showing that they do not have a general defect in lymphocyte trafficking. We conclude that under physiological conditions, CCR4 is a nonredundant, necessary component of skin-specific lymphocyte trafficking.     

Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.     

Constitutive association of cell surface CCR5 and CXCR4 in the presence of CD4

Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1alpha. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine-chemokine receptor interactions and HIV-1 coreceptor functions.     

Synergy between CD40 ligation and IL-4 on fibroblast proliferation involves IL-4 receptor signaling.

Fibrosis can be an undesired consequence of activated cellular immune responses. The purpose of this work was to determine whether CD40 ligation and the pro-fibrotic cytokine IL-4 interact in regulating fibroblast proliferation and collagen production, and, if so, the mechanisms used. This study found that the combination of IL-4 and ligation of CD40 on the fibroblast cell surface had synergistic effects in stimulating fibroblast proliferation. In contrast, CD40 ligation negated the inhibitory effects of IFN-gamma on fibroblast proliferation. Western blotting analyses of fibroblast crude lysates revealed that a potential mechanism of the synergy between CD40 ligation and IL-4 was the phosphorylation of proteins at 130 kDa and, to a lesser degree, at 95, 85, and 75 kDa. Immunoprecipitation-Western blotting experiments showed that phosphorylation levels of IL-4Ralpha, Janus kinase 1, insulin receptor substrate 1, and insulin receptor substrate 2, factors with molecular mass close to the observed 130 kDa major phosphorylation band, increased in response to the combined CD40 ligation and IL-4 action. In contrast, there was no evidence that synergy was mediated by an increased expression of IL-4Ralpha chain, CD40, or the autocrine profibrotic cytokines IL-6 and TGF-beta. These findings suggest that CD40-CD40 ligand contacts between fibroblasts and cells secreting IL-4 may promote the profibrotic effects of IL-4 by affecting signal transduction and reducing the anti-fibrotic effects of IFN-gamma.