Hemofiltrate CC chemokine 1[9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages

Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.     

Interaction of Chemokine Receptor CCR5 with its Ligands: Multiple Domains for HIV-1 gp120 Binding and a Single Domain for Chemokine Binding

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α, and MIP-1β, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1α, or MIP-1β. This mAb inhibited most of the RANTES and MIP-1α chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH₂-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1–derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH₂-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.     

Agonist-induced internalization of CC chemokine receptor 5 as a mechanism to inhibit HIV replication

The chemokine G protein-coupled receptor CC chemokine receptor 5 (CCR5) is used as an entry gate by CCR5-tropic and dual- or CCR5/CXC chemokine receptor 4-tropic strains of HIV to enter the human host cells. Thus, CCR5 antagonists (i.e., maraviroc) have been proven to be clinically effective by preventing the interaction between viral glycoprotein 120 and CCR5 and thus impeding viral entry into host cells. However, the emergence of HIV strains resistant to CCR5 antagonists has been reported in vitro and in vivo, where the virus has adapted to enter the cells via antagonist-bound CCR5. An alternative strategy that should obviate this mode of viral resistance would entail the ablation of the CCR5 portal for HIV entry from the cell surface through agonist-induced receptor internalization. Although this protective effect has been demonstrated clearly with natural CCR5 ligands, the chemoattractant properties of these chemokines have precluded them from further consideration in terms of drug development. Thus, we sought to explore the possibility of developing novel small molecules and selective CCR5 agonists devoid of eliciting chemotaxis. Indeed, the CCR5 agonists described herein were found to induce profound down-modulation of CCR5 (and not CXC chemokine receptor 4) from the cell surface and its sustained sequestration in the intracellular compartment without inducing chemotaxis in vitro. The bioactivity profile of these novel CCR5 agonists is exemplified by the compound (R)-2-(4-cyanophenyl)-N-(1-(1-(N,1-diphenylmethylsulfonamido)propan-2-yl)piperidin-4-yl)acetamide (ESN-196) that potently inhibits HIV-1 infection in human peripheral blood mononuclear cells and macrophages in vitro with potencies comparable to that of maraviroc and moreover demonstrates full activity against a maraviroc-resistant HIV-1 RU570 strain.     

Preclinical evaluation of synthetic -2 RANTES as a candidate vaginal microbicide to target CCR5

A potential strategy that can be used to combat the worldwide AIDS epidemic is the development of a vaginal microbicide that prevents the sexual transmission of human immunodeficiency virus type 1 (HIV-1). Certain CC chemokines, including RANTES, MIP-1alpha, and MIP-1beta, might facilitate the development of such microbicides since they potently suppress HIV-1 infection by binding to CCR5, the viral coreceptor used by most sexually transmitted strains of HIV-1 to enter host cells. In this study, we evaluated whether a CCR5-specific fragment of RANTES that lacks two N-terminal residues (-2 RANTES) and possesses especially potent HIV-1 suppressive activity has toxicity profiles conducive to the advancement of testing in candidate microbicide formulations. Analyses were carried out with a synthetic version of the chemokine, which was formulated with either Novasomes 7474, a nonphospholipid liposome, or methylcellulose gel. Dialysis studies demonstrated that the formulated -2 RANTES was released from both vehicles and retained anti-HIV-1 activity. Preclinical toxicity studies carried out with Swiss Webster mouse and New Zealand White rabbit vaginal irritation models demonstrated minimal inflammation and minimal adverse changes in cervicovaginal tissue integrity after short-term (10 min) and long-term (24 h) exposure to formulations containing up to 1 mg/ml of -2 RANTES. Similarly, no toxicity was observed with formulations of bioactive murine RANTES in the Swiss Webster mouse vaginal irritation model. Overall, these preclinical studies suggest that -2 RANTES is suitable for further testing as a candidate anti-HIV-1 microbicide.     

The LD78β isoform of MIP-1α is the most potent CCR5 agonist and HIV-1–inhibiting chemokine

LD78alpha and LD78beta are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Two molecular forms of natural LD78beta (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78alpha and LD78beta only differ in 3 amino acids, both LD78beta variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78alpha. On the contrary, LD78beta was only 2-fold more efficient than LD78alpha in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor-transfected cells, both molecular forms of LD78beta proved to be much more potent than LD78alpha in inducing an intracellular calcium rise through CCR5. Compared with LD78alpha and RANTES, this preferential binding of LD78beta to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78beta is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.     

Protecting from R5-tropic HIV: individual and combined effectiveness of a hammerhead ribozyme and a single-chain Fv antibody that targets CCR5

The CCR5 chemokine receptor is important for most clinical strains of HIV to establish infection. Individuals with naturally occurring polymorphisms in the CCR5 gene who have reduced or absent CCR5 are apparently otherwise healthy, but are resistant to HIV infection. With the goal of reducing CCR5 and protecting CCR5+ cells from R5-tropic HIV, we used Tag-deleted SV40-derived vectors to deliver several anti-CCR5 transgenes: 2C7, a single-chain Fv (SFv) antibody; VCKA1, a hammerhead ribozyme; and two natural CCR5 ligands, MIP-1alpha and MIP-1beta, modified to direct these chemokines, and hence their receptor to the endoplasmic reticulum. These transgenes were delivered using recombinant, Tag-deleted SV40-derived vectors to human CCR5+ cell lines and primary cells: monocyte-derived macrophages and brain microglia. All transgenes except MIP-1alpha decreased CCR5, as assayed by immunostaining, Northern blotting, and cytofluorimetry (FACS). Individually, all transgenes except MIP-1alpha protected from low challenge doses of HIV. At higher dose HIV challenges, protection provided by all transgenes diminished, the SFv and the ribozyme being most potent. Vectors carrying these two transgenes were used sequentially to deliver combination anti-CCR5 genetic therapy. This approach gave approximately additive reduction in CCR5, as measured by FACS and protected from higher dose HIV challenges. Reducing cell membrane CCR5 using anti-CCR5 transgenes, alone or in combinations, may therefore provide a degree of protection from R5-tropic strains of HIV.     

Bone marrow CD34(+) cells and megakaryoblasts secrete beta-chemokines that block infection of hematopoietic cells by M-tropic R5 HIV

CD34(+) cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34(+) cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34(+) cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34(+) cells and CD34(+)KIT(+) cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1alpha, and MIP-1beta and that IFN-gamma stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34(+)KIT(+) cells, even by RT-PCR. Conditioned media from CD34(+) cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34(+) cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34(+) cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.     

The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains.

Infection of target cells by HIV-1 requires initial binding interactions between the viral envelope glycoprotein gp120, the cell surface protein CD4, and one of the members of the seven-transmembrane G protein-coupled chemokine receptor family. Most primary isolates (R5 strains) use chemokine receptor CCR5, but some primary syncytium-inducing, as well as T cell line-adapted, strains (X4 strains) use the CXCR4 receptor. Signaling from both CCR5 and CXCR4 is mediated by pertussis toxin (PTX)-sensitive G(i) proteins and is not required for HIV-1 entry. Here, we show that the PTX holotoxin as well as its binding subunit, B-oligomer, which lacks G(i)-inhibitory activity, blocked entry of R5 but not X4 strains into primary T lymphocytes. Interestingly, B-oligomer inhibited virus production by peripheral blood mononuclear cell cultures infected with either R5 or X4 strains, indicating that it can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1beta or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1beta or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity is mediated by signaling events that involve PKC. B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.     

Macrophage-tropic HIV induces and exploits dendritic cell chemotaxis

Immature dendritic cells (iDCs) express the CC chemokine receptor (CCR)5, which promotes chemotaxis toward the CC chemokines regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta. By contrast, mature DCs downregulate CCR5 but upregulate CXC chemokine receptor (CXCR)4, and as a result exhibit enhanced chemotaxis toward stromal cell-derived factor (SDF)-1alpha. CCR5 and CXCR4 also function as coreceptors for macrophage-tropic (M-tropic) and T cell-tropic (T-tropic) human immunodeficiency virus (HIV)-1, respectively. Here, we demonstrate chemotaxis of iDCs toward M-tropic (R5) but not T-tropic (X4) HIV-1. Furthermore, preexposure to M-tropic HIV-1 or its recombinant envelope protein prevents migration toward CCR5 ligands. The migration of iDCs toward M-tropic HIV-1 may enhance formation of DC-T cell syncytia, thus promoting viral production and destruction of both DC and T helper lymphocytes. Therefore, disturbance of DC chemotaxis by HIV-1 is likely to contribute to immunosuppression in primary infection and AIDS. In addition, migration of iDCs toward HIV-1 may aid the capture of R5 HIV-1 virions by the abundant DC cell surface protein DC-specific intercellular adhesion molecule (ICAM)3-grabbing nonintegrin (DC-SIGN). HIV-1 bound to DC cell-specific DC-SIGN retains the ability to infect replication-permissive T cells in trans for several days. Consequently, recruitment of DC by HIV-1 could combine with the ability of DC-SIGN to capture and transmit the virus to T cells, and so facilitate dissemination of virus within an infected individual.     

CCR1 and CCR5 promote hepatic fibrosis in mice

Hepatic fibrosis develops as a response to chronic liver injury and almost exclusively occurs in a proinflammatory environment. However, the role of inflammatory mediators in fibrogenic responses of the liver is only poorly understood. We therefore investigated the role of CC chemokines and their receptors in hepatic fibrogenesis. The CC chemokines MIP-1α, MIP-1β, and RANTES and their receptors CCR1 and CCR5 were strongly upregulated in 2 experimental mouse models of fibrogenesis. Neutralization of CC chemokines by the broad-spectrum CC chemokine inhibitor 35k efficiently reduced hepatic fibrosis, and CCR1- and CCR5-deficient mice displayed substantially reduced hepatic fibrosis and macrophage infiltration. Analysis of fibrogenesis in CCR1- and CCR5-chimeric mice revealed that CCR1 mediates its profibrogenic effects in BM-derived cells, whereas CCR5 mediates its profibrogenic effects in resident liver cells. CCR5 promoted hepatic stellate cell (HSC) migration through a redox-sensitive, PI3K-dependent pathway. Both CCR5-deficient HSCs and CCR1- and CCR5-deficient Kupffer cells displayed strong suppression of CC chemokine–induced migration. Finally, we detected marked upregulation of RANTES, CCR1, and CCR5 in patients with hepatic cirrhosis, confirming activation of the CC chemokine system in human fibrogenesis. Our data therefore support a role for the CC chemokine system in hepatic fibrogenesis and suggest distinct roles for CCR1 and CCR5 in Kupffer cells and HSCs.     

Lack of good correlation of serum CC-chemokine levels with human immunodeficiency virus-1 disease stage and response to treatment

Three CC-chemokines-MIP-1alpha (CCL3), MIP-1beta (CCL4), and RANTES (CCL5)-are natural ligands for the human immunodeficiency virus-1 (HIV-1) coreceptor CCR5. To determine correlations between CC-chemokines and HIV-1 disease stage or response to treatment, we examined serum levels of MIP-1alpha, MIP-1beta, and RANTES in 60 infected patients during 18 months while they were taking highly active antiretroviral therapy (HAART). Our results demonstrate that serum levels of MIP-1alpha and RANTES were increased in HIV-1-infected individuals compared with those in healthy controls. We found no significant differences among 4 clinical stages of HIV-1 infection in the serum levels of three CC-chemokines. Longitudinal HAART analyses revealed a pronounced decline in serum MIP-1alpha levels over time. We found no difference in this decline between HAART responders and nonresponders. These findings indicate that production of MIP-1alpha and RANTES changes during HIV-1 infection and treatment; however, our results suggest that serum levels of CC-chemokines should not be used as biomarkers for HIV-1 disease stage or response to treatment.     

A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock

CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4(-/-)) mice by gene targeting. CCR4(-/-) mice developed normally. Splenocytes and thymocytes isolated from the CCR4(-/-) mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1alpha. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4(-/-) mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4(+/+) mice. After high dose LPS treatment, serum levels of tumor necrosis factor alpha, interleukin 1beta, and MIP-1alpha were reduced in CCR4(-/-) mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4(-/)- mice by flow cytometry also revealed a significant decrease in the F4/80(+) cell population. This may reflect a defect in the ability of the CCR4(-/-) macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.     

Endogenous levels of C-C chemokines MIP-1α, MIP-1β, and RANTES do not reflect the disease course in HIV-seropositive individuals

It has been shown that the beta (C-C) chemokines macrophage inflammatory protein-1alpha/beta (MIP-1alpha/beta) and RANTES, which are released by CD8+ T cells, are potent inhibitors of HIV viral replication in vitro. To investigate whether serum concentrations of these chemokines reflect such a protective effect in vivo, we measured these in peripheral blood of 60 HIV seropositive patients, 10 healthy subjects, and 10 disease controls. Values were compared with the CDC disease stages and immunological surrogate markers of disease progression, such as CD4+ count, beta2-microglobulin and 5'-neopterin serum levels. In addition, HIV RNA was measured in sera. All three chemokines were not significantly different between HIV patients and healthy individuals, nor were differences of chemokine levels between the CDC stages significant. Instead, disease controls exhibited significantly more MIP-1alpha and MIP-1beta than normals or HIV patients. Furthermore, within the HIV-seropositive subjects we did not observe any relationship with the surrogate markers of HIV disease, CD4+ count, CD4+/CD8+ ratio, beta2-microglobulin, and 5'-neopterin (all correlations NS). HIV viral load did not correlate with the measured chemokine concentrations (r < 0.1, NS). In conclusion, endogenous levels of MIP-1alpha, MIP-1beta, and RANTES do not reflect the hypothesized protective effect on disease progression in HIV infection. Thus, despite potential beneficial effects of the investigated chemokines, other factors may equally contribute to HIV replication control in vivo.     

Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties

Hemofiltrate CC chemokine (HCC)-1 is a recently described human chemokine that is constitutively expressed in numerous tissues and is present at high concentrations in normal plasma. Using a cell line expressing CC chemokine receptor (CCR)5 as a bioassay, we isolated from human hemofiltrate an HCC-1 variant lacking the first eight amino acids. HCC-1[9-74] was a potent agonist of CCR1, CCR3, and CCR5 and promoted calcium flux and chemotaxis of T lymphoblasts, monocytes, and eosinophils. It also blocked entry of HIV-1 strains using CCR5 as coreceptor. Limited tryptic digestion of HCC-1 generated the active variant. Conditioned media from several tumor cell lines activated HCC-1 with a high efficiency, and this activity could be inhibited by serine protease inhibitors. Our results indicate that HCC-1 represents a nonfunctional precursor that can be rapidly converted to the active chemokine by proteolytic processing. This process represents an additional mechanism by which tumor cells might generate chemoattractant molecules and recruit inflammatory cells. It might also affect HIV-1 replication in infected individuals and play an important role in AIDS pathogenesis.     

Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis

During the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1alpha), CCR2 (MCP-1), CCR5 (RANTES, MIP-1alpha, MIP-1beta), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1(-/-) mice. We found that neutrophil accumulation in CCR1(-/-) mice was comparable to that in wild-type animals but that renal recruitment of CD4(+) and CD8(+) T cells and macrophages increased significantly. Moreover, CCR1(-/-) mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1(-/-) mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-gamma and TNF-alpha. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1alpha, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.     

Differential sensitivities of pyrogenic chemokine fevers to CC chemokine receptor 5 antibodies

Macrophage inflammatory protein (MIP)-1beta and RANTES (regulated on activation, normal T-cells expressed and secreted) are members of the CC-family of chemokines. Although these two peptides are structurally and functionally related to one another, each exhibits distinct features, which allows it to independently regulate specific aspects of the host inflammatory response. They evoked intense and functionally different febrile responses when applied directly on pyrogen-sensitive cells located in the in the preoptic area of the anterior hypothalamus (POA). The present experiments were carried out to test the central role of CCR5, a functional receptor for MIP-1beta and RANTES, in the febrile responses induced by these chemokines when injected directly into the POA. The microinjection of an equimolecular dose (50 pg) of either MIP-1beta or RANTES into the POA induced a rapid onset; monophasic fever in rats that persisted for a long period. The microinjection of 2.0 microg specific neutralizing antibodies against CCR5 (anti-CCR5) into the POA fails to affect the effects on body temperature induced by MIP-1beta. However, pretreatment with the same dose of anti-CCR5 suppressed the febrile response induced by RANTES given at the same site. The microinjection of control IgG or anti-CCR5 does not affect basal temperature, when administered alone at the same hypothalamic site. The present experiments show that hypothalamic CCR5 are functionally involved in the febrile response induced by RANTES, but not by MIP-1beta. They also suggest the existence of functionally different components in the presumptive primary locus of the thermoregulatory controller, in which both chemotactic cytokines, together other mediators, could play a relevant role in the complex process of fever pathogenesis.     

Role of CCR5 in IFN-gamma-induced and cigarette smoke-induced emphysema

Th1 inflammation and remodeling characterized by tissue destruction frequently coexist in human diseases. To further understand the mechanisms of these responses, we defined the role(s) of CCR5 in the pathogenesis of IFN-gamma-induced inflammation and remodeling in a murine emphysema model. IFN-gamma was a potent stimulator of the CCR5 ligands macrophage inflammatory protein-1alpha/CCL-3 (MIP-1alpha/CCL-3), MIP-1beta/CCL-4, and RANTES/CCL-5, among others. Antibody neutralization or null mutation of CCR5 decreased IFN-gamma-induced inflammation, DNA injury, apoptosis, and alveolar remodeling. These interventions decreased the expression of select chemokines, including CCR5 ligands and MMP-9, and increased levels of secretory leukocyte protease inhibitor. They also decreased the expression and/or activation of Fas, FasL, TNF, caspase-3, -8, and -9, Bid, and Bax. In accordance with these findings, cigarette smoke induced pulmonary inflammation, DNA injury, apoptosis, and emphysema via an IFN-gamma-dependent pathway(s), and a null mutation of CCR5 decreased these responses. These studies demonstrate that IFN-gamma is a potent stimulator of CC and CXC chemokines and highlight the importance of CCR5 in the pathogenesis of IFN-gamma-induced and cigarette smoke-induced inflammation, tissue remodeling, and emphysema. They also demonstrate that CCR5 is required for optimal IFN-gamma stimulation of its own ligands, other chemokines, MMPs, caspases, and cell death regulators and the inhibition of antiproteases.     

Cloning, expression, and characterization of the human eosinophil eotaxin receptor

Although there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of beta- chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein- coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and RANTES with Kds of 0.1, 2.7 and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-1 alpha or MIP-1 beta. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a Ca(2+) - flux. Competition binding studies on primary eosinophils gave binding affinities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-1 alpha.     

Aminooxypentane-RANTES Induces CCR5 Internalization but Inhibits Recycling: A Novel Inhibitory Mechanism of HIV Infectivity

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH₂-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.