Identification of human macrophage inflammatory proteins 1alpha and 1beta as a native secreted heterodimer

Chemokines are secreted proteins that function as chemoattractants for leukocytes. The chemokines macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha and MIP-1beta) now have been shown to be secreted from activated human monocytes and peripheral blood lymphocytes (PBLs) as a heterodimer. Immunoprecipitation and immunoblot analysis revealed that antibodies to either MIP-1alpha or MIP-1beta precipitated a protein complex containing both MIP-1alpha and MIP-1beta under normal conditions from culture supernatants and lysates of these cells. Mass spectrometry of the complexes, precipitated from the culture supernatants of monocytes and PBLs, revealed the presence of NH(2)-terminal truncated MIP-1alpha (residues 5-70) together with either intact MIP-1beta or NH(2)-terminal truncated MIP-1beta (residues 3-69), respectively. The secreted MIP-1alpha/beta heterodimers were dissociated into their component monomers under acidic conditions. Exposure of monocytes or PBLs to monensin induced the accumulation of heterodimers composed of NH(2)-terminal truncated MIP-1alpha and full-length MIP-1beta in the Golgi complex. The mixing of recombinant chemokines in vitro demonstrated that heterodimerization of MIP-1alpha and MIP-1beta is specific and that it occurs at physiological conditions, pH 7.4, and in the range of nanomolar concentrations. The data presented here provide the first biochemical evidence for the existence of chemokine heterodimers under natural conditions. Formation of heterodimers of MIP-1alpha/beta may have an impact on intracellular signaling events that contribute to CCR5 and possibly to other chemokine receptor functions.     

Regulated production and molecular diversity of human liver and activation-regulated chemokine/macrophage inflammatory protein-3 alpha from normal and transformed cells

Liver and activation-regulated chemokine (LARC), also designated macrophage inflammatory protein-3alpha (MIP-3alpha), Exodus, or CCL20, is a C-C chemokine that attracts immature dendritic cells and memory T lymphocytes, both expressing CCR6. Depending on the cell type, this chemokine was found to be inducible by cytokines (IL-1beta) and by bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as measured by a specific ELISA. Although coinduced with monocyte chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and IL-1beta in diploid fibroblasts, leukocytes produced LARC/MIP-3alpha only in response to LPS. However, in myelomonocytic THP-1 cells LARC/MIP-3alpha was better induced by phorbol ester, whereas in HEp-2 epidermal carcinoma cells IL-1beta was the superior inducer. The production levels of LARC/MIP-3alpha (1-10 ng/ml) were, on the average, 10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable to those of other less abundantly secreted chemokines. Natural LARC/MIP-3alpha protein isolated from stimulated leukocytes or tumor cell lines showed molecular diversity, in that NH(2)- and COOH-terminally truncated forms were purified and identified by amino acid sequence analysis and mass spectrometry. In contrast to other chemokines, including MCP-1 and IL-8, the natural processing did not affect the calcium-mobilizing capacity of LARC/MIP-3alpha through its receptor CCR6. Furthermore, truncated natural LARC/MIP-3alpha isoforms were equally chemotactic for lymphocytes as intact rLARC/MIP-3alpha. It is concluded that in addition to its role in homeostatic trafficking of leukocytes, LARC/MIP-3alpha can function as an inflammatory chemokine during host defense.     

Macrophage inflammatory protein-1

Macrophage inflammatory protein (MIP)-1alpha was identified 15 years ago as the first of now four members of the MIP-1 CC chemokine subfamily. These proteins termed CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CCL9/10 (MIP-1delta), and CCL15 (MIP-1gamma) according to the revised nomenclature for chemokines are produced by many cells, particularly macrophages, dendritic cells, and lymphocytes. MIP-1 proteins, which act via G-protein-coupled cell surface receptors (CCR1, 3, 5), e.g. expressed by lymphocytes and monocytes/macrophages (MPhi), are best known for their chemotactic and proinflammatory effects but can also promote homoeostasis. The encouraging results of preclinical studies in murine models of inflammation, i.e. asthma, arthritis, or multiple sclerosis, have led to the development of potent CCR3 and 5 antagonists, some of which are currently being tested in first clinical trials.     

Amino-terminal processing of MIP-1beta/CCL4 by CD26/dipeptidyl-peptidase IV

CD26 is a membrane-bound ectopeptidase with dipeptidyl peptidase IV (DPPIV) activity that has diverse functional properties in T cell physiology and in regulation of bioactive peptides. We have previously reported that activated human peripheral lymphocytes (PBL) secrete an amino-terminal truncated form of macrophage inflammatory protein (MIP)-1beta/(3-69) with novel functional specificity for CCR1, 2, and 5. In this report, we show that the full length MIP-1beta is processed by CD26/DPPIV to the truncated form and that cleavage can be blocked by DPPIV inhibitory peptides derived from HIV Tat(1-9) or the thromboxane A2 receptor, TAX2-R(1-9). Addition of Tat(1-9) or TAX2-R(1-9) peptides to PBL cultures partially blocks endogenous MIP-1beta processing. The kinetics of conversion of MIP-1beta from intact to MIP-1beta(3-69) in activated PBLs correlates with cell surface expression of CD26. Our results suggest that NH2-terminal processing of MIP-1beta and possibly other chemokines may depend on the balance between CD26/DPPIV enzymatic activity and cellular and viral proteins that modulate enzyme function.     

Differential recognition and scavenging of native and truncated macrophage-derived chemokine (macrophage-derived chemokine/CC chemokine ligand 22) by the D6 decoy receptor

The promiscuous D6 receptor binds several inflammatory CC chemokines and has been recently proposed to act as a chemokine-scavenging decoy receptor. The present study was designed to better characterize the spectrum of CC chemokines scavenged by D6, focusing in particular on CCR4 ligands and analyzing the influence of NH(2)-terminal processing on recognition by this promiscuous receptor. Using D6 transfectants, it was found that D6 efficiently bound and scavenged most inflammatory CC chemokines (CCR1 through CCR5 agonists). Homeostatic CC chemokines (CCR6 and CCR7 agonists) were not recognized by D6. The CCR4 agonists CC chemokine ligand 17 (CCL17) and CCL22 bound to D6 with high affinity. CCL17 and CCL22 have no agonistic activity for D6 (chemotaxis and calcium fluxes), but were rapidly scavenged, resulting in reduced chemotactic activity on CCR4 transfectants. CD26 mediates NH(2) terminus processing of CCL22, leading to the production of CCL22 (3-69) and CCL22 (5-69) that do not interact with CCR4. These NH(2)-terminal truncated forms of CCL22 were not recognized by D6. The results presented in this study show that D6 recognizes and scavenges a wide spectrum of inflammatory CC chemokines, including the CCR4 agonists CCL22 and CCL17. However, this promiscuous receptor is not engaged by CD26-processed, inactive, CCL22 variants. By recognizing intact CCL22, but not its truncated variants, D6 expressed on lymphatic endothelial cells may regulate the traffic of CCR4-expressing cells, such as dendritic cells.     

Macrophage inflammatory protein 1alpha inhibits postentry steps of human immunodeficiency virus type 1 infection via suppression of intracellular cyclic AMP

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.     

Interleukin-8-mediated heterologous receptor internalization provides resistance to HIV-1 infectivity. Role of signal strength and receptor desensitization

Human immunodeficiency virus type 1 (HIV-1) entry into CD4(+) cells requires the chemokine receptors CCR5 or CXCR4 as co-fusion receptors. We have previously demonstrated that chemokine receptors are capable of cross-regulating the functions of each other and, thus, affecting cellular responsiveness at the site of infection. To investigate the effects of chemokine receptor cross-regulation in HIV-1 infection, monocytes and MAGIC5 and rat basophilic leukemia (RBL-2H3) cell lines co-expressing the interleukin-8 (IL-8 or CXCL8) receptor CXCR1 and either CCR5 (ACCR5) or CXCR4 (ACXCR4) were generated. IL-8 activation of CXCR1, but not the IL-8 receptor CXCR2, cross-phosphorylated CCR5 and CXCR4 and cross-desensitized their responsiveness to RANTES (regulated on activation normal T cell expressed and secreted) (CCL5) and stromal derived factor (SDF-1 or CXCL12), respectively. CXCR1 activation internalized CCR5 but not CXCR4 despite cross-phosphorylation of both. IL-8 pretreatment also inhibited CCR5- but not CXCR4-mediated virus entry into MAGIC5 cells. A tail-deleted mutant of CXCR1, DeltaCXCR1, produced greater signals upon activation (Ca(2+) mobilization and phosphoinositide hydrolysis) and cross-internalized CXCR4, inhibiting HIV-1 entry. The protein kinase C inhibitor staurosporine prevented phosphorylation and internalization of the receptors by CXCR1 activation. Taken together, these results indicate that chemokine receptor-mediated HIV-1 cell infection is blocked by receptor internalization but not desensitization alone. Thus, activation of chemokine receptors unrelated to CCR5 and CXCR4 may play a cross-regulatory role in the infection and propagation of HIV-1. Since DeltaCXCR1, but not CXCR1, cross-internalized and cross-inhibited HIV-1 infection to CXCR4, the data indicate the importance of the signal strength of a receptor and, as a consequence, protein kinase C activation in the suppression of HIV-1 infection by cross-receptor-mediated internalization.     

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.     

Induction of rapid and extensive beta-chemokine synthesis in macrophages by human immunodeficiency virus type 1 and gp120, independently of their coreceptor phenotype

Human immunodeficiency virus type 1 (HIV-1) interacts with its target cells through CD4 and a coreceptor, generally CCR5 or CXCR4. Macrophages display CD4, CCR5, and CXCR4 that are competent for binding and entry of virus. Virus binding also induces several responses by lymphocytes and macrophages that can be dissociated from productive infection. We investigated the responses of macrophages to exposure to a series of HIV-1 species, R5 species that productively infect and X4 species that do not infect macrophages. We chose to monitor production of several physiologically relevant factors within hours of treatment to resolve virally induced effects that may be unlinked to HIV-1 production. Our novel findings indicate that independently of their coreceptor phenotype and independently of virus replication, exposure to certain R5 and X4 HIV-1 species induced secretion of high levels of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, RANTES, and tumor necrosis factor alpha. However two of the six R5 species tested, despite efficient infection, were unable to induce rapid chemokine production. The acute effects of virus on macrophages could be mimicked by exposure to purified R5 or the X4 HIV-1 envelope glycoprotein gp120. Depletion of intracellular Ca(2+) or inhibition of protein synthesis blocked the chemokine induction, implicating Ca(2+)-mediated signal transduction and new protein synthesis in the response. The group of viruses able to induce this chemokine response was not consistent with coreceptor usage. We conclude that human macrophages respond rapidly to R5 and X4 envelope binding by production of high levels of physiologically active proteins that are implicated in HIV-1 pathogenesis.     

Macrophage inflammatory protein-1

Macrophage inflammatory protein-1 (MIP-1) and MIP-1β are highly related members of the CC chemokine subfamily. Despite their structural similarities, MIP-1 and MIP-1β show diverging signaling capacities. Depending on the MIP-1 subtype and its NH₂-terminal processing, one or more of the CC chemokine receptors CCR1, CCR2, CCR3 and CCR5 are recognized. Since both human MIP-1 subtypes (LD78 and LD78β) and MIP-1β signal through CCR5, the major co-receptor for M-tropic HIV-1 strains, these chemokines are capable of inhibiting HIV-1 infection in susceptible cells. In this review, different aspects of human and mouse MIP-1 and MIP-1β are discussed, including their protein and gene structures, their regulated production, their receptor usage and biological activities and their role in several pathologies including HIV-1 infection.     

The seventh transmembrane domain of cc chemokine receptor 5 is critical for MIP-1beta binding and receptor activation: role of MET 287

CC chemokine receptor 5 (CCR5) is a high-affinity receptor for macrophage inflammatory protein (MIP)-1beta and functions as the major coreceptor for entry of macrophage-tropic (M-tropic) human immunodeficiency virus type 1 (HIV-1). To evaluate the role of transmembrane domains (TM) in the receptor function of CCR5, the seventh transmembrane domain (TM7) was examined in a series of chimeric receptor constructs including CCR5TM (CCR5 backbone/CCR5 TM7 replaced with CCR1 TM7) and mutants of CCR5TM. The CCR5TM chimera exhibited a dramatic reduction in receptor activation, as well as little or no MIP-1beta binding. Further mutational analysis revealed that Met 287 in TM7 of CCR5 is a critical molecular determinant for both MIP-1beta binding and receptor activation. Interestingly, all of the chimeric/mutated receptors were biologically active in an HIV-1 coreceptor fusion assay, demonstrating that chemokine binding is independent of HIV-1 coreceptor activity.     

The LD78beta isoform of MIP-1alpha is the most potent CC-chemokine in inhibiting CCR5-dependent human immunodeficiency virus type 1 replication in human macrophages

The CC-chemokines RANTES, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta are natural ligands for the CC-chemokine receptor CCR5. MIP-1alpha, also known as LD78alpha, has an isoform, LD78beta, which was identified as the product of a nonallelic gene. The two isoforms differ in only 3 amino acids. LD78beta was recently reported to be a much more potent CCR5 agonist than LD78alpha and RANTES in inducing intracellular Ca2+ signaling and chemotaxis. CCR5 is expressed by human monocytes/macrophages (M/M) and represents an important coreceptor for macrophage-tropic, CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains to infect the cells. We compared the antiviral activities of LD78beta and the other CC-chemokines in M/M. LD78beta at 100 ng/ml almost completely blocked HIV-1 replication, while at the same concentration LD78alpha had only weak antiviral activity. Moreover, when HIV-1 infection in M/M was monitored by a flow cytometric analysis using p24 antigen intracellular staining, LD78beta proved to be the most antivirally active of the chemokines. RANTES, once described as the most potent chemokine in inhibiting R5 HIV-1 infection, was found to be considerably less active than LD78beta. LD78beta strongly downregulated CCR5 expression in M/M, thereby explaining its potent antiviral activity.     

Association of chemokine-mediated block to HIV entry with coreceptor internalization.

Chemokines inhibit entry of HIV into CD4(+) T cells more effectively than into macrophages or transfected adherent cells. Here, we tested whether chemokine receptor internalization could account for cell type differences in the effectiveness of chemokines. Infection of CEM T cells expressing stably transduced wild-type CCR5 was much more readily inhibited by chemokine than were transduced HOS cells. This response correlated with the efficiency of CCR5 internalization. A mutated CCR5, termed M7-CCR5, in which the Ser/Thr phosphorylation sites in the cytoplasmic tail were changed to Ala, did not internalize in response to MIP-1alpha. M7-CCR5 was expressed at slightly higher levels than wild-type on stably transduced cell lines and was somewhat more potent as an HIV-1 coreceptor. The mutated receptor mobilized intracellular Ca(2+) in response to chemokine to a level 4-fold higher than did the wild type CCR5. Unexpectedly, the receptor was desensitized as efficiently as wild type, suggesting that desensitization does not require cytoplasmic tail phosphorylation. Entry of R5 HIV-1 reporter virus into cells stably expressing M7-CCR5 was largely resistant to blocking by MIP-1alpha. As much as 80% of entry inhibition was attributed to receptor internalization. Aminooxypentane (AOP)-MIP-1alpha was able to induce a low level of M7-CCR5 internalization in HOS and to weakly inhibit HIV-1 entry. Introduction of dominant negative dynamin into HOS cells reduced the ability of chemokine to inhibit infection. The inefficiency of internalization of chemokine receptors in some cell types could allow virus to replicate in vivo in the presence of endogenous chemokine. Last, M7-CCR5 is a useful tool for discriminating coreceptor internalization from binding site masking in the evaluation of small molecule inhibitors of HIV-1 entry.     

pH-independent endocytic cycling of the chemokine receptor CCR5

Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin-coated pits and delivered to recycling endosomes. Subsequently, ligand- free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G-protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH(4)Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the beta(2)-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1beta (CCL4) only exhibit pH-dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor-agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP-1beta dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.     

Tat protein is an HIV-1-encoded beta-chemokine homolog that promotes migration and up-regulates CCR3 expression on human Fc epsilon RI+ cells

Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. HIV-1 Tat protein is a potent chemoattractant for basophils and lung mast cells obtained from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Tat protein induced a rapid and transient Ca(2+) influx in basophils and mast cells, analogous to beta-chemokines. Tat protein neither induced histamine release from human basophils and mast cells nor increased IL-3-stimulated histamine secretion from basophils. The chemotactic activity of Tat protein was blocked by preincubation of FcepsilonRI(+) cells with anti-CCR3 Ab. Preincubation of Tat with a mAb anti-Tat (aa 1-86) blocked the migration induced by Tat. In contrast, a mAb specific for the basic region (aa 46-60) did not inhibit the chemotactic effect of Tat protein. Tat protein or eotaxin desensitized basophils to a subsequent challenge with the autologous or the heterologous stimulus. Preincubation of basophils with Tat protein up-regulated the level of CCR3 mRNA and the surface expression of the CCR3 receptor. Tat protein is the first identified HIV-1-encoded beta-chemokine homologue that influences the directional migration of human FcepsilonRI(+) cells and the expression of surface receptor CCR3 on these cells.     

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC(50), 1.8 to 7.3 microM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.     

Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-alpha/beta-induced inflammatory responses and antiviral defense in liver

IFN-alpha/beta-mediated functions promote production of MIP-1alpha (or CCL3) by mediating the recruitment of MIP-1alpha-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-gamma delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-alpha/beta and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1alpha during infection and was dependent on IFN-alpha/beta effects for induction. Resident F4/80(+) liver leukocytes were identified as primary IFN-alpha/beta responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1alpha and IFN-gamma in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.     

Chemokine receptor CCR5 deficiency exacerbates cerulein-induced acute pancreatitis in mice

Acute pancreatitis (AP) is an inflammatory disease involving the production of different cytokines and chemokines and is characterized by leukocyte infiltration. Because the chemokine receptor CCR5 and its ligands [the CC chemokines CCL3/MIP-1alpha, CCL4/MIP-1beta, and CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES)] regulate leukocyte chemotaxis and activation, we investigated the expression of CCR5 ligands and the role of CCR5 and its ligands in experimental AP in mice. AP was induced by hourly intraperitoneal injections of cerulein in CCR5-deficient (CCR5(-/-)) or wild-type (WT) mice. Induction of AP by cerulein resulted in an early increase of pancreatic CCL2, CCL3, and CCL4 mRNA expression, whereas CCL5 mRNA expression occurred later. CCR5(-/-) mice developed a more severe pancreatic injury than WT mice during cerulein-induced AP, as assessed by a more pronounced increase in serum amylase and lipase levels and by more severe pancreatic edema, inflammatory infiltrates (mainly neutrophils), and necrosis. CCR5(-/-) mice also exhibited increased production of CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-1beta during the course of cerulein-induced AP. In vivo simultaneous neutralization of CC chemokines with monoclonal antibodies in CCR5(-/-) mice reduced the severity of cerulein-induced AP, indicating a role of CC chemokines in exacerbating the course of AP in the absence of CCR5. Moreover, simultaneous neutralization of CCR5 ligands in WT mice also reduced the severity of cerulein-induced AP. In conclusion, lack of the chemokine receptor CCR5 exacerbates experimental cerulein-induced AP and leads to increased levels of CC chemokines and a more pronounced pancreatic inflammatory infiltrate, suggesting that CCR5 expression can modulate severity of AP.     

Chemokine receptor expression and function in CD4+ T lymphocytes with regulatory activity

We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4(+) T cells (Th(IL-10)) releasing high levels of IL-10, low IFN-gamma, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting Th(IL-10), including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting Th(IL-10), in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated Th(IL-10) exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently Th(IL-10) than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory Th(IL-10) lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated Th(IL-10) cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.