Chemokines MIP-1β Research Articles

An in vitro study to elucidate the effects of product Nkabinde on immune response in peripheral blood mononuclear cells of healthy donors.

Introduction: A significant number of the South African population still rely on traditional medicines (TM) as their primary healthcare due to their belief in their holistic healing and immune-boosting properties. However, little to no scientific data is available on the effects of most TM products on cytokine and cellular biomarkers of the immune response. Here, we evaluated the impact of traditional medicine [Product Nkabinde (PN)] in inducing cellular and cytokine biomarkers of inflammation in peripheral blood mononuclear cells (PBMCs) from eight healthy volunteers. Methods: PN was supplied by a local Traditional Health Practitioner (THP). The IC50 (half maximum concentration) of the standardized extract on isolated PBMCs was established using the cell viability assay over 24h of incubation. Luminex and flow cytometry assays were used to measure cytokine and cellular levels in PBMCs stimulated with PN and/or PHA over 24, 48, and 72h, respectively. Results: The IC50 concentration of PN in treated PBMCs was established at 325.3μg/mL. In the cellular activation assay, the percentages of CD38-HLA-DR + on total CD4+ T cells were significantly increased in PBMCs stimulated with PN compared to unstimulated controls after 24h (p = 0.008). PN significantly induced the production of anti-inflammatory IL-10 (p = 0.041); proinflammatory cytokines IL-1α (p = 0.003), TNF-α (p < 0.0001); and chemokine MIP-1β (p = 0.046) compared to the unstimulated control after 24h. At 48h incubation, the production of proinflammatory cytokines IL-1α (p = 0.034) and TNF-α (p = 0.011) were significantly induced following treatment with PN. Conclusion: We conclude that the PN possesses in vitro immunomodulatory properties that may influence immune and inflammatory responses. More studies using PN are needed to further understand key parameters mediating induction, expression, and regulation of the immune response in the context of pathogen-associated infections.

Biological Evaluation of the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A for Disease Profiling of 48 Immune Factors

Abstract Cytokine, chemokine and growth factor research plays a significant role in achieving a deeper understanding of the immune system and disease. We recently developed a MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A multiplex assay for simultaneous measurement of picogram levels of 48 human immune factors using Luminex® xMAP® technology in small volumes of serum, plasma or cell culture samples. Distinctly different stimulant-dependent cytokine responses were observed for challenged human PBMCs. Both LPS and Con-A induced similarly the secretion of 13 analytes, whereas LPS preferentially induced 10 analytes, including TNFα, IL-1α and IL-1β. Con-A preferentially induced 16 analytes including Th17 cell cytokines IL-17A and IL-17F. Plasma samples, from active (n=20) and benign (n=18) ovarian cancer patients (Dr. Richard Moore, U of Rochester Medical Center) were tested at UPMC. Fourteen analytes were significantly elevated in active patients vs. benign patients including: cytokines TNFα, IL-5, M-CSF; chemokines MCP-3, Fractalkine; Growth Factors FLT-3L, PDGF-BB (p&amp;lt;0.05); cytokines IL-6, IL-18 (p&amp;lt;0.02); cytokines IL-7, IL-27, IL-10, IL-13 (key role in cancer pathogenesis); chemokine MIP-1β (p&amp;lt;0.01). Residual serum and plasma sepsis patient samples (n=16) were compared to those from healthy controls (n=20) and 7 analytes were significantly elevated in sepsis patients: cytokines IL-1RA, IL-6 (p&amp;lt;0.05), IL-7, IL-27, MIP-1β (p&amp;lt;0.02), M-CSF; chemokine MIG (p&amp;lt;0.01). Twelve other analytes trended higher in sepsis samples. Thus, the Human Cytokine/Chemokine/Growth Factor Panel A is a useful tool for profiling 48 immune factors both in cell culture or blood samples for the study of human disease and inflammation.

Semen IgM, IgG1, and IgG3 Differentially Associate With Pro-Inflammatory Cytokines in HIV-Infected Men.

Genital inflammation significantly increases the risk for HIV infection. The seminal environment is enriched in pro-inflammatory cytokines and chemokines. Here, we investigated the interplay between semen cytokines and humoral immunity to understand whether the characteristics of semen antibodies are associated with genital inflammation. In 36 HIV-infected and 40 HIV-uninfected mens' semen, HIV-specific antibodies (gp120, gp41, p66, and p24), immunoglobulin (Ig) subclasses, isotypes and cytokines, using multiplex assays, were measured. Semen IgG1, IgG3, and IgM were significantly higher in HIV-infected compared to HIV-uninfected men (p < 0.05). In HIV-uninfected men, pro-inflammatory cytokines IL-6, IL-8, and MCP-1 significantly correlated with IgG1 and total IgG (IgG1+IgG2+IgG3+IgG4) (both r≥0.55; p≤0.001). Total IgG in HIV-infected men correlated to HIV-specific antibodies in the semen irrespective of antiretroviral (ARV) use. In HIV-infected, ARV-treated men, p66 and gp41-specific antibodies were inversely correlated with IL-6 and MIP-1α (both r≥−0.65, p≤0.03). In HIV-infected, ARV-naïve men, p24 and gp120-specific antibodies correlated significantly with pro-inflammatory TNF-α (r≥0.44, p≤0.03), while p24 antibodies correlated significantly with chemokine MIP-1β (r = 0.45; p = 0.02). Local cytokines/chemokines were associated with the mucosal-specific Ig subclasses which likely effect specific antibody functions. Together, these data inform on mucosal-specific immunity that may be elicited in the male genital tract (MGT) in future vaccines and/or combination HIV prevention strategies.

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1β/CC-Chemokine Ligand 4 (MIP-1β/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 μs molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1β. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1β, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1β. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1β interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.

Oral Serum-Derived Bovine Immunoglobulin/Protein Isolate Has Immunomodulatory Effects on the Colon of Mice that Spontaneously Develop Colitis.

Dietary immunoglobulin concentrates prepared from animal plasma can modulate the immune response of gut-associated lymphoid tissue (GALT). Previous studies have revealed that supplementation with serum-derived bovine immunoglobulin/protein isolate (SBI) ameliorates colonic barrier alterations in the mdr1a-/- genetic mouse model of IBD. Here, we examine the effects of SBI on mucosal inflammation in mdr1a-/- mice that spontaneously develop colitis. Wild type (WT) mice and mice lacking the mdr1a gene (KO) were fed diets supplemented with either SBI (2% w/w) or milk proteins (Control diet), from day 21 (weaning) until day 56. Leucocytes in mesenteric lymph nodes (MLN) and in lamina propria were determined, as was mucosal cytokine production. Neutrophil recruitment and activation in MLN and lamina propria of KO mice were increased, but were significantly reduced in both by SBI supplementation (p < 0.05). The increased neutrophil recruitment and activation observed in KO mice correlated with increased colon oxidative stress (p < 0.05) and SBI supplementation reduced this variable (p < 0.05). The Tact/Treg lymphocyte ratios in MLN and lamina propria were also increased in KO animals, but SBI prevented these changes (both p < 0.05). In the colon of KO mice, there was an increased production of mucosal pro-inflammatory cytokines such as IL-2 (2-fold), IL-6 (26-fold) and IL-17 (19-fold), and of chemokines MIP-1β (4.5-fold) and MCP-1 (7.2-fold). These effects were significantly prevented by SBI (p < 0.05). SBI also significantly increased TGF-β secretion in the colon mucosa, suggesting a role of this anti-inflammatory cytokine in the modulation of GALT and the reduction of the severity of the inflammatory response during the onset of colitis.

Alterations in the secretory pattern of dermal dendritic cells following melanin uptake

Under a variety of circumstances, melanin occurs in the dermal compartment of the skin, being mostly observed in cells that have been termed melanophages, some of which have been identified as dermal dendritic cells. We analysed changes in the expression and secretion pattern of cytokines by dendritic cells after the uptake of melanin from various sources. Dendritic cells were derived from human primary blood monocytes or from the human monocytic cell line THP-1. Melanin uptake increased the secretion of the chemokines MIP-1β (CCL4) and MCP-1 (CCL2). The higher MIP-1β secretion was accompanied by higher MIP-1β gene expression. Elevation of MIP-1β secretion was dependent on the uptake of melanin but could not be induced by the phagocytosis of latex beads, indicating that the phagocytic process itself was not sufficient to increase the secretion of this cytokine. The data thus show that the uptake of melanin changes the cytokine expression and secretion pattern of dendritic-like cells.

Staphylococcus aureus protein A mediates innate immune responses during nasal colonization in humans (49.25)

Abstract Mucosa of the anterior human nares can harbor Staphylococcus aureus (SA), an etiologic agent that causes severe infections. Nasal carriage of SA is a complex host-pathogen interaction involving bacterial and host mediators that influence innate host defenses. We have revealed that the exoproteome of SA nasal carrier strains contains unique determinants, which enable SA to colonize the nares. In the current study, we assess how one of these bacterial mediators, Staphylococcal protein A (Spa), affects host innate immune response to SA nasal carriage. Functional disruption of spa was performed in an SA nasal carrier strain using Targetron insertion of a group II intron. Subsequent in vitro growth kinetics analysis and ex vivo culture on human nasal epithelial cells (HNECs) revealed no differences between wild type and spa-disrupted (Δspa) SA strains. However, the Δspa mutant induced lower expression of pro-inflammatory cytokines including IL6, IL8 and TNFα from HNECs compared to the levels elicited by the wild type strain. Additionally, IL17, a crucial regulator of mucosal immunity, was induced at significantly lower levels in HNECs inoculated with Δspa SA compared to those inoculated with the wild type SA strain. Furthermore, expression of the chemokines MIP1β and MCP1, as well as HL60 neutrophil migration, were significantly reduced in conditioned media from HNECs inoculated with Δspa mutants. Spa is likely involved in mediating the mucosal response to SA colonizing the nose.

Simultaneous evaluation of the circulating levels of both Th1 and Th2 chemokines in patients with autoimmune Addison's disease.

Chemokines play a key role in the recruitment of the immune cells into the autoimmune process. Thus, the simultaneous evaluation of circulating levels of Th1-related chemokines, such as CX chemokine ligand 10 (CXCL10) and macrophage inflammatory proteins 1α (CCL3/MIP-1α), and Th2-related chemokines, such as macrophage inflammatory proteins 1 β (CCL4/MIP-1β) could be useful in the approach to some autoimmune diseases, including autoimmune Addison's disease (AAD). To evaluate plasmatic levels of MIP-1α, MIP-1β, CXCL10 and adrenocortical antibodies in patients with AAD under treatment with corticosteroids. Twelve women and 5 men (group 1) were divided in 2 subgroups: 9 subjects with isolated AAD (group 1a) and 8 with AAD associated with chronic autoimmune thyroiditis (group 1b). MIP-1α, MIP- 1β and CXCL10 were evaluated in the serum of all patients and in 20 healthy controls, using a system for microarray suspension. The levels of MIP-1α, MIP-1β and CXCL10 resulted significantly increased vs controls (p<0.001). An inverse significant correlation between the serum levels of MIP- 1β and the duration of the disease was observed. High levels of MIP-1α and MIP-1β associated with increased levels of CXCL10 in AAD seem to indicate a role of these chemokines in the autoimmune pathology of adrenal gland through the recruitment in loco of Th1 and Th2 cells. The simultaneous measurement of Th1-related chemokines (CXCL10 and MIP-1α) and of Th2-related chemokine MIP-1β in the serum of patients with AAD would sustain a novel preliminary hypothesis on the immune microenvironment of chronic autoimmune inflammation within adrenal glands.

Endothelin-1–Induced Macrophage Inflammatory Protein-1β Expression in Monocytic Cells Involves Hypoxia-Inducible Factor-1α and AP-1 and Is Negatively Regulated by microRNA-195

Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Our studies show that placenta growth factor levels are high in SCD, and placental growth factor induces the release of the vasoconstrictor endothelin-1 (ET-1) from pulmonary microvascular endothelial cells. In this study, we observed that ET-1 increased the expression of the chemokines MIP-1β or CCL4. ET-1-induced MIP-1β mRNA expression in THP-1 cells and human peripheral blood monocytes occurred via the activation of PI3K, NADPH oxidase, p38 MAPK, and JNK-1 but not JNK-2. ET-1-induced MIP-1β expression involved hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia, as demonstrated by silencing with HIF-1α small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1β promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-κB binding motifs in the proximal MIP-1β promoter (-1053/+43 bp) were mutated. Furthermore, ET-1 significantly downregulated the expression of a key microRNA, microRNA-195a, which showed a complementary binding site in the 3' untranslated region of MIP-1β mRNA. Moreover, ET-1-induced MIP-1β mRNA expression in either THP-1 cells or peripheral blood monocytes was reduced upon expression of microRNA-195a. Conversely, transfection of monocytes with anti-microRNA-195a oligonucleotide augmented several-fold ET-1-induced MIP-1β expression. Taken together, these studies showed that ET-1-mediated MIP-1β gene expression is regulated via hypoxia-response elements, AP-1, and NF-κB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1β RNA. These studies provide what we believe are new avenues, based on targets of HIF-1α and microRNAs, for ameliorating inflammation in SCD.

Oral tolerance reduces Th17 cells as well as the overall inflammation in the central nervous system of EAE mice

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory immune response directed against myelin antigens of the central nervous system. In its murine model, EAE, Th17 cells play an important role in disease pathogenesis. These cells can induce blood-brain barrier disruption and CNS immune cells activation, due to the capacity to secrete high levels of IL-17 and IL-22 in an IL-6+TGF-β dependent manner. Thus, using the oral tolerance model, by which 200μg of MOG 35–55 is given orally to C57BL/6 mice prior to immunization, we showed that the percentage of Th17 cells as well as IL-17 secretion is reduced both in the periphery and also in the CNS of orally tolerated animals. Altogether, our data corroborates with the pathogenic role of IL-17 and IFN-γ in EAE, as its reduction after oral tolerance, leads to an overall reduction of pro-inflammatory cytokines, such as IL-1α, IL-6, IL-9, IL-12p70 and the chemokines MIP-1β, RANTES, Eotaxin and KC in the CNS. It is noteworthy that this was associated to an increase in IL-10 levels. Thus, our data clearly show that disease suppression after oral tolerance induction, correlates with reduction in target organ inflammation, that may be caused by a reduced Th1/Th17 response.

Chicken scavenger receptors and their ligand-induced cellular immune responses

The scavenger receptors (SRs) comprise structurally and functionally divergent groups of cell surface and secreted proteins that play an important role in innate immune defenses. Searching translated chicken genomic databases revealed many proteins homologous to mammalian SRs. SR mediated immune functions (oxidative burst, degranulation, phagocytosis, nitric oxide (NO) production, and cytokine expression) were evaluated in chicken heterophils, peripheral blood mononuclear cells (PBMC), and a chicken macrophage cell line (HD11) using various SR class A and B ligands. Results showed that the SR-A ligands, fucoidan, poly(I) and poly(G), but not SR-B ligands, phosphatidylserine and LDL, stimulated dose-dependent NO production in HD11 cells. However, SR-A ligands failed to induce NO in chicken monocytes. Quantitative RT-PCR indicated that SR ligands differentially regulated the gene expression of cytokines and chemokine in HD11 cells with a strong up-regulation of the cytokines IL-1β and IL-6 and the chemokine MIP-1β, but had no effect on IL-4, IL-12, IFN-γ, and IFN-β. SR-B ligands did not alter expression of these genes. SR-A ligands had no stimulatory effect on functional response in heterophils. However, LDL, a SR-B ligand stimulated oxidative burst in both heterophils and PBMC. Additionally, results indicate that SRs are involved in bacterial binding in macrophages.

The Human CC Chemokine MIP-1β Dimer Is Not Competent to Bind to the CCR5 Receptor

Chemokine dimerization has been the subject of much interest in recent years as evidence has accumulated that different quaternary states of chemokines play different biological roles; the monomer is believed to be the receptor-binding unit, whereas the dimer has been implicated in binding cell surface glycosaminoglycans. However, although several studies have provided evidence for this paradigm by making monomeric chemokine variants or dimer-impaired chemokines, few have provided direct evidence of the receptor function of a chemokine dimer. We have produced a covalent dimer of the CC chemokine macrophage inflammatory protein-1beta (MIP-1beta) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1beta-A10C). This variant was shown to be a nondissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of glycosaminoglycan binding, MIP-1beta-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1beta-A10C neither binds nor activates the MIP-1beta receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide cross-link by incubation with 1 mm dithiothreitol. This work provides the first definitive evidence that the CC chemokine MIP-1beta dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.

Macrophage tolerance induced by stimulation with Toll-like receptor 7/8 ligands

The engagement of Toll-like receptors (TLRs) results in resistance to subsequent challenge with respective ligands in macrophages. Studies have shown that stimulation by ligands for TLR2, TLR4, TLR5 and TLR9 induces this state of hypo-responsiveness (homo-tolerance) towards subsequent stimulation with the same ligands. However, whether homo-tolerance is induced by the ligands of TLR7/8 has not been previously determined. We found that ligands for TLR7/8, namely ss-RNA from HIV and an imidazoquinoline compound, R848, induced macrophage tolerance, as judged by the production of the chemokine MIP-1β. IRAK-1 phosphorylation was also inhibited in the tolerant cells after subsequent stimulation with R848, although no significant differences were observed in the protein levels of TLR7 between tolerant and non-tolerant cells. These results indicate that macrophage tolerance induced by TLR7/8 ligands is regulated at least at the level of IRAK-1 activation.

Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1β in monocyte culture, whereas T H1 cytokines (IL-2 and IFN-γ) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-γ and the chemokine MIP-1β, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T H1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.

Cutting Edge: Neutrophil Granulocyte Serves as a Vector forLeishmaniaEntry into Macrophages

Macrophages (MF) are the final host cells for multiplication of the intracellular parasite Leishmania major (L. major). However, polymorphonuclear neutrophil granulocytes (PMN), not MF, are the first leukocytes that migrate to the site of infection and encounter the parasites. Our previous studies indicated that PMN phagocytose but do not kill L. major. Upon infection with Leishmania, apoptosis of human PMN is delayed and takes 2 days to occur. Infected PMN were found to secrete high levels of the chemokine MIP-1beta, which attracts MF. In this study, we investigated whether MF can ingest parasite-infected PMN. We observed that MF readily phagocytosed infected apoptotic PMN. Leishmania internalized by this indirect way survived and multiplied in MF. Moreover, ingestion of apoptotic infected PMN resulted in release of the anti-inflammatory cytokine TGF-beta by MF. These data indicate that Leishmania can misuse granulocytes as a "Trojan horse" to enter their final host cells "silently" and unrecognized.

Serum levels of chemokines correlate with disease activity in patients with retinal vasculitis

Retinal vasculitis (RV) is characterised pathologically by migration of leucocytes across the blood–retinal barrier leading to oedema and photoreceptor cell dysfunction. Chemokines are a family of small molecules involved in leucocyte migration. In this study, levels of chemokines were measured in serum from patients with RV and correlated with disease activity and drug treatment. Serum samples ( n=100; 25 active, 75 inactive) were obtained from 50 patients with RV, and levels of the chemokines MIP-1α, macrophage inflammatory protein-1β (MIP-1β) and monocyte chemoattractant protein-1 (MCP-1) were measured by ELISA. For longitudinal analysis levels of the same chemokines were measured in six consecutive serum samples from 10 of the above patients. Chemokine levels were correlated with disease activity and current drug treatment for each sample. Sera from 20 healthy individuals were used as control samples. Serum levels of MIP-1β were significantly raised in patients with RV, whether active or not, compared to healthy controls ( P=0.04). Levels of MIP-1β and MCP-1 correlated with disease activity in some patients and with prednisolone levels in patients on this treatment alone. MIP-1α levels were not detectable in all samples but were present in significantly more samples from patients with active or inactive RV compared with healthy controls. Serum levels of the chemokines MIP-1β and MIP-1α, but not MCP-1 were raised in patients with retinal vasculitis. Longitudinal analysis suggested that MIP-1β and MCP-1 levels were controlled by drug treatment, particularly prednisolone. These data demonstrate that chemokines are involved in the pathogenesis of RV and may act as novel therapeutic targets.

Natural Truncation of the Chemokine MIP-1β/CCL4 Affects Receptor Specificity but Not Anti-HIV-1 Activity

Activated lymphocytes synthesize and secrete substantial amounts of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha/CCL3 and MIP-1 beta/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1 beta secreted by activated human peripheral blood lymphocytes (MIP-1 beta(3-69)) lacks the two NH(2)-terminal amino acids of the full-length protein. This truncated form of MIP-1 beta has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1 beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1 beta(3-69) showed that the truncated protein not only shares the ability of intact MIP-1 beta to induce Ca(2+) signaling through CCR5, but unlike the full-length protein, it also triggers a Ca(2+) response via CCR1 and CCR2b. These results demonstrate that NH(2)-terminally truncated MIP-1 beta functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.

RANTES-induced chemokine cascade in dendritic cells.

Dendritic cells (DC) are the most potent APCs and the principal activators of naive T cells. We now report that chemokines can serve as activating agents for immature DC. Murine bone marrow-derived DC respond to the CC chemokine RANTES (10-100 ng/ml) by production of proinflammatory mediators. RANTES induces rapid expression of transcripts for the CXC chemokines KC and macrophage inflammatory protein (MIP)-2, the CC chemokines MIP-1beta and MIP-1alpha, and the cytokines TNF-alpha and IL-6. Synthesis of KC, IL-6, and TNF-alpha proteins were also demonstrated. After 4 h, autoinduction of RANTES transcripts was observed. These responses are chemokine specific. Although DC demonstrated weak responses to eotaxin, DC failed to respond to other chemokines including KC, MIP-2, stromal-derived factor-1alpha, MIP-1beta, MIP-1alpha, monocyte chemoattractant protein-1, T cell activation gene 3, or thymus-derived chemotactic agent 4. In addition, RANTES treatment up-regulated expression of an orphan chemokine receptor termed Eo1. Chemokine induction was also observed after treatment of splenic DC and neonatal microglia with RANTES, but not after treatment of thymocytes or splenocytes depleted of adherent cells. TNF-alpha-treated DC lose responsiveness to RANTES. DC from mice deficient for CCR1, CCR3, and CCR5 respond to RANTES, indicating that none of these receptors are exclusively used to initiate the chemokine cascade. RANTES-mediated chemokine amplification in DC may prolong inflammatory responses and shape the microenvironment, potentially enhancing acquired and innate immune responses.

CC chemokine MIP-1 beta can function as a monomer and depends on Phe13 for receptor binding.

The reported structures of many CC chemokines show a conserved dimer interface along their N-terminal region, raising the possibility that the quaternary arrangement of these small immune proteins might influence their function. We have produced and analyzed several mutants of MIP-1 beta having a range of dimer K(d) values in order to determine the significance of dimerization in receptor binding and cellular activation. NMR and analytical ultracentrifugation were used to analyze the oligomeric state of the mutants. Functional relevance was determined by receptor binding affinity and the ability to invoke intracellular calcium release from CHO cells transfected with the MIP-1 beta receptor CCR5. The monomeric N-terminally truncated mutant MIP(9) was able to bind the CCR5 receptor with a K(i) of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability to tightly bind (K(i) = 480 pM) and to activate (EC(50) = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP-1 beta, as a key determinant for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic side chain to be important for both binding to CCR5 and chemokine dimerization.