Fcgamma receptor I- and III-mediated macrophage inflammatory protein 1alpha induction in primary human and murine microglia

Microglial cell phagocytic receptors may play important roles in the pathogenesis and treatment of several neurological diseases. We studied microglial Fc receptor (FcR) activation with respect to the specific FcgammaR types involved and the downstream signaling events by using monoclonal antibody (MAb)-coated Cryptococcus neoformans immune complexes as the stimuli and macrophage inflammatory protein 1alpha (MIP-1alpha) production as the final outcome. C. neoformans complexed with murine immunoglobulin G (IgG) of gamma1, gamma2a, and gamma3, but not gamma2b isotype, was effective in inducing MIP-1alpha in human microglia. Since murine gamma2b binds to human FcgammaRII (but not FcgammaRI or FcgammaRIII), these results indicate that FcgammaRI and/or FcgammaRIII is involved in MIP-1alpha production. Consistent with this, an antibody that blocks FcgammaRII (IV.3) failed to inhibit MIP-1alpha production, while an antibody that blocks FcgammaRIII (3G8) did. An anti-C. neoformans MAb, 18B7 (IgG1), but not its F(ab')(2), induced extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase kinase phosphorylation, and MIP-1alpha release was suppressed by the ERK inhibitor U0126. C. neoformans plus 18B7 also induced degradation of I-kappaBalpha, and MIP-1alpha release was suppressed by the antioxidant NF-kappaB inhibitor pyrrolidine dithiocarbamate. To confirm the role of FcR more directly, we isolated microglia from wild-type and various FcR-deficient mice and then challenged them with C. neoformans plus 18B7. While FcgammaRII-deficient microglia showed little difference from the wild-type microglia, both FcgammaRI alpha-chain- and FcgammaRIII alpha-chain-deficient microglia produced less MIP-1alpha, and the common Fc gamma-chain-deficient microglia showed no MIP-1alpha release. Taken together, our results demonstrate a definitive role for FcgammaRI and FcgammaRIII in microglial chemokine induction and implicate ERK and NF-kappaB as the signaling components leading to MIP-1alpha expression. Our results delineate a new mechanism for microglial activation and may have implications for central nervous system inflammatory diseases.     

MIP-1alpha[CCL3] acting on the CCR1 receptor mediates neutrophil migration in immune inflammation via sequential release of TNF-alpha and LTB4

In the present study, we investigated the involvement of macrophage-inflammatory protein-1alpha (MIP-1alpha)[CC chemokine ligand 3 (CCL3)], MIP-1beta[CCL4], regulated on activation, normal T expressed and secreted (RANTES)[CCL5], and CC chemokine receptors (CCRs) on neutrophil migration in murine immune inflammation. Previously, we showed that ovalbumin (OVA)-triggered neutrophil migration in immunized mice depends on the sequential release of tumor necrosis factor alpha (TNF-alpha) and leukotriene B(4)(LTB(4)). Herein, we show increased mRNA expression for MIP-1alpha[CCL3], MIP-1beta[CCL4], RANTES[CCL5], and CCR1 in peritoneal cells harvested from OVA-challenged, immunized mice, as well as MIP-1alpha[CCL3] and RANTES[CCL5] but not MIP-1beta[CCL4] proteins in the peritoneal exudates. OVA-induced neutrophil migration response was muted in immunized MIP-1alpha[CCL3](-/-) mice, but it was not inhibited by treatment with antibodies against RANTES[CCL5] or MIP-1beta[CCL4]. MIP-1alpha[CCL3] mediated neutrophil migration in immunized mice through induction of TNF-alpha and LTB(4) synthesis, as these mediators were detected in the exudates harvested from OVA-challenged immunized wild-type but not MIP-1alpha[CCL3](-/-) mice; administration of MIP-1alpha[CCL3] induced a dose-dependent neutrophil migration, which was inhibited by treatment with an anti-TNF-alpha antibody in TNF receptor 1 (p55(-/-))-deficient mice or by MK 886 (a 5-lipoxygenase inhibitor); and MIP-1alpha[CCL3] failed to induce LTB(4) production in p55(-/-) mice. MIP-1alpha[CCL3] used CCR1 to promote neutrophil recruitment, as OVA or MIP-1alpha[CCL3] failed to induce neutrophil migration in CCR1(-/-) mice, in contrast to CCR5(-/-) mice. In summary, we have demonstrated that neutrophil migration observed in this model of immune inflammation is mediated by MIP-1alpha[CCL3], which via CCR1, induces the sequential release of TNF-alpha and LTB(4). Therefore, whether a similar pathway mediates neutrophil migration in human immune-inflammatory diseases, the development of specific CCR1 antagonists might have a therapeutic potential.     

Neutralization of macrophage inflammatory protein 2 (MIP-2) and MIP-1alpha attenuates neutrophil recruitment in the central nervous system during experimental bacterial meningitis

Chemokines are low-molecular-weight chemotactic cytokines that have been shown to play a central role in the perivascular transmigration and accumulation of specific subsets of leukocytes at sites of tissue damage. Using in situ hybridization (ISH), we investigated the mRNA induction of macrophage inflammatory protein 2 (MIP-2), MIP-1alpha, monocyte chemoattractant protein 1 (MCP-1), and RANTES. Challenge of infant rats' brains with Haemophilus influenzae type b intraperitoneally resulted in the time-dependent expression of MIP-2, MIP-1alpha, MCP-1, and RANTES, which was maximal 24 to 48 h postinoculation. Immunohistochemistry showed significant increases in neutrophils and macrophages infiltrating the meninges, the ventricular system, and the periventricular area. The kinetics of MIP-2, MIP-1alpha, MCP-1, and RANTES mRNA expression paralleled those of the recruitment of inflammatory cells and disease severity. Administration of anti-MIP-2 or anti-MIP-1alpha antibodies (Abs) resulted in significant reduction of neutrophils. Administration of anti-MCP-1 Abs significantly decreased macrophage infiltration. Combined studies of ISH and immunohistochemistry showed that MIP-2- and MIP-1alpha-positive cells were neutrophils and macrophages. MCP-1-positive cells were neutrophils, macrophages, and astrocytes. Expression of RANTES was localized predominantly to resident astrocytes and microglia. The present study indicates that blocking of MIP-2 or MIP-1alpha bioactivity in vivo results in decreased neutrophil influx. These data are also the first demonstration that the C-C chemokine MIP-1alpha is involved in neutrophil recruitment in vivo.     

MIP-3alpha, MIP-3beta and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells.

We demonstrate here that the CC chemokines macrophage inflammatory protein-3alpha (MIP-3alpha), macrophage inflammatory protein-3beta (MIP-3beta) and the CX3C chemokine fractalkine induce the chemotaxis of interleukin-2 (IL-2)-activated natural killer (IANK) cells. In addition, these chemokines enhance the binding of [gamma-35S]guanine triphosphate ([gamma-35S]GTP) to IANK cell membranes, suggesting that receptors for these chemokines are G protein-coupled. Our results show that MIP-3alpha receptors are coupled to Go, Gq and Gz, MIP-3beta receptors are coupled to Gi, Gq and Gs, whereas fractalkine receptors are coupled to Gi, and Gz. All three chemokines induced a robust calcium response flux in IANK cells. Cross-desensitization experiments show that MIP-3alpha, MIP-3beta or fractalkine use receptors not shared by each other or by the CC chemokine regulated on activation, normal, T-cell expressed, and secreted (RANTES), the CXC chemokines stromal-derived factor-1alpha (SDF-1alpha) and interferon-inducible protein-10 (IP-10), or the C chemokine lymphotactin.     

Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19.

Macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19 are members of the CC chemokine subfamily which exert their effects through specific receptors, CCR6 and CCR7, respectively. Previously, we have reported that human neutrophils have the capacity to produce a number of chemokines, including IL-8/CXCL8, GROalpha/CXCL1, IP-10/CXCL10, and MIG/CXCL9. Herein, we show that neutrophils also have the ability to express and release MIP-3alpha/CCL20 and MIP-3beta/CCL19 when cultured with either LPS or TNF-alpha. We also report that MIP-3alpha/CCL20 and MIP-3beta/CCL19 production by LPS-stimulated neutrophils is negatively modulated by IL-10. Remarkably, we found that supernatants harvested from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells (DC), but also triggered rapid integrin-dependent adhesion of CCR6- and CCR7-expressing lymphocytes to purified VCAM-1 and ICAM-1, respectively. Importantly, both chemotaxis and rapid integrin-dependent adhesion were dramatically suppressed by anti-MIP-3alpha/CCL20 and anti-MIP-3beta/CCL19 neutralizing antibodies, indicating that MIP-3alpha/CCL20 and MIP-3beta/CCL19 present in the supernatants were both biologically active. As these chemokines are primarily chemotactic for DC and specific lymphocyte subsets, the ability of neutrophils to produce MIP-3alpha/CCL20 and MIP-3beta/CCL19 might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to the regulation of the immune response.     

Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor

Macrophage inflammatory protein (MIP)-1alpha is a chemokine that is associated with Th1 cytokine responses. Expression and antibody blocking studies have implicated MIP-1alpha in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). We examined the role of MIP-1alpha and its CCR5 receptor in the induction of EAE by immunizing C57BL / 6 mice deficient in either MIP-1alpha or CCR5 with myelin oligodendrocyte glycoprotein (MOG). We found that MIP-1alpha-deficient mice were fully susceptible to MOG-induced EAE. These knockout animals were indistinguishable from wild-type mice in Th1 cytokine gene expression, the kinetics and severity of disease, and infiltration of the central nervous system by lymphocytes, macrophages and granulocytes. RNase protection assays showed comparable accumulation of mRNA for the chemokines interferon-inducible protein-10, RANTES, macrophage chemoattractant protein-1, MIP-1beta, MIP-2, lymphotactin and T cell activation gene-3 during the course of the disease. CCR5-deficient mice were also susceptible to disease induction by MOG. The dispensability of MIP-1alpha and CCR5 for MOG-induced EAE in C57BL / 6 mice supports the idea that differential chemokine expression patterns represent differences in disease mechanism that underlie various models of EAE, and possibly distinct patterns of pathology seen in MS.     

Monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete MIP-1alpha and TNF-alpha and inhibit poly-IC-induced IFN-alpha in vitro

Ebola virus infection of humans is associated with high levels of circulating inflammatory chemokines and cytokines. We demonstrate that direct infection of human PBMC results in the induction of MCP-1, MIP-1alpha, RANTES, and TNF-alpha as early as 24 h p.i. in response to live virus. Monocyte-derived macrophages infected with live Ebola-virus secreted MIP-1alpha and TNF-alpha specifically while RANTES and MCP-1 were secreted by with both live or inactivated virus stimulation and do not require viral replication. Type I interferons (IFN-alpha and -beta), IL-1beta and IL-10, were not induced by Ebola virus. Furthermore, live virus infection of both PBMCs and monocytes-derived macrophages inhibited IFN-alpha induced by double-stranded RNA in vitro. These data provide the first direct evidence of a role for macrophages in the pathogenesis to Ebola virus and suggest that Ebola virus can inhibit cellular antiviral mechanisms mediated by type I interferons.     

Binding of RANTES, MCP-1, MCP-3, and MIP-1alpha to cells in human skin.

Based on their ability to induce leukocyte chemotaxis and adhesion to endothelial cells (ECs), chemokines have been implicated in driving inflammatory leukocyte emigration. Recently, it was suggested that chemokines can accomplish their pro-emigratory role more effectively while being bound to the luminal surface of the ECs. Previously, such binding was demonstrated in situ in human skin for the prototype alpha-chemokine interleukin (IL)-8. Here we used an in situ binding assay to investigate the binding characteristics of several beta-chemokines in intact human skin. RANTES, MCP-1, and MCP-3 bound, similar to IL-8, in a specific saturable manner to the ECs of venules and small veins but not arteries or capillaries. RANTES inhibited MCP-1 and MCP-3 binding and vice versa, indicating that the EC binding sites are shared among these beta-chemokines; moreover, IL-8 and RANTES cross-competed for each other's binding, suggesting that the same chemokine binding sites are used by members of alpha- and beta-chemokine subfamilies. Conversely, MIP-1alpha did not bind to the ECs and did not compete for binding of RANTES. Analogous to IL-8, all of the tested beta-chemokines bound to the resident dermal cells. As a novel aspect of chemokine interaction with cells in normal skin, we observed specific, saturable binding of RANTES, MCP-1, and MCP-3 but not MIP-1alpha or IL-8 to the ECs of dermal afferent lymphatic vessels. RANTES, MCP-1, and MCP-3 also cross-competed for each other's binding to lymphatics, suggesting a common binding site with a novel chemokine binding profile. We suggest that the chemokine binding to the ECs of lymphatics may be involved in the process of leukocyte entry into the afferent lymphatic vessels.     

Altered intracellular expression of the chemokines MIP-1alpha,MIP-1beta and IL-8 by peripheral blood CD4+ and CD8+ T cells in mild allergic asthma

BACKGROUND: The ability of chemokines to regulate Th1 and Th2 responses suggests a role in the pathogenesis of atopic disorders such as allergic asthma where Th2 response dominance has been observed. Although the impact of allergic asthma on local chemokine production in the lung has been the subject of investigation, little is know about the influence of disease progression on peripheral chemokine production. We now report use of whole blood culture and flow cytometry to assess the influence of mild allergic asthma on peripheral T-cell chemokine expression. METHODS: Study participants included patients with mild allergic asthma (n = 7) and nonasthmatic controls (n = 7). Following in vitro stimulation of peripheral venous blood with phorbol 12-myristate acetate (PMA) and ionomycin, flow cytometry was used to estimate the percentage of CD4+ and CD8+ T cells producing a number of chemokines, including macrophage inflammatory proteins MIP-1alpha and MIP-1beta, RANTES (regulated on activation, T-cell expressed and secreted), monocytic chemotactic protein-1 (MCP)-1, and interleukin (IL)-8, or the cytokines interferon (IFN)-gamma and IL-4. Serum levels of MIP-1alpha, MIP-1beta, RANTES, MCP-1, IL-8, IFN-gamma and IL-4 were also assessed by quantitative ELISA. RESULTS: Intracellular expression of MIP-1beta by CD4+ and CD8+ T cells from allergic asthmatics was significantly reduced in comparison to that observed for nonasthmatics (median = 2.29% (1.75-3.50) vs 4.57% (3.38-6.64), P = 0.05; 14.20% (13.18-17.88) vs 44.10% (30.38-48.70), P = 0.01). Similarly, intracellular expression of MIP-1alpha by CD8+ T cells from allergic asthmatics was also significantly lower (3.67% (1.17-5.42) vs 17.10% (4.97-20.43), P = 0.05). Conversely, IL-8 expression by both CD4+ and CD8+ T cells from allergic asthmatics demonstrated significant enhancement (9.93% (7.77-11.28) vs 4.14% (3.61-7.11), P = 0.05; 8.40% (6.97-10.04) vs 4.98% (3.37-6.08), P = 0.05). Examination of intracellular IFN-gamma and IL-4 revealed no significant difference in the expression of either cytokine by CD4+ T-cells from allergic asthmatics and nonasthmatics. In contrast, expression of IFN-gamma was significantly reduced in CD8+ T-cells from allergic asthmatics (24.60% (21.08-32.50) vs 48.40% (41.50-55.28), P = 0.01). CONCLUSIONS: The occurrence in mild allergic asthma of peripheral T-cell chemokine expression suggestive of a diminished Th1 response, coinciding with marginal change in cytokine profiles indicative of a Th2 response bias, confirms the importance of chemokine involvement in the etiology of allergic asthma. The ability to use whole blood culture to estimate chemokine expression in T cell subsets may ultimately provide a practical means to evaluate disease status and to monitor early intervention therapies which target chemokines.     

Contrasting roles for RANTES and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in a murine model of allergic peritonitis.

Cell accumulation and CC chemokine production were assessed in the peritoneal cavity of ovalbumin (OVA)-sensitized mice following antigen challenge. Intraperitoneal challenge with OVA induced a significant eosinophil influx from 6 h post-challenge with increased numbers persisting at 24 h. At 6 h there was also a marked presence of neutrophils. Messenger RNA expression and protein levels for the chemokines RANTES and MIP-1 alpha were measured in the cell pellets and supernatants, respectively, from peritoneal washes following OVA challenge. RANTES mRNA was detected from 2 h to 4 h following OVA injection, whereas mRNA for MIP-1 alpha was only detectable at 4 h. RANTES protein was first detected from 4 h after OVA injection and by 24 h the protein levels had increased further. Basal levels of MIP-1 alpha were detected in peritoneal washes. These levels peaked at 2 h after OVA challenge and rapidly declined to basal levels by 6 h. A functional role for the chemokines was assessed using neutralizing polyclonal antibodies. Co-injection of OVA with anti-RANTES antibodies resulted in a significant inhibition of eosinophil infiltration into the cavity at 6 h and 24 h (63% and 52% inhibition, respectively) without significantly influencing the number of neutrophils present. In contrast, injection of anti-MIP-1 alpha antibodies only inhibited neutrophil migration at the 6 h time point by 44% without significantly affecting the accumulation of eosinophils. These results demonstrate an important role for RANTES in mediating eosinophil influx in allergic inflammation and a contrasting role for MIP-1 alpha in mediating neutrophil recruitment.     

Role of the autocrine chemokines MIP-1alpha and MIP-1beta in the metastatic behavior of murine T cell lymphoma

The ESb-MP T-cell line is a highly malignant murine lymphoma, which preferentially metastasizes toward the kidney. This could be a result of the local production of monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES), which are chemotactic for ESb-MP cells. Here, we demonstrate that ESb-MP cells are already responsive to the chemotactic activity of macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta from 1 ng/ml onward. Moreover, upon stimulation with lipopolysaccharide (LPS) or virus, ESb-MP cells themselves produce significant amounts of MIP-1 ( approximately 200 ng/ml). Indeed, the major autocrine chemoattractants, isolated from ESb-MP cells, were intact MIP-1alpha and MIP-1beta. Pretreatment with LPS or addition of MIP-1 inhibited the in vitro migration of ESb-MP cells toward various chemokines. Moreover, compared with untreated lymphoma cells, LPS-treated cells produced significantly less metastasis in mice. The results represented here suggest that the role of chemokines in attracting tumor cells at secondary sites depends on a balance between autocrine-produced and tissue-derived chemokines. This delicate balance should be considered in the design of antichemokine strategies in different tumor types.     

Inhibition of M-tropic HIV-1 infection by the fd phage-gene 3 protein with MIP-1alpha-binding activity

CCR5 is a chemokine receptor with seven transmembrane-domains. It is expressed on T cells and macrophages and functions as the principal co-receptor for macrophage (M)-tropic strains of HIV-1. The anti-CCR5 monoclonal antibody (mAb) 2D7 inhibits the binding and chemotaxis of the three natural beta-chemokine ligands of CCR5, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES, to CCR5(+) cells. The mAb also efficiently blocks the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro.In this study, we attempted to determine the peptide motif recognized with the 2D7 mAb. We isolated phage clones by panning a phage display library using 2D7 and identified three peptide motifs. One of these phage clones (M23) showed a marked inhibitory activity on HIV-1 infection. The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the g3p of the M23 phage clone (M23-g3p). The M23-g3p was purified by fast-performance liquid chromatography (FPLC). We show here that (1) M23-g3p was specifically recognized with anti-CCR5 mAb; (2) M23-g3p showed inhibitory activity on the infectivity of M-tropic but not T-tropic HIV-1 strains; (3) M23-g3p bound to MIP-1alpha, MIP-1beta, and RANTES but not MCP-1. These results suggested that the M23-g3p might mimic the CCR5-binding domain shared by beta-chemokines, MIP-1alpha, MIP-1beta, and RANTES as well as the HIV-1 infection.     

Fcgamma receptor signaling in primary human microglia: differential roles of PI-3K and Ras/ERK MAPK pathways in phagocytosis and chemokine induction

Cryptococcus neoformans monoclonal antibody immune complex (IC) induces beta-chemokines and phagocytosis in primary human microglia via activation of Fc receptor for immunoglobulin G (FcgammaR). In this report, we investigated microglial FcgammaR signal-transduction pathways by using adenoviral-mediated gene transfer and specific inhibitors of cell-signaling pathways. We found that Src inhibitor PP2 and Syk inhibitor piceatannol inhibited phagocytosis, macrophage-inflammatory protein-1alpha (MIP-1alpha) release, as well as phosphorylation of extracellular-regulated kinase (ERK) and Akt, consistent with Src/Syk involvement early in FcgammaR signaling. Constitutively active mitogen-activated protein kinase kinase (MEK) induced MIP-1alpha, and Ras dominant-negative (DN) inhibited IC-induced ERK phosphorylation and MIP-1alpha production. These results suggest that the Ras/MEK/ERK pathway is necessary and sufficient in IC-induced MIP-1alpha expression. Neither Ras DN nor the MEK inhibitor U0126 inhibited phagocytosis. In contrast, phosphatidylinositol-3 kinase (PI-3K) inhibitors Wortmannin and LY294002 inhibited phagocytosis without affecting ERK phosphorylation or MIP-1alpha production. Conversely, Ras DN or U0126 did not affect Akt phosphorylation. Together, these results demonstrate distinct roles played by the PI-3K and Ras/MEK/ERK pathways in phagocytosis and MIP-1alpha induction, respectively. Our results demonstrating activation of functionally distinct pathways following microglial FcgammaR engagement may have implications for human central nervous system diseases.     

Role of MCP-1 and MIP-1alpha in retinal neovascularization during postischemic inflammation in a mouse model of retinal neovascularization

Macrophages are important participants in neovascularization. This study was designed to examine the role of the monocyte/macrophage chemotactic proteins, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) in a mouse model of oxygen-induced ischemic retinopathy and to determine whether the morphology and distribution of macrophages/microglia are concomitantly altered. The MCP-1, MIP-1alpha mRNA levels increased at 3 h after ischemia. MCP-1, MIP-1alpha, and vascular endothelial growth factor protein levels were also increased markedly and were maximal on days 1, 0.5, and 1, respectively, after ischemia. In situ hybridization showed that MCP-1 and MIP-1alpha were localized in the hypoxic inner retina. Immunostaining demonstrated that the macrophages/microglia in the retina had morphological changes with enlarged processes, and some were closely associated with neovascular tufts at postnatal day 17. Coadministration of the neutralizing antibodies against MCP-1 and MIP-1alpha inhibited retinal neovascularization by 30%. Our data suggest that MCP-1 and MIP-1alpha are involved in the induction of retinal neovascularization and play a role in the inflammation induced by the ischemic retinopathy, possibly by modulating or attracting macrophages/microglia.     

Lipopolysaccharide from an Escherichia coli htrB msbB mutant induces high levels of MIP-1 alpha and MIP-1 beta secretion without inducing TNF-alpha and IL-1 beta

OBJECTIVE: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines. METHODS: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS). The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay. RESULTS: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines. Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS. Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs. This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion. Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS. CONCLUSION: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.     

A chimeric MIP-1alpha/RANTES protein demonstrates the use of different regions of the RANTES protein to bind and activate its receptors

Human RANTES (CCL5) and MIP-1alpha (CCL3) bind and activate several CC chemokine receptors. RANTES is a high-affinity ligand for CCR1 and CCR5, and it binds CCR3 with moderate affinity and CCR4 with low affinity. MIP-1alpha has similar binding characteristics to RANTES except that it does not bind to CCR3. Here we have generated a chimera of human MIP-1alpha and RANTES, called MIP/RANTES, consisting of the eight amino terminal residues of MIP-1alpha preceding the CC motif, and the remainder of the sequence is RANTES. The chimera is able to induce chemotaxis of human monocytes. MIP/RANTES has >100-fold reduction in binding to CCR1 and does not bind to CCR3 but retains full, functional binding to CCR5. It has equivalent affinity for CCR5 to MIP-1alpha and RANTES, binding with an IC(50) of 1.12 nM, and is able to mobilize calcium and induce endocytosis of CCR5 in PBMC in a manner equi-potent to RANTES. It also retains the ability to inhibit R5 using HIV-1 strains. Therefore, we conclude that the amino terminus of RANTES is not involved in CCR5 binding, but it is essential for CCR1 and CCR3.     

Simian/human immunodeficiency virus(89.6) expressing the chemokine genes MIP-1alpha,RANTES, or lymphotactin

We constructed replication competent, attenuated, nef-deleted SHIV(89.6) that express the rhesus macaque chemokine genes MIP-1alpha, RANTES, or LTN from the nef region. The chemokine inserts were stable during several passages in CEMx174 cells and the viruses grew well in activated rhesus PBMC. Expression of virally encoded MIP-1alpha, RANTES, or LTN was detected in culture fluids from infected HOS CD4(+) CXCR4(+) cells, that were used because they have a low background production of these chemokines. The in vitro growth kinetics of all nef-deleted SHIV(89.6) were slower than the parental strain in both CEMx174 cells and rhesus PBMC. Rhesus macaques were susceptible to SHIV(89.6-MIP-1alpha), SHIV(89.6-RANTES), SHIV(89.6-LTN), and nef-deleted control SHIV(89.6-dLTN) infection via the intrarectal route using standard virus doses, and intact viruses were reisolated from infected animals throughout the interval of acute infection. SHIV expressing the chemokine genes MIP-1alpha, RANTES, or LTN may help determine the in vivo roles for these chemokines in modulating virus replication and disease.     

Single-cell analysis of the murine chemokines MIP-1alpha,MIP-1beta,RANTES and ATAC/lymphotactin by flow cytometry

Upon stimulation, leukocytes secrete chemokines to attract distinct effector cell populations to the site of inflammation. Only a few data are available about the phenotype and the frequencies of cells expressing particular chemokines. To date, the expression of individual chemokines is mainly analyzed at the mRNA level or via ELISA. Both techniques do not allow the analysis of chemokines at the level of single cells. We have established the intracellular flow-cytometric detection of the murine chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, regulated on activation normal T cell expressed and secreted (RANTES) and activation-induced, T cell-derived and chemokine-related cytokine (ATAC)/lymphotactin. For detection of the nonclassical chemokine ATAC, we generated the novel mAb MTAC-2. Using this assay, we analyzed for the first time the frequency and kinetics of the expression of these murine chemokines in lymphocyte subpopulations. We show that these chemokines are differentially expressed by NK cells, naive and memory CD4(+) and CD8(+) T cells. Our results emphasize that the analysis of chemokine expression at the single-cell level is required to understand the functional role of specialized lymphocyte subpopulations in vivo.