MIP-3alpha neutralizing monoclonal antibody protects against TNBS-induced colonic injury and inflammation in mice

A characteristic feature of human inflammatory bowel disease, particularly Crohn's disease, is the presence of activated CD4(+) T cells. Recently, we have shown that colonic epithelial cell production of macrophage inflammatory protein (MIP)-3alpha, a CD4 T cell-directed chemokine, is elevated in inflammatory bowel disease. However, the functional relevance of MIP-3alpha production during intestinal inflammation is poorly understood. The aim of this study was to determine whether MIP-3alpha production is increased during murine 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and to examine the effect of anti-MIP-3alpha neutralizing monoclonal antibody administration in this model. We found that the administration of TNBS significantly increased colonic MIP-3alpha protein levels in Balb/c mice. Consistent with this, a marked increase in the number of CCR6-bearing lamina propria CD4(+) and CD8(+) T cells was also observed in TNBS-treated animals. Treatment of mice with an anti-MIP-3alpha neutralizing monoclonal antibody significantly reduced TNBS-mediated increases in colonic weight-to-length ratio, mucosal ulceration, histological damage, and myeloperoxidase activity. TNBS-mediated increases in the number of CCR6-bearing lamina propria T cells were also substantially reduced by anti-MIP-3alpha neutralizing monoclonal antibody treatment. Taken together, our findings indicate that blockade of MIP-3alpha bioactivity can significantly reduce TNBS-mediated colonic injury and T cell recruitment, suggesting a role for this chemokine in the pathophysiology of intestinal inflammation.     

Role for macrophage inflammatory protein 2 (MIP-2), MIP-1alpha,and interleukin-1alpha in the delayed-type hypersensitivity response to viral antigen

BALB/c mice sensitized to herpes simplex virus type 1 (HSV-1) develop a vigorous delayed-type hypersensitivity (DTH) response upon intradermal virus antigen challenge. Although CD4(+) T cells are a key mediator of this response, neutrophils are the most abundant cells at the antigen challenge site both initially and at the peak of the reaction. We investigated what role, if any, neutrophils play in the DTH to a viral antigen. We show here that antibody-mediated depletion of neutrophils 1 day before antigen challenge significantly suppressed ear swelling and markedly reduced cellular influx. Additionally, neutrophil depletion was associated with decreased expression of macrophage inflammatory protein 2 (MIP-2) and MIP-1alpha, as well as with a >60-fold increase in HSV-1 replication. Neutralizing antibodies to neutrophil chemoattractants MIP-2 or MIP-1alpha but not KC significantly suppressed DTH and sharply reduced neutrophil accumulation in the ear pinna. Purified bone marrow-derived neutrophils exposed to interleukin-1alpha (IL-1alpha) produced chemokines in an 8-h assay. Administration of neutralizing antibody to IL-1alpha significantly reduced ear swelling and suppressed the levels of MIP-2, MIP-1alpha, MIP-1beta, and RANTES. We conclude that neutrophils are a critical component of the DTH response to viral antigen. They are recruited to the DTH test site by MIP-2 and MIP-1alpha, where they can be activated by IL-1alpha. The infiltrating cells also help suppress virus replication in immunized mice.     

Cytokine control of memory B cell homing machinery

The germinal center (GC) is a pivotal site for the development of B cell memory. Whereas GC B cells do not chemotax to most chemokines and do not express the adhesion receptors L-selectin, alpha(4)beta(7), and cutaneous lymphocyte Ag (CLA), memory B cells respond to various chemotactic signals and express adhesion receptors. In this study, we show that CD40 ligand, IL-2, and IL-10 together drive this transition of GC B cells to memory phenotype in vitro, up-regulating memory B cell markers, chemotactic responses to CXC ligand (CXCL)12, CXCL13, and CCL19, and expression of adhesion receptors L-selectin, alpha(4)beta(7), and CLA. Moreover, addition of IL-4 modulates this transition, preventing chemotactic responses to CXCL12 and CXCL13 (but not to CCL19), and inhibiting the re-expression of L-selectin, but not of CLA or alpha(4)beta(7). CCR7 expression, responsiveness to CCL19, and L-selectin/alpha(4)beta(7) phenotype are coordinately regulated. Thus, IL-2/IL-10 and IL-4 play important and distinctive roles in developing the migratory capacities of memory B cells.     

The importance of IL-1 beta and TNF-alpha,and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1 beta, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-alpha was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1 beta or TNF-alpha, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1 beta, TNF-alpha, or MIP-1 alpha. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R(-/-) mice and TNFR(-/-) mice was not observed at all, but severe arthritis was developed in IL-6(-/-) mice. These results suggest that IL-1 beta and TNF-alpha play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.     

CD8+ T cells are a biologically relevant source of macrophage inflammatory protein-1 alpha in vivo

Chemokines are small proteins that direct the migration of leukocytes to inflammatory foci. Many cell types, including macrophages, fibroblasts, endothelial cells, and lymphocytes, produce chemokines in vitro, but biologically relevant sources of chemokines in vivo have not been well characterized. To investigate the pertinent sources of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in vivo, we used MIP-1 alpha-deficient (MIP-1 alpha-/-) mice as donors and as recipients in adoptive transfer experiments after a lethal infection with Listeria monocytogenes (LM). Unexpectedly, we found that the production of MIP-1 alpha by CD8+ T cells was critical in this system, as the cells from MIP-1 alpha-/- mice primed with LM were significantly less effective in protecting naive mice against a lethal infection by LM than were the CD8+ T cells from wild-type (wt) mice. This requirement for donor T cell production of MIP-1 alpha was confirmed by the observation that wt donor T cells do not mediate protection when coadministered with an anti-MIP-1 alpha polyclonal antiserum. Production of MIP-1 alpha by the recipient mice was not required for protection, because wt and MIP-1 alpha-/- recipients were equally well protected by wt T cells. A 2- to 3-fold decrease in the number of transferred lymphocytes was seen in the spleens of mice receiving T cells from MIP-1 alpha-/- mice compared with those receiving wt T cells. In addition, CD8+ T cells from MIP-1 alpha-/- mice had a reduced ability to kill LM-infected target cells in vitro. These findings demonstrate that T cell production of MIP-1 alpha is required for clearance of an intracellular pathogen in vivo.     

Aborted germinal center reactions and B cell memory by follicular T cells specific for a B cell receptor V region peptide

A fundamental problem in immunoregulation is how CD4(+) T cells react to immunogenic peptides derived from the V region of the BCR that are created by somatic mechanisms, presented in MHC II, and amplified to abundance by B cell clonal expansion during immunity. BCR neo Ags open a potentially dangerous avenue of T cell help in violation of the principle of linked Ag recognition. To analyze this issue, we developed a murine adoptive transfer model using paired donor B cells and CD4 T cells specific for a BCR-derived peptide. BCR peptide-specific T cells aborted ongoing germinal center reactions and impeded the secondary immune response. Instead, they induced the B cells to differentiate into short-lived extrafollicular plasmablasts that secreted modest quantities of Ig. These results uncover an immunoregulatory process that restricts the memory pathway to B cells that communicate with CD4 T cells via exogenous foreign Ag.     

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1 alpha at 4 degrees C for 1 h was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1 alpha, and the pulsing effect with MIP-1 alpha could not be overcome by subsequent exposure of cells to MIP-1 beta. Also, pulse exposure of cells to MIP-1 beta blocked the activity of subsequently added MIP-1 alpha. For specificity, the action of a nonrelated myelosuppressive factor H-ferritin, was compared. MIP-1 alpha and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM, and CFU-GM. MIP-1 beta did not block the suppressive activity of H-ferritin. Also, hemin and an inactive recombinant human H-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1 alpha. These results show that MIP-1 beta's ability to block the action of MIP-1 alpha is specific. In addition, the results suggest that MIP-1 alpha and MIP-beta can, through rapid action, modulate early myeloid progenitor cell proliferation.     

Cutting edge: molecular mechanisms of synergy between CD40 and the B cell antigen receptor: role for TNF receptor-associated factor 2 in receptor interaction

Optimal Ag-specific B lymphocyte activation requires both recognition of Ag by the B cell Ag receptor (BCR) and contact-mediated interactions with Ag-specific Th lymphocytes. One of these interactions involves ligation of B cell CD40 by T cell-expressed CD154. CD40 signaling is crucial for Ab production, isotype switching, up-regulation of surface molecules, development of germinal centers, and the humoral memory response. The signaling pathways emanating from the BCR and CD40 are able to cooperate, but the molecular mechanisms responsible for this interaction are incompletely understood. The present study explored the roles of signaling motifs in the CD40 cytoplasmic tail in this synergy. We find that threonine in the PXQXT motif in the TNFR-associated factor-2 binding site is critical for synergistic effects of CD40 and BCR signals, independent of its phosphorylation. Furthermore, data suggest an indirect role for TNFR-associated factor-2 in the cooperative signaling.     

Silent development of memory progenitor B cells

T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. In the experiments we report, plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during the period of germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only 4 days was sufficient. Our results support a model of memory B cell development in which extensive affinity/specificity maturation can take place within a B cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not take place.     

Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines.

Macrophage inflammatory protein (MIP)-3alpha is a chemokine involved in the migration of T cells and immature dendritic cells. To study the contribution of proinflammatory cytokines and chemokines to the recruitment of these cells in rheumatoid arthritis (RA) synovium, we looked at the effects of the monocyte-derived cytokines IL-1beta and TNF-alpha and the T cell-derived cytokine IL-17 on MIP-3alpha production by RA synoviocytes. Addition of IL-1beta, IL-17, and TNF-alpha induced MIP-3alpha production in a dose-dependent manner. At optimal concentrations, IL-1beta (100 pg/ml) was much more potent than IL-17 (100 ng/ml) and TNF-alpha (100 ng/ml). When combined at lower concentrations, a synergistic effect was observed. Conversely, the anti-inflammatory cytokines IL-4 and IL-13 inhibited MIP-3alpha production by activated synoviocytes, but IL-10 had no effect. Synovium explants produced higher levels of MIP-3alpha in RA than osteoarthritis synovium. MIP-3alpha-producing cells were located in the lining layer and perivascular infiltrates in close association with CD1a immature dendritic cells. Addition of exogenous IL-17 or IL-1beta to synovium explants increased MIP-3alpha production. Conversely, specific soluble receptors for IL-1beta, IL-17, and TNF-alpha inhibited MIP-3alpha production to various degrees, but 95% inhibition was obtained only when the three receptors were combined. Similar optimal inhibition was also obtained with IL-4, but IL-13 and IL-10 were less active. These findings indicate that interactions between monocyte and Th1 cell-derived cytokines contribute to the recruitment of T cells and dendritic cells by enhancing the production of MIP-3alpha by synoviocytes. The inhibitory effect observed with cytokine-specific inhibitors and Th2 cytokines may have therapeutic applications.     

Differential effects of IL-27 on human B cell subsets

IL-27 is a novel heterodimeric cytokine of the IL-12 family that plays an important role in the regulation of T cell responses. Its role on human B cells has not been previously studied. In this study, we show that both chains of the IL-27 receptor complex, IL-27R and gp130, are constitutively expressed at the surface of naive and memory human tonsillar B cells, and are induced on germinal center B cells following CD40 stimulation. In naive B cells, IL-27 induced strong STAT1 and STAT3 phosphorylation, whereas it induced moderate STAT1 and low STAT3 activation in memory B cells. IL-27 induced T-bet expression in naive and memory B cells stimulated by CD40 or surface Ig engagement, but induced significant IL-12Rbeta2 surface expression in anti-Ig-stimulated naive B cells only. In anti-Ig-stimulated naive or memory B cells, IL-27 also induced CD54, CD86, and CD95 surface expression. In addition, IL-27 increased proliferation of anti-Ig-activated naive B cells and of anti-CD40-activated naive and germinal center B cells, but not of CD40-activated memory B cells. These data indicate that the B cell response to IL-27 is modulated during B cell differentiation and varies depending on the mode of B cell activation.     

Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

Isolation and characterization of an IL-1-dependent IL-4-producing bovine CD4+ T cell clone.

An Ag-specific interleukin 1 (IL-1)-dependent bovine CD4+ Th cell clone, termed 300B1, was isolated and found to resemble the previously described IL-1-dependent murine CD4+ Th2 cell clone, D10.G4.1. Both the 300B1 and the D10.G4.1 T cell clones proliferated to bovine (Bo) IL-1 beta, human (Hu) IL-1 alpha and IL-1 beta, and murine IL-1 alpha when cells were costimulated with concanavalin A (Con A). Proliferation of the 300B1 clone, when costimulated with Con A, appeared to be IL-1-specific in that proliferation could not be promoted by BoIL-2, HuIL-3, HuIL-4, HuIL-5, or HuIL-6. The 300B1 clone produced interferon-gamma (IFN-gamma), but not IL-2 following stimulation with either Con A, Con A plus phorbol 12-myristate 13-acetate or Ag plus antigen-presenting cells. Upon stimulation with Con A, the 300B1 clone expressed IL-4 mRNA and produced an autocrine growth factor (AGF) that could be inhibited by anti-HuIL-4 but not by anti-HuIL-2 Ab. The clonal derivation of the 300B1 clone was confirmed by isolating five 300B1 subclones, all of which produced IFN-gamma and an AGF but not IL-2. Collectively, these results suggest the IL-1-dependent bovine 300B1 Th cell clone produces IL-4, but not IL-2, as an AGF. Furthermore, the bovine Th cell clone appeared to share many characteristics of previously described murine Th2 cell clones except that the bovine clone produced IFN-gamma.     

Nitric oxide regulates MIP-1alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response

BACKGROUND: Chemokines and chemokine receptors have been shown to play a critical role in HIV infection. Chemokine receptors have been identified as coreceptors for viral entry into susceptible target cells, and several members of the beta chemokine subfamily of cytokines, MIP-1alpha, MIP-1beta, and RANTES, have been identified as the major human immunodeficiency virus (HIV)-suppressive factors produced by activated CD8+ T lymphocytes. In macrophages, HIV-1 infection itself was shown to upregulate the production of MIP-1alpha and MIP-1beta. In the present study, we address the mechanisms by which HIV-1 infection regulates beta chemokine responses in macrophages and lymphocytes. MATERIAL AND METHODS: To address whether nitric oxide (NO), generated as a consequence of HIV-1 infection, regulates beta chemokine responses in monocyte/macrophages and/or macrophage-depleted peripheral blood mononuclear cells (PBMCs) these two cell populations were isolated from HIV seronegative donors, placed in culture, and infected with HIV-1 in either the presence or absence of exogenous activators (e.g. lipopolysaccharide, phytohemagglutinin), inhibitors of nitric oxide synthase (NOS), or chemical donors of NO. Cultures were analyzed for beta chemokine responses by ELISA and RNase protection. RESULTS: LPS-induced MIP-1alpha release is enhanced in HIV-1-infected, as compared to uninfected, monocyte/macrophage cultures, and this enhancing effect is partially blocked by the addition of inhibitors of NOS, and can be reproduced by chemical generators of NO even in the absence of HIV-1 infection. A similar strategy was used to demonstrate a role for NO in HIV-1-mediated induction of MIP-1alpha in unstimulated macrophage cultures. NOS inhibitors also decreased MIP-1alpha and MIP-1beta production by phytohemagglutinin-stimulated monocyte-depleted PBMC cultures. CONCLUSIONS: These results indicate that NO amplifies MIP-1alpha responses in activated macrophages and lymphocytes, and suggests that this pleiotropic molecule might function as an enhancing signal that regulates secretion of beta chemokines during HIV-1 infection. These findings reveal a novel mechanism by which NO might regulate the anti-HIV activity of immune cells.     

Stimulation of the B cell receptor, CD86 (B7-2), and the beta 2-adrenergic receptor intrinsically modulates the level of IgG1 and IgE produced per B cell

Our findings using B cells from either wild-type, CD86-deficient, or beta 2-adrenergic receptor (beta2AR)-deficient mice suggest three mechanisms by which the level of IgG1 and IgE production can be increased on a per cell basis. Trinitrophenyl-specific B cells enriched from unimmunized mouse spleens were pre-exposed to Ag and/or the beta 2AR ligand terbutaline for 24 h before being activated by either a beta 2AR-negative Th2 cell clone or CD40 ligand/Sf9 cells and IL-4 in the presence or absence of an anti-CD86 Ab. Data suggest that the first mechanism involves a B cell receptor (BCR)-dependent up-regulation of CD86 expression that, when CD86 is stimulated, increases the amount of IgG1 and IgE produced in comparison to unstimulated cells. The second mechanism involves a BCR- and beta 2AR-dependent up-regulation of CD86 to a level higher than that induced by stimulation of either receptor alone that, when CD86 is stimulated, further increases the amount of IgG1 and IgE produced. The third mechanism is BCR-independent and involves a beta 2AR-dependent increase in the ability of a B cell to respond to IL-4. Flow cytometric and limiting dilution analyses suggest that the increase in IgG1 and IgE occurs independently from the isotype switching event. These findings suggest that the BCR, the beta 2AR, and CD86 are involved in regulating IL-4-dependent IgG1 and IgE production.     

Increased expression of MIP-1 alpha and MIP-1 beta mRNAs in the brain correlates spatially and temporally with the spongiform neurodegeneration induced by a murine oncornavirus

The chimeric murine oncornavirus FrCas(E) causes a rapidly progressive paralytic disease associated with spongiform neurodegeneration throughout the neuroaxis. Neurovirulence is determined by the sequence of the viral envelope gene and by the capacity of the virus to infect microglia. The neurocytopathic effect of this virus appears to be indirect, since the cells which degenerate are not infected. In the present study we have examined the possible role of inflammatory responses in this disease and have used as a control the virus F43. F43 is an highly neuroinvasive but avirulent virus which differs from FrCas(E) only in 3' pol and env sequences. Like FrCas(E), F43 infects large numbers of microglial cells, but it does not induce spongiform neurodegeneration. RNAase protection assays were used to detect differential expression of genes encoding a variety of cytokines, chemokines, and inflammatory cell-specific markers. Tumor necrosis factor alpha (TNF-alpha) and TNF-beta mRNAs were upregulated in advanced stages of disease but not early, even in regions with prominent spongiosis. Surprisingly there was no evidence for upregulation of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6 or of the microglial marker F4/80 at any stage of this disease. In contrast, increased levels of the beta-chemokines MIP-1 alpha and -beta were seen early in the disease and were concentrated in regions of the brain rich in spongiosis, and the magnitude of responses was similar to that observed in the brains of mice injected with the glutamatergic neurotoxin ibotenic acid. MIP-1alpha and MIP-1beta mRNAs were also upregulated in F43-inoculated mice, but the responses were three- to fivefold lower and occurred later in the course of infection than was observed in FrCas(E)-inoculated mice. These results suggest that the robust increase in expression of MIP-1 alpha and MIP-1 beta in the brain represents a correlate of neurovirulence in this disease, whereas the TNF responses are likely secondary events.     

Examination of the function of RANTES, MIP-1alpha, and MIP-1beta following interaction with heparin-like glycosaminoglycans

Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca(2+) flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1alpha, RANTES (regulated on activation normal T cell expressed), and MIP-1beta). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wild-type cells in order to produce the same intracytoplasmic Ca(2+) flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1alpha than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1beta. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1alpha, RANTES, or MIP-1beta. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

The microenvironment of human Peyer's patches inhibits the increase in CD38 expression associated with the germinal center reaction

Analysis of B cells in the human tonsils identified CD38 expression as a hallmark of germinal center (GC) B cells. However, the signals responsible for the in vivo induction of CD38 have not been determined. The primary site for generation of memory and plasma cells in the gastrointestinal tract is the GCs of Peyer's patches (PP). PP and intestinal mucosa, but not tonsils or oral mucosa, express mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The ligand for MAdCAM-1, integrin alpha(4)beta(7), is expressed on naive B cells and memory B cells that traffic to the gastrointestinal tract. In this study we determine that, unlike tonsil, human PP GC B cells do not express significant levels of CD38. PP B cells can be induced to express CD38 upon culture with CD40 ligand, anti-B cell receptor, and IFN-gamma. However, coculture of tonsil naive B cells with an Ab directed against integrin beta(7) inhibits IFN-gamma-induced CD38 hyperexpression. The absence of CD38 on PP GCs suggests that there are tissue-specific pathways of B cell development that differ between tonsil and PP. The differential expression pattern of MAdCAM-1, together with the observation that ligation of beta(7) can inhibit the induction of CD38 expression, suggests that ligation of alpha(4)beta(7) in vivo may contribute to a PP-specific GC phenotype.     

Macrophage-inflammatory protein-1alpha receptor expression on normal and chronic myeloid leukemia CD34+ cells

We have assessed expression of MIP-1alpha binding sites on the surface of CD34+ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34+ (15.7 +/- 6.2% in NBM and 9 +/- 4% in CML), which has specific macrophage-inflammatory protein-1alpha (MIP-1alpha) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34+ Thy+ stem cells. However, more than 80% of methanol-fixed CD34+ cells do bind MIP-1alpha, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34+, MIP-1alpha-R+ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1alpha receptor implicated in HIV infection, CCR5, revealed that very few CD34+ cells expressed these receptors and that expression was confined to the CD34+ Thy- progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1alpha are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.