Activation of B Cell Locomotion In Vitro

This project looks at the signals that induce locomotion in resting B cell populations and in germinal centre B cells, both from human tonsil. Signals that induce locomotion in blood B cells compared with high-density tonsil B cells were also studied. Polarization studies of the response of cells from immunized mice to antigen are also included. B cells were purified from tonsil by established procedures to yield a high- density fraction (resting cells out of cycle) and low-density (activated) fraction. Germinal centre cells were present in, and were purified from, the latter fraction. Cells from these fractions were assayed for locomotor activity. Two methods were used to study the locomotor activity of B cells; (1) Polarization assay. Measurement of shape-change from a spherical to a polarized shape on stimulation with an attractant. (2) Invasion of collagen gels. Lymphocytes overlaid on a collagen gel containing an attractant will migrate into the gel in larger numbers than into control gels. Previous studies of T cells showed that the full development of the capacity for locomotion and chemotaxis in lymphocytes requires two stages, (a) Resting cells require to be cultured with a growth activator and move from G0 into the G1 phase of cell cycle. After overnight culture, a locomotor population of cells is obtained, (b) These cells are now capable of responses to chemoattiactants and show immediate (<30 min) polarization and locomotion when incubated in their presence. (1) High density B cells. These are small surface IgM+ and surface IgD+ cells which are not in cycle. When freshly purified from the tonsil, very few of these cells show locomotor capacities. The results presented here demonstrate that culture overnight in IL-4, aCD40 or lL-13 induces locomotor shape change in a high proportion of high-density B cells. The proportion of polarized cells increases slowly over a period of 24-48 hours suggesting that locomotor capacity is activated as the cells pass from G0 to the G1 phase of growth. Anti-IL-4 and anti-IL-13 inhibit the locomotion induced by their respective cytokines. IFN-gamma inhibits the locomotion response induced by IL-4. Culture in combination of IL-4 and aCD40 stimulates polarization of more cells than culture in either alone. A combination of IL-4, aCD40, and aIgM stimulates polarization of still more cells (up to 60-70% of the population). Adding aIgM to cultures with aCD40, or with IL-4 does not increase the polarization significantly compared with either aCD40 or IL-4 alone. In addition to study of the effect of locomotor activators on locomotion in overnight culture, the immediate (<30min.) effects of attractants on locomotion of the resting B cell fraction were studied, using either cells direct from the tonsil or cultured B cells. The polyclonal activators, algD and aIgM, were tested in short term assays (30 min. incubation) on freshly isolated cells and on cells cultured in IL-4. Both populations showed immediate polarization to anti-Ig, but cultured cells responded more strongly than cells direct from the tonsil. The optimum attractant concentration of anti-Ig was 100ng-1mug/ml. There was no response to the appropriate isotype controls, mouse IgG2a and sheep Ig. Cells cultured in IL-4 also polarized in a short-term assay to aCD40 (100ng-1mug/ml) within 30 minutes but not to isotype control mouse IgG 1. The results suggest that in contrast to IL-4 and IL-13, anti-CD40 acts not only as a locomotor activator but also as a chemoattractant. Cells direct from the tonsil showed no chemoattractant response to anti-CD40. To measure locomotion itself, cells cultured in IL-4 were layered on top of collagen gels incorporating aIgM, aIgD, aCD40, and HBSS alone, and were allowed to invade for 18 hours. The number of cells invading gels incorporating any of the three stimuli was greater than that invading gels containing medium alone. FACS analysis on invaded cells show that 75 +/- 12% of cells were IgM+ and 82% IgD+. The gel invasion assay selects the locomotor population and demonstrates clearly that small resting IgM+ and IgD+ B cells not only change- shape in response to anti-IgM and anti-IgD, but also show invasive locomotion in response to these antibodies. (Abstract shortened by ProQuest.)

Interleukin-33 promoting Th1 lymphocyte differentiation dependents on IL-12

No abstract available.The pro-Th2 cytokine IL-33 is now emerging as an important Th1 cytokine-IFN-γ inducer in murine CD4+ T cells that is essential for protective cell-mediated immunity against viral infection in mice. However, whether IL-33 can promote human Th1 cell differentiation and how IL-33 polarizes Th1 cells is less understood. We assessed the ability of IL-33 to induce Th1 cell differentiation and IFN-γ production in vitro and in vivo. We report here that IL-33 alone had no ability in Th1 cell polarization. However it potentiated IL-12-mediated Th1 cell differentiation and IFN-γ production in TCR-stimulated murine and human CD4+ T cells in vitro and in vivo. IL-33 promoted Th1 cell development via MyD88 and synergized with IL-12 to enhance St2 and IL-12R expression in CD4+ T cells. These data therefore provide a novel mechanism for Th1 cell differentiation and optimal induction of a Type 1 response. Thus, IL-33 is capable of inducing IL-12-dependent Th1 cell differentiation in human and mouse CD4+ T cells

IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice

Background&lt;p&gt;&lt;/p&gt; The initiation and regulation of pulmonary fibrosis are not well understood. IL-33, an important cytokine for respiratory diseases, is overexpressed in the lungs of patients with idiopathic pulmonary fibrosis.&lt;p&gt;&lt;/p&gt; Objectives&lt;p&gt;&lt;/p&gt; We aimed to determine the effects and mechanism of IL-33 on the development and severity of pulmonary fibrosis in murine bleomycin-induced fibrosis.&lt;p&gt;&lt;/p&gt; Methods&lt;p&gt;&lt;/p&gt; Lung fibrosis was induced by bleomycin in wild-type or Il33r (St2)−/− C57BL/6 mice treated with the recombinant mature form of IL-33 or anti–IL-33 antibody or transferred with type 2 innate lymphoid cells (ILC2s). The development and severity of fibrosis was evaluated based on lung histology, collagen levels, and lavage cytology. Cytokine and chemokine levels were quantified by using quantitative PCR, ELISA, and cytometry.&lt;p&gt;&lt;/p&gt; Results&lt;p&gt;&lt;/p&gt; IL-33 is constitutively expressed in lung epithelial cells but is induced in macrophages by bleomycin. Bleomycin enhanced the production of the mature but reduced full-length form of IL-33 in lung tissue. ST2 deficiency, anti–IL-33 antibody treatment, or alveolar macrophage depletion attenuated and exogenous IL-33 or adoptive transfer of ILC2s enhanced bleomycin-induced lung inflammation and fibrosis. These pathologic changes were accompanied, respectively, by reduced or increased IL-33, IL-13, TGF-β1, and inflammatory chemokine production in the lung. Furthermore, IL-33 polarized M2 macrophages to produce IL-13 and TGF-β1 and induced the expansion of ILC2s to produce IL-13 in vitro and in vivo.&lt;p&gt;&lt;/p&gt; Conclusions&lt;p&gt;&lt;/p&gt; IL-33 is a novel profibrogenic cytokine that signals through ST2 to promote the initiation and progression of pulmonary fibrosis by recruiting and directing inflammatory cell function and enhancing profibrogenic cytokine production in an ST2- and macrophage-dependent manner

Anti-Toll-like receptor 2 and 4 antibodies suppress inflammatory response in mice

Toll-like receptors (TLRs) 2 and 4 recognize different endogenous and exogenous agonists and play a distinct role in infection and inflammation. However, their ultimate effect in a given infectious and inflammatory disease is less understood. We produced polyclonal anti-murine TLR2 and TLR4 antibodies and investigated their effect on cutaneous leishmaniasis and inflammatory arthritis. Administration of these antibodies to susceptible BALB/c mice, infected in the footpad with Leishmania major, reduced footpad swelling but not the parasite load compared with mice treated with control IgG. The antibodies synergistically reduced leishmanial-specific T-cell proliferation, T helper type 1 and type 2 cytokine production, specific IgG1 and IgG2a antibody synthesis, and T-cell receptor and co-stimulatory molecule expression on dendritic cells in infected mice. We then tested the effect of these antibodies on collagen-induced arthritis (CIA) in DBA/1 mice, a classic model of chronic inflammation. Both antibodies markedly suppressed the development of clinical parameters with concomitant reduction of pro-inflammatory cytokine production. These data therefore suggest that anti-TLR2 and 4 antibodies may have a synergistic therapeutic effect on inflammatory disease in vivo

Chemoattraction of human T cells by IL-18

Cell locomotion is crucial to the induction of an effective immune response. We report here the chemoattraction of CD4� T cells by IL-18, a member of the IL-1 cytokine family. Recombinant IL-18 increased the proportion of T cells in polarized morphology in vitro and stimulated their subsequent invasion into collagen gels in an IL-18 concentration gradient-dependent manner. Immunofluorescent microscopy studies determined that the major cell type responding to IL-18 was IL-18R�CD4�. Importantly, synovial CD4� T cells from patients with rheumatoid arthritis responded to IL-18, adopting polarized morphology and gel invasion without further activation ex vivo, indicating the physiologic relevance of our observations. Finally, injection of rIL-18 into the footpad of DBA/1 mice led to local accumulation of inflammatory cells. These data therefore demonstrate for the first time lymphocyte chemoattractant properties of a member of the IL-1 cytokine family and its relevance in inflammatory disease

IL-33 activates B1 cells and exacerbates contact sensitivity

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor alpha, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33-activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33-activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2(-/-) mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity

A proinflammatory role of IL-18 in the development of spontaneous autoimmune disease

Serum from patients with systemic lupus erythematosus (SLE) contained significantly higher concentrations of IL-18 than normal individuals. MRL/lpr mice, which develop spontaneous lupus-like autoimmune disease, also had higher serum levels of IL-18 than wild-type MRL/�� mice. Daily injections of IL-18 or IL-18 plus IL-12 resulted in accelerated proteinuria, glomerulonephritis, vasculitis, and raised levels of proinflammatory cytokines in MRL/lpr mice. IL-18-treated MRL/lpr mice also developed a “butterfly” facial rash resembling clinical SLE. In contrast, MRL/lpr mice treated with IL-18 plus IL-12 did not develop a facial rash. The facial lesion in the IL-18-treated mice showed epidermal thickening with intense chronic inflammation accompanied by increased apoptosis, Ig deposition, and early systemic Th2 response compared with control or IL-12 plus IL-18-treated mice. These data therefore show that IL-18 is an important mediator of lupus-like disease and may thus be a novel target for therapeutic intervention of spontaneous autoimmune diseases