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

IL-13 expression by blood T cells and not eosinophils is increased in asthma compared to non-asthmatic eosinophilic bronchitis

<p>Abstract</p> <p>Background</p> <p>In asthma interleukin (IL)-13 is increased in the airway compared with non-asthmatic eosinophilic bronchitis. Whether this differential expression is specific to the airway or is more generalised is uncertain.</p> <p>Methods</p> <p>We sought to examine IL-13 expression in peripheral blood T-cells and eosinophils in asthma and non-asthmatic eosinophilic bronchitis. Peripheral blood CD3+ cell and eosinophil intracellular IL-13 expression from subjects with asthma, non-asthmatic eosinophilic bronchitis and healthy controls was assessed. The effect of priming by asthmatic serum on the release of IL-13 by peripheral blood mononuclear cells from healthy subjects was examined and the serum from these subjects was analysed for a range of chemokines and cytokines.</p> <p>Results</p> <p>The median (IQR)% intracellular IL-13 expression by CD3+ cells was increased in asthma [5.3 (2.7–9.8)%; n = 12] compared to non-asthmatic eosinophilic bronchitis [1.1 (0.5–3)%; n = 7] and healthy controls [1.7 (0.2–3%); n = 9] (p = 0.02), but was not significantly different in eosinophils across the groups. IL-13 released from healthy peripheral blood mononuclear cells (n = 10) was increased by asthmatic serum [117 (47.8–198)pg/ml] compared to control [78.5 (42.6–128)pg/ml; p = 0.02), but was not affected by non-asthmatic serum.</p> <p>Conclusion</p> <p>Our findings support the view that IL-13 expression is increased in peripheral blood-derived T cells in asthma and that asthmatic serum up-regulates IL-13 release from healthy peripheral blood mononuclear cells.</p

Constitutive, but Not Challenge-Induced, Interleukin-10 Production Is Robust in Acute Pre-Pubescent Protein and Energy Deficits: New Support for the Tolerance Hypothesis of Malnutrition-Associated Immune Depression Based on Cytokine Production in vivo

The tolerance model of acute (i.e., wasting) pre-pubescent protein and energy deficits proposes that the immune depression characteristic of these pathologies reflects an intact anti-inflammatory form of immune competence that reduces the risk of autoimmune reactions to catabolically released self antigens. A cornerstone of this proposition is the finding that constitutive (first-tier) interleukin(IL)-10 production is sustained even into the advanced stages of acute malnutrition. The IL-10 response to inflammatory challenge constitutes a second tier of anti-inflammatory regulation and was the focus of this investigation. Weanling mice consumed a complete diet ad libitum, a low-protein diet ad libitum (mimicking incipient kwashiorkor), or the complete diet in restricted daily quantities (mimicking marasmus), and their second-tier IL-10 production was determined both in vitro and in vivo using lipopolysaccharide (LPS) and anti-CD3 as stimulants of innate and adaptive defences, respectively. Both early (3 days) and advanced (14 days) stages of wasting pathology were examined and three main outcomes emerged. First, classic in vitro systems are unreliable for discerning cytokine production in vivo. Secondly, in diverse forms of acute malnutrition declining challenge-induced IL-10 production may provide an early sign that anti-inflammatory control over immune competence is failing. Thirdly, and most fundamentally, the investigation provides new support for the tolerance model of malnutrition-associated inflammatory immune depression

The Impact of T Cell Intrinsic Antigen Adaptation on Peripheral Immune Tolerance

Overlapping roles have been ascribed for T cell anergy, clonal deletion, and regulation in the maintenance of peripheral immunological tolerance. A measurement of the individual and additive impacts of each of these processes on systemic tolerance is often lacking. In this report we have used adoptive transfer strategies to tease out the unique contribution of T cell intrinsic receptor calibration (adaptation) in the maintenance of tolerance to a systemic self-antigen. Adoptively transferred naïve T cells stably calibrated their responsiveness to a persistent self-antigen in both lymphopenic and T cell–replete hosts. In the former, this state was not accompanied by deletion or suppression, allowing us to examine the unique contribution of adaptation to systemic tolerance. Surprisingly, adapting T cells could chronically help antigen-expressing B cells, leading to polyclonal hypergammaglobulinemia and pathology, in the form of mild arthritis. The helper activity mediated by CD40L and cytokines was evident even if the B cells were introduced after extended adaptation of the T cells. In contrast, in the T cell–replete host, neither arthritis nor autoantibodies were induced. The containment of systemic pathology required host T cell–mediated extrinsic regulatory mechanisms to synergize with the cell intrinsic adaptation process. These extrinsic mechanisms prevented the effector differentiation of the autoreactive T cells and reduced their precursor frequency, in vivo

Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells

Surfactant Protein D (SP-D) is an integral molecule of the innate immunity secreted by the epithelial cells lining the mucosal surfaces. Its C-type lectin domain offers pattern recognition functions while it binds to putative receptors on immune cells to modify cellular functions. Activated PBMCs and increased serum levels of SP-D are observed under a range of pathophysiological conditions including infections. Thus, we speculated if SP-D can modulate systemic immune response via direct interaction with activated PBMCs. Here, we have examined interaction of a recombinant fragment of human SP-D (rhSP-D) on PHA-activated PBMCs. We observed a significant downregulation of TLR2, TLR4, CD11c and CD69 upon rhSP-D treatment. rhSP-D inhibited production of Th1 (TNF-α and IFN-γ) and Th17 (IL-17) cytokines along with IL-6. Interestingly, levels of IL-2, Th2 (IL-4) and regulatory (IL-10 and TGF-β) cytokines were unaltered. Differential expression of co-stimulatory CD28 and co-inhibitory CTLA4 expression along with their ligands CD80 and CD86 revealed selective up-regulation of CTLA4 at both mRNA and protein level. In addition, rhSP-D induced apoptosis only in the activated but not in non-activated PBMCs. Blockade of CTLA4 inhibited rhSP-D mediated apoptosis, confirming an involvement of CTLA4 in induction of apoptosis. We conclude that SP-D restores immune homeostasis: it regulates expression of immunomodulatory receptors and cytokines, which is followed by apoptosis induction of immune-activated cells. These findings appear to suggest a general role for SPD in immune surveillance against activated immune cells

Beneficial autoimmunity at body surfaces – immune surveillance and rapid type 2 immunity regulate tissue homeostasis and cancer

Epithelial cells line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation or toxins cause activation of epithelial cells with release of cytokines and chemokines as well as alterations in the expression of cell surface ligands. Such display of epithelial stress is rapidly sensed by tissue resident immunocytes, which can directly interact with self-moieties on epithelial cells and initiate both local and systemic immune responses. Epithelial cells are thus key drivers of immune surveillance at body surface tissues. However, epithelial cells have a propensity to drive type 2 immunity (rather than type 1) upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis