Antibodies to Human Thymic Epithelium Form Part of the Murine Autoreactive Repertoire

Monoclonal antibody (mAb) MR6 recognises a 200 kDa glycoprotein, gp200-MR6, which is expressed at high levels on the surface of human thymic cortical epithelium. In order to produce further mAbs against the gp200-MR6 molecule, mice were immunised with purified human gp200-MR6, hybridomas produced and supernatants screened for MR6-like reactivity on human thymic sections. Surprisingly this conventional hybridoma technique failed to produce stable hybridoma cells producing MR6-like antibodies. However, antibodies with specificitie other than MR6-like were obtained. Three such antibodies (1B2, 3A3 and 4B3) were analysed further. Expression of 1B2-antigen, 3A3-antigen and 4B3-antigen was analysed on skin, tonsil and thymic sections, on cultured thymic epithelial cells (TEC), thymocytes and peripheral blood mononuclear cells (PBMC), and found to be expressed by both lymphocytes and epithelial cell populations. Furthermore, the antigens were also expressed on mouse thymic, epithelial cells. The regulation of expression of these antigens was analysed following mitogen or cytokine stimulation of PBMC and cultured TEC, respectively. Expression on T cells was clearly affected by mitogens that mimic activation through the T cell receptor and expression on cultured TEC was affected by T cell-derived cytokines. Thus, the shared epithelial- lymphocyte molecules identified in this study may play a role in the cross-talk between the developing thymocytes and their epithelial microenvironment. The production of mAbs with specificities other than that of purified gp200-MR6 indicates that a wide range of B cells with specificity for components of the human thymic microenvironment exist in the normal mouse. These may detect epitopes that are shared with common pathogens to which the animals are exposed. Alternatively, they may be autoreactive B cells that are normally silent in the absence of T cell help. This help may be provided by T cells specific for human gp200-MR6, or nonspecifically by polyclonal activation induced by the adjuvant

Impact of dibenzocyclooctadiene lignans from Schisandra chinensis on the redox status and activation of human innate immune system cells

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Docosahexaenoic Acid Modulates NK Cell Effects on Neutrophils and Their Crosstalk.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadNatural killer (NK) cells and neutrophils engage in crosstalk that is important in inflammation and likely also for resolution of inflammation. NK cells activate neutrophils and induce their infiltration to the inflamed sites but may also influence their apoptosis and their subsequent efferocytosis by macrophages. Several studies indicate that docosahexaenoic acid (DHA) can inhibit NK cell cytotoxicity but the effects of DHA on the ability of NK cells to engage in crosstalk with neutrophils and affect their functions have not been described. This study explored the kinetics of the effects of NK cells and NK cells pre-treated with DHA on neutrophil surface molecule expression and apoptosis, as well as the ability of NK cells to affect other neutrophil functions. In addition, the study explored the effects of neutrophils on NK cell phenotype and function. Primary NK cells were pre-incubated with or without DHA, then stimulated and co-cultured with freshly isolated neutrophils. When co-cultured with NK cells, neutrophils had higher expression levels of CD11b and CD47; secreted more IL-8, IL-1ra, and CXCL10; had increased phagocytic ability; and their apoptosis was increased early after initiation of the co-culture while dampened at a later time-point. Pre-incubation of NK cells with DHA attenuated NK cell-induced upregulation of CD11b and CD47 on neutrophils, had minor effects on NK cell induction of cytokine/chemokine secretion or their phagocytic ability. Neutrophils also affected the function of NK cells, lowering the frequency of NKp46+ and CXCR3+ NK cells and increasing the concentrations of IFN-γ, TNF-α, and GM-CSF in the co-cultures. Pre-incubation of NK cells with DHA further decreased the frequency of NKp46+ NK cells in the co-culture with neutrophils and decreased the concentrations of IFN-γ, CCL3 and GM-CSF. These findings indicate that NK cells have mostly pro-inflammatory effects on neutrophils and that DHA can attenuate some of these pro-inflammatory effects. Neutrophils had both anti- and pro-inflammatory effects on NK cells. When NK cells had been pre-treated with DHA, the anti-inflammatory effects were increased and some of the pro-inflammatory effects attenuated. Overall, the results suggest that DHA may lead to a more anti-inflammatory microenvironment for NK cell and neutrophil crosstalk.Icelandic Research Fund University of Iceland Research Fund Landspitali University Hospital Research Fund Memorial Fund of Helga Jonsdottir and Sigurlidi Kristjansso

Acute phase inflammation is characterized by rapid changes in plasma/peritoneal fluid N-glycosylation in mice.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.Murine zymosan-induced peritonitis is a widely used model for studying the molecular and cellular events responsible for the initiation, persistence and/or resolution of inflammation. Among these events, it is becoming increasingly evident that changes in glycosylation of proteins, especially in the plasma and at the site of inflammation, play an important role in the inflammatory response. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based glycosylation profiling, we investigated the qualitative and quantitative effect of zymosan-induced peritonitis on N-glycosylation in mouse plasma and peritoneal fluid. Our results show that both N-glycomes exhibit highly similar glycosylation patterns, consisting mainly of diantennary and triantennary complex type N-glycans with high levels (>95 %) of galactosylation and sialylation (mostly NeuGc) and a medium degree of core fucosylation (30 %). Moreover, MS/MS structural analysis, assisted by linkage-specific derivatization of sialic acids, revealed the presence of O-acetylated sialic acids as well as disialylated antennae ("branching sialylation") characterized by the presence of α2-6-linked NeuGc on the GlcNAc of the NeuGcα2-3-Galβ1-3-GlcNAc terminal motif. A significant decrease of (core) fucosylation together with an increase of both α2-3-linked NeuGc and "branching sialylation" were observed in N-glycomes of mice challenged with zymosan, but not in control mice injected with PBS. Importantly, substantial changes in glycosylation were already observed 12 h after induction of peritonitis, thereby demonstrating an unexpected velocity of the biological mechanisms involved.Dutch Arthritis Association (Reumafonds) LLP-24 Innovative Medicines Initiative Joint Undertaking (IMI JU)/ 115142-2 Netherlands Genomic Initiative/93511033 info:eu-repo/grantAgreement/EC/FP7/278535info:eu-repo/grantAgreement/EC/FP7/27853

Immunomodulating effects of lichen-derived polysaccharides on monocyte-derived dendritic cells

To access Publisher full text version of this article. Please click on the hyperlink in Additional Links fieldMany naturally occurring polysaccharides from fungi and lichens have been found to have immunomodulating activity. However, the majority of these studies have focused on their effects on the innate arm of the immune system. Although dendritic cells (DCs) belong to the innate immune system, they play an important role as a bridge between the innate and the adaptive immune response. In this study, the effects of 11 chromatographically purified and well-characterised lichen polysaccharides (of different structural types) on the maturation of DCs were tested by analysing the secretion of IL-12p40 and IL-10 by human monocyte-derived dendritic cells in vitro. Four of the polysaccharides upregulated IL-10 secretion by the dendritic cells, as compared with unstimulated cells, the beta-glucans lichenan and Ths-2 and the heteroglycans Pc-4 and thamnolan. IL-12p40 secretion was significantly upregulated by the beta-glucan lichenan and the heteroglycans Pc-2, Pc-4, thamnolan and Ths-4, while the mature dendritic cells stimulated with the heteroglycan Pc-1 secreted significantly less IL-12p40 than the unstimulated cells. Proportional index (PI) was used to determine the relationship between the IL-12p40 and IL-10 secretion. The PI of all the beta-glucans, i.e. lichenan, pustulan and Ths-2, and the heteroglycan thamnolan was significantly lower than the PI observed for the unstimulated cells, which was mainly due to increased IL-10 secretion. Therefore, these polysaccharides could be considered suitable candidates in tolerance and anti-inflammatory studies, as IL-10 is one of the major cytokines involved in tolerance and anti-inflammatory responses

Immunomodulating polysaccharides from the lichen Thamnolia vermicularis var. subuliformis

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldThree heteroglycans Ths-4, Ths-5 and thamnolan and a beta-glucan, Ths-2, isolated from the lichen Thamnolia vermicularis var. subuliformis were tested for in vitro immunomodulating activities and shown to have various influences on the immune system. All the polysaccharides except Ths-4 caused a stimulation of rat spleen cell proliferation. In contrast, Ths-4 caused cell death early in the culture, probably due to over-stimulation. Moreover, the galactofuranomannans, Ths-4, Ths-5 and the beta-glucan Ths-2, induced rat spleen cells to secrete IL-10 significantly above background levels. In addition, Ths-4 and Ths-5 stimulated significant TNF-alpha secretion by rat peritoneal macrophages. The galactofuranomannans Ths-4 and Ths-5 have similar structures apart from the molecular weight. Thus, it may be concluded that the molecular size might influence the potency but not the pattern of activity for Ths-4 and Ths-5. The galactofuranorhamnan thamnolan had less mitogenic effect than Ths-5 and Ths-2 and neither induced IL-10 secretion by rat spleen cells nor TNF-alpha secretion by peritoneal macrophages to significant levels. This shows that thamnolan with its unusual galactofuranorhamnan structure differs from the other Thamnolia polysaccharides in its immunomodulatory activity

Two circulating neutrophil populations in acute inflammation in mice.

Recent studies indicate that neutrophils are heterogeneous and may have an immunosuppressive role in addition to their well-known phagocytic and bactericidal function. This study examined neutrophil subpopulations in the circulation, peritoneum, spleen and bone marrow from mice at various time points after induction of acute inflammation. MATERIAL, TREATMENT AND METHODS: Female C57BL/6 mice were injected intraperitoneally with lipopolysaccharide (LPS). Blood, peritoneal, spleen and bone marrow cells were collected and counted and expression of surface molecules and chemokine receptors analyzed with flow cytometry. Chemokine and cytokine concentrations in serum and peritoneal fluid were determined by ELISA. Neutrophil numbers in the circulation decreased following administration of LPS but reached similar numbers to those prior to inflammation at 8 h. At that time point, two distinct neutrophil populations were present in the circulation. These two neutrophil populations differed in size, granularity and expression of CD11b and Ly6G. Few neutrophils were recruited into the peritoneum until 24 h after administration of LPS at a time when the neutrophils in the circulation had increased their expression of the chemokine receptor CXCR2. Induction of acute inflammation leads to the appearance of two circulating neutrophil subpopulations, which may differ in their activation state and function.Icelandic Research Fund University of Iceland Research Fun

Murine antigen-induced inflammation--a model for studying induction, resolution and the adaptive phase of inflammation.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageMurine zymosan-induced peritonitis is the model most frequently used to study resolution of inflammation. However, the antigen-induced peritonitis model may be better suited for studying resolution of inflammation and the adaptive phase that follows. The objective of this study was to provide an evaluation of the kinetics of cells and mediators during induction, resolution and the adaptive immune phases of a murine antigen-induced inflammation. Female C57BL/6 mice were immunized twice subcutaneously with mBSA and three weeks after the initial immunization they were injected intraperitoneally (i.p.) with mBSA, which induced peritonitis. Peritoneal cells were counted and expression of surface molecules and chemokine receptors analyzed with flow cytometry. Chemokine and cytokine concentrations in peritoneal fluid were determined by ELISA. Two neutrophil populations, differing in size and granularity and slightly in expression of surface molecules, were observed in the peritoneal cavity after induction of inflammation. Macrophages disappeared from the peritoneal cavity following i.p. administration of mBSA but appeared again as they differentiated from recruited monocytes and peaked in numbers at 48 h. At that time point, two distinct populations of macrophages were present in the peritoneal cavity; one with high expression of F4/80, also expressing the atypical chemokine receptor D6 as well as CCR7; the other expressing low levels of F4/80 and also expressing CD11c and CD138. Eosinophils appeared in the peritoneum 3h following i.p. administration of mBSA and peaked at 48 h. At that time point they had upregulated their expression of CCR3 but decreased their expression of CD11b. Peritoneal levels of CCL11 peaked at 6h and may have led to recruitment of the eosinophils. NK cells and T cells peaked at 48 h, whereas B cells peaked at 5 days, with the majority being B1 cells. Peritoneal concentrations of pro-inflammatory cytokines (IL-β and IL-6) and chemokines (CCL2 and CCL3) peaked at 3h, whereas IL-1ra peaked at 6h, sTNF-R at 24h and sIL-6R and TGF-β at 48 h. The results show kinetic alterations in cell populations and mediators in a murine model that may be an excellent model to study initiation and resolution of inflammation and the following adaptive phase.Icelandic Research Fund University of Iceland Research Fund Landspitali University Hospital Research Fun

Dietary fish oil decreases the proportion of classical monocytes in blood in healthy mice but increases their proportion upon induction of inflammation.

Fish oil can have beneficial effects in health and disease. In healthy individuals, reduction of the inflammatory status may be of benefit, whereas in patients with systemic inflammation, such as sepsis, it is important to diminish the immunosuppression that is thought to contribute to poor outcome. The objective of this study was to determine the effects of dietary fish oil on monocytes/macrophages in blood, bone marrow, spleen, and peritoneum and chemokine concentrations in blood and peritoneum in healthy mice and mice with endotoxin-induced inflammation. Mice were fed a Western-type diet without fish oil (C) or with 2.8% fish oil (FO) for 6 wk and then either killed (healthy mice) or injected i.p. with endotoxin (LPS) and killed after 3, 8, 12, 24, or 48 h. Blood, bone marrow, spleen, and peritoneal lavage were collected. Expression of cell surface molecules and chemokine receptors was analyzed by flow cytometry and chemokine concentrations measured by ELISA. Healthy mice in the FO group had lower proportions of classical monocytes in blood than healthy mice in the C group. LPS administration increased the proportion of classical monocytes in blood in mice in the FO group but not in those in the C group. Healthy mice in the FO group had lower serum concentrations of CCL2 than mice in the C group, but in inflamed mice, CCL2 concentrations were higher in the FO group than in the C group. These results indicate that dietary fish oil can attenuate the inflammatory status in homeostasis but intensify the immune response upon inflammation.Icelandic Research Fund University of Icelan