J. Guay

Intravenous immunoglobulin attenuates airway inflammation through induction of forkhead box protein 3–positive regulatory T cells

BACKGROUND Intravenous immunoglobulin (IVIG) is a frequently used disease-modifying therapy for a large spectrum of autoimmune and inflammatory conditions, yet its mechanisms of action are incompletely understood. Using a robust murine model of antigen-driven allergic airways disease, we have demonstrated that IVIG markedly improves ovalbumin (OVA)-induced airway hyperresponsiveness characterized by 4- to 6-fold enhancement in regulatory T (Treg) cells in pulmonary and associated lymphoid tissues. OBJECTIVE We sought to determine whether IVIG induces antigen-specific Treg cells and to address cellular interactions that lead to induction of Treg cells by IVIG. METHODS C57Bl/6 mice were sensitized and challenged by means of intranasal OVA exposure. IVIG or albumin control was administered 24 hours before challenge. Treg cells were tracked by using green fluorescent protein (GFP)-forkhead box protein 3 (Foxp3) knock-in reporter mice (Foxp3(GFP)), and Treg cell and dendritic cell (DC) phenotypes and activities were elucidated by using coculture and flow cytometry. RESULTS IVIG therapy of OVA-sensitized and OVA-challenged mice induced antigen-specific forkhead box protein 3 (Foxp3)-positive Treg cells from non-Treg cell precursors. The induced Treg cells home specifically to the lungs and draining lymph nodes and have greatly potentiated suppressive activity compared with that seen in Treg cells purified from control mice. Induction of Treg cells is mediated by tolerogenic DCs generated after IVIG exposure. Compared with albumin-treated, OVA-exposed mice, IVIG-primed DCs express altered Notch ligands, including increased Delta-4 and reduced Jagged-1 levels, reflecting decreased T(H)2 polarization. Furthermore, IVIG-primed DCs can stimulate Treg cell differentiation from uncommitted Foxp3(-)CD4(+) T cells ex vivo, and adoptive transfer of IVIG-primed DCs abrogates airway hyperresponsiveness and induces Treg cells. CONCLUSION The anti-inflammatory effects of IVIG therapy can be mediated by the immunomodulation of DCs, creating a bridge that induces antigen-specific, highly suppressive Treg cells.

Children with atopic histories exhibit impaired lipopolysaccharide-induced Toll-like receptor-4 signalling in peripheral monocytes

Background The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll‐like receptor (TLR)‐4 on CD4+ cells in nasal mucosa.

B lymphocytes in inflammatory airway diseases.

B lymphocytes are key players in all facets of adaptive immune responses and are responsible for the production of IgE antibodies, initiators of allergic hypersensitivity reactions. Recent evidence indicates that B cells may be a crucial player in allergic and inflammatory airway pathology, directly populating upper and lower airway tissues. This review examines human and animal studies that directly demonstrated the presence of B lymphocytes in airway tissues and elaborates on their function as antibody‐secreting cells, antigen‐presenting cells and producers of inflammatory and regulatory cytokines. B lymphocytes appear to contribute to multiple facets of immune homeostasis in inflammatory diseases of the upper and lower airways.

Intravenous immunoglobulin attenuates airway hyperresponsiveness in a murine model of allergic asthma.

Background Intravenous immunoglobulin (IVIG) has potent anti‐inflammatory and immune‐modulating properties. IVIG has been utilized as a steroid‐sparing agent in severe asthma, but the results of clinical trials have been conflicting.

5-oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes.

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.

Expression of Semaphorin4C by Th2-stimulated B cells

Background Semaphorins are a family of proteins implicated in neurogenesis, angiogenesis and axonal migration. Previous work from our laboratory has shown that Th2stimulated B cells express a unique semaphorin, SEMA4C. Given the roles of other semaphorins, we hypothesized that SEMA4C was expressed on Th2stimulated B cells and might play a role in B cell trafficking and tissue homing therefore being expressed stronger in terminally differentiated B cells, such as memory and plasma cells. Experiment and results B cells were purified from tonsils removed from children undergoing routine tonsillectomy. They were cultured for 24 hours or 7 days at a concentration of 5X10 4 cells/mL in complete medium in the presence of aCD40 with either IFNg, IL-4, IL-21 or both IL-4 and IL-21. RNA was extracted, cDNA was made, and SEMA4C mRNA was amplified by qPCR and compared to the housekeeping gene GAPDH. After 24h culture, SEMA4C mRNA expression was detected only in IL-4stimulated cells but after 7 days, the expression was much stronger in both conditions containing IL-21, and decreased in IL-4-only stimulated cells. B cells were also similarly cultured for 72 hours at a concentration of 0.5X10 6 cells/mL in chamber slides. Slides were stained for SEMA4C with a fluorescent Alexa488 antibody and then visualized using fluorescent microscopy. As expected, stimulation with IL-21 yielded much higher SEMA4C expression than IL-4 alone whereas IFNg did not induce significant staining. Figures 1 and 2. Conclusion SEMA4C is induced in B cells upon Th2 stimulation but increases further upon follicle-like differentiation triggered by IL-21. This data confirms that SEMA4C is specific to terminally-differentiated B cells and supports our hypothesis that it might play a role in B-cell trafficking and homing in peripheral tissue follicles. SEMA4C could therefore be a key player in local allergic inflammation.

Atopy affects LPS responsiveness and TLR-4 expression in children peripheral mononuclear cells

Background Lipopolysaccharide (LPS) exposure in early life is associated with a lower incidence of atopy. We sought to determine whether atopy regulates TLR-4 expression and LPS-induced signal transduction on peripheral immune cells e.g. CD4 T ‘helper’ T cells, monocytes and B cells of a large pediatric cohort.