Institut für Ernährungsmedizin

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Einfluss der IgG-Subklassen sowie der immunmodulatorischen Aminosäuren Arginin und Glutamin auf die Aktivierung menschlicher Darmmastzellen
(2011) Lechowski, Sandra; Lorentz, Axel
Mast cells are key effector cells in allergic diseases, like food allergy. Activation of mast cells occurs mainly by crosslinking of immunoglobulin E (IgE) receptor FcεRI. The role of allergen specific immunoglobulin G (IgG) subclasses IgG1, IgG2, IgG3, and IgG4 in activation of intestinal mast cells is not yet fully clarified. On the one hand, mast cells can be activated via IgG1 and by the mechanisms of ?IgG-supercrosslinking?. On the other hand, IgG1 and IgG4 are up regulated during immunotherapy and might be able to attenuate allergic reactions. In this work, we investigated the effect of IgG subclasses on IgE dependent and IgE-independent mediator release of human intestinal mast cells. Mast cells, isolated and purified from human intestinal tissue, were cultured with their growth factor stem cell factor (SCF) in combination with interferon γ (IFN-γ) or with interleukin 4 (IL-4). IFN-γ is known to up regulate expression of the activating IgG receptor FcγRI on mast cells, derived from peripheral blood. IL-4 is known to enhance IgE triggered mediator release from human intestinal mast cells. Expression of FcγRI, FcγRII, FcγRIII and FcεRI was analysed by flow cytometry. IgG subclasses should be isolated from human sera by affinity chromatography. However, the required purity of at least 95 % for each subclass could not be achieved. Thus, intestinal mast cells were stimulated with human myeloma IgG1-4 and their specific anti-IgG1-4 antibodies in combination with IgE/anti-IgE and the release of inflammatory mediators was measured. We could show that human intestinal mast cells cultured with IFN-γ express FcγRI, while FcγRII and FcγRIII were only weakly expressed. Mast cells cultured with IL-4 do not express FcγR. IgG subclasses themselves did not activate intestinal mast cells neither in culture with IFN-γ, nor cultured with IL-4. In combination with IgE/anti-IgE, the IgG subclasses IgG1/anti IgG1, IgG2/anti IgG2 and IgG4/anti IgG4 tended to decrease the release of pre-stored β-hexosaminidase and de novo synthesized leukotriene C4 (LTC4) but the reduction was not significant. To conclude, IFN-γ induced in human intestinal mast cells the expression of FcγRI, but this did not result in stimulation of the cells by IgG. Thus, human intestinal mast cells do not respond or only to a small extend to IgG subclasses and differ from mast cells derived from peripheral blood which could be activated via IgG1. The second part of the work examines the effect of the immunomodulatory amino acids arginine and glutamine on IgE-dependent activation of human intestinal mast cells. Background was the measured anti-inflammatory effect of combined pharmacological doses of both amino acids on intestinal biopsies from patients with Crohn?s disease. Beside allergic diseases, mast cells play a role in chronic intestinal inflammation. Therefore, they could be involved in the described effect. Human intestinal mast cells were incubated over night with combined physiological doses or pharmacological doses of arginine (0.1 mmol/L or 2 mmol/l) and glutamine (0.6 mmol/l or 10 mmol/l). Mast cells were stimulated with IgE/anti-IgE and release of β-hexosaminidase and LTC4, induction of cytokines and activation of signaling molecules was analysed. In human intestinal mast cells, cultured in SCF and IL-4, combined pharmacological concentrations of arginine and glutamine led to decreased release of LTC4 about 40 ± 22 % and to reduced induction of cytokines like CCL2 (monocyte chemotactic protein 1), CCL4 (macrophage inflammatory protein 1 beta), CXCL8 (IL-8), and TNF (tumor growth factor alpha) about an average of 58 ± 35 % compared to physiological doses of arginine and glutamine. The anti-inflammatory effects of arginine and glutamine were associated with decreased activation levels of MAP kinases like ERK1/2, p38, JNK pan und MEK1/2 about 38 ± 17 % as well as serine/threonine kinase Akt about 23 ± 20 %. Therewith, arginine and glutamine reduce activation levels of signaling molecules known to be involved in IgE dependent mast cell cytokine expression. Here, the results show that arginine and glutamine exert anti-inflammatory effects on human intestinal mast cells in vitro. Further investigations are necessary to monitor these findings in vivo prior to use arginine and glutamine as immunomodulatory agents in inflammatory and allergic diseases.

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