Dávid Medgyesi

Dual β-adrenergic modulation in the immune system : Stimulus-dependent effect of isoproterenol on MAPK activation and inflammatory mediator production in macrophages

This is the first study to demonstrate that the interaction between beta-adrenoceptor activation, and the production of inflammatory mediators can be modulated in opposite ways by two inflammatory stimuli, namely, protein kinase C (PKC)-activating phorbol myristyl acetate (PMA) and lipopolysaccharide (LPS). We provided evidence that isoproterenol treatment, when combined with phorbol ester increased the production of tumor necrosis factor-alpha, interleukin-12, and nitric oxide in murine macrophages, as well as in human monocytes and differentiated PLB-985 cells, while in agreement with earlier findings, it decreased inflammatory mediator production in combination with LPS stimulation. The contrasting effect on inflammatory mediator production, shown for the PMA and LPS activated cells was accompanied by parallel changes in activation of ERK1/2 and p38 MAPKs. Thus, isoproterenol significantly increased MAPK activation (phosphorylation) in PMA-treated cells and, conversely, it decreased the activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 in LPS-stimulated cells. The opposing effects of isoproterenol on LPS-induced versus PMA-induced mediator production and the concurrent changes in MAPK activation highlight the role of this kinase pathway in macrophage activation and provide new insights regarding the flexible ways through which beta-adrenoceptor stimulation can modulate the inflammatory response in macrophages. Our results challenge the dogma that beta-adrenoceptor signaling is only immunosuppressive, and offer potential opportunities for new therapeutic approaches in the treatment of inflammatory and autoimmune diseases.

Functional mapping of the FcγRII binding site on human IgG1 by synthetic peptides

Receptors specific for the Fc part of IgG (FcγR) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory FcγRmay result in autoimmunity. Thus, the modulation of IgG‐FcγR interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to FcγRII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P234 and S298 were synthesized and used in binding and functional experiments. Binding of the peptides to FcγR was assayed in vitro and ex vivo, and peptides foundto interact were functionally tested. The shortest effective peptide was T256–P271, which bound to soluble recombinant FcγRIIb with Kd=6×106 M–1. The biotinylated peptides R255–P271 and T256–P271 complexed by avidin exhibited functional activity; they induced FcγRIIb‐mediated inhibition of the BCR‐triggered Ca2+ response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF‐α and IL‐6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the FcγRII‐binding part of IgG1.

B cell receptor-induced Ca2+ mobilization mediates F-actin rearrangements and is indispensable for adhesion and spreading of B lymphocytes

B cells acquire membrane‐bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane‐bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen‐tethered surface, which is followed by the formation of radial lamellipodia‐like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR‐induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PLCγ and CRAC‐mediated calcium mobilization are critical regulators of these processes. Measuring calcium and actin dynamics in live cells, we found that a generation of actin‐based membrane protrusion is strongly linked to the dynamics of a cytoplasmic‐free calcium level. Finally, we demonstrate that PLCγ and CRAC channels regulate the activity of actin‐severing protein cofilin, linking BCR‐induced calcium signaling to the actin dynamics.

Grb2 associated binder 2 couples B-cell receptor to cell survival

B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Ig-beta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.

CD16/32-specific biotinylated 2.4G2 single-chain Fv complexed with avidin–FITC enhances FITC-specific humoral immune response in vivo in a CD16-dependent manner

Fcgamma receptors (FcgammaRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcgammaRs may facilitate antigen presentation and dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcgammaRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcgammaRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcgammaRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin-FITC, and tested its effect on the FITC-hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11b(high) and CD11c(high) cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC-dextran) or TD (FITC-keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcgammaRIII might be a key mediator of this mechanism.

Grb2‐Associated Binder 1 (Gab1) Adaptor/Scaffolding Protein Regulates Erk Signal in Human B Cells

Abstract:  RNA silencing experiments showed that knocking down Gab1 adaptor protein in BL41 human Burkitt lymphoma cells significantly reduced B cell receptor (BCR)–induced Erk phosphorylation, indicating that Gab1 plays a pivotal role in regulating Erk activity in B cells.