Physiological up-regulation of inhibitory receptors Fc gamma RII and CR1 on memory B cells is lacking in SLE patients.

Under physiological conditions immune complexes (IC) are efficiently cleared from the circulation and meanwhile provide important feedback signals for the immune system via Fc gamma Rs and complement receptors. Dysregulation of these mechanisms have been implicated in conditions where IC concentrations reach pathological levels and inflict diseases, like systemic lupus erythematosus (SLE). Our aim was to compare distinct sub-populations of CD19(+) B cells of healthy individuals and SLE patients with regard to their expression of Fc gamma R type II (Fc gamma RII, CD32), complement receptor type 1 (CR1, CD35) and complement receptor type 2 (CR2, CD21) and sIgG/IgM. The following four groups of peripheral CD19(+) B cells were investigated: IgM(+)/CD27(-) naive, IgM(+)/CD27(+) and IgM(-)/CD27(+) memory cells and CD27(high) plasmablasts. We demonstrate that the expression of the inhibitory receptors Fc gamma RII and CR1 is up-regulated on peripheral memory B cells of healthy controls, whereas this up-regulation is considerably impaired on the memory B cells of SLE patients. This reduction affects both the IgM(+) and switched memory B cells. We found a striking difference between the expression of complement receptors CD21 and CD35; namely, no up-regulation of CD21 occurred on the memory B cells of healthy donors, and its decreased expression in SLE patients was characteristic for both the CD27(-) naive and the CD27(+) memory B-cell populations. Our results clearly demonstrate that the previously reported reduced expression of IC-binding receptors is mainly due to the disturbed memory compartment; however, the higher frequency of CD19(+)/CD27(high)/sIg(low) plasmablasts expressing minimal levels of these receptors also contributes to this diminution.

Altered expression of Fcgamma and complement receptors on B cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by B cell hyper-reactivity, autoantibody production, immune complex (IC) deposition, and multiple organ damage. The contribution of IC and B cell-mediated changes in the pathogenesis of SLE is well established, however, the exact role of IC-binding receptors expressed on B cells, Fcgamma receptors, and complement receptors CR1 and CR2 in these pathological processes is unclear. Development of lupus-like symptoms in mice defective for the inhibitory Fc-gammaRIIb and genetic association of certain FcgammaR genes with SLE demonstrate a significant role for these receptors but reports indicating alterations of Fcgamma or complement receptor-mediated B cell functions in human SLE are relatively few. The present review highlights a selected set of data including our own discussing the significance of animal models, genetics, and functional alterations of these IC-binding receptors in the etiopathogenesis of SLE.

[On the way to understand the mechanism of immune tolerance]. / Közeledünk az immunológiai tolerancia mechanizmusának megértése felé.

This introductory paper deals with mechanisms playing important role in the development of immunological tolerance. Discusses the problems of "immunological self" and "non-self", the importance of the roles of AIRE gene products, as well as the role of dendritic cells and regulatory T-cells (T-reg cells) in immune tolerance and autoimmunity.

The role of CR2 in autoimmunity.

Complement activation is one of the most powerful mechanisms taking place during inflammation and immune responses. Over the last 30 years increasing evidence has proven the role of C3 and receptors for its activation fragments in the initiation and regulation of immune responses. Since complement also has a basic importance in the maintenance of immune homeostasis, abnormalities affecting complement proteins and their receptors may lead to pathological conditions. Autoimmune conditions develop as a result of a range of genetic and environmental factors. Findings obtained from animal models support the notion that malfunctioning of complement receptors, particularly CR2, might be involved in the breakdown of tolerance and excessive antibody production by auto reactive B-cell clones. In addition to B cells, activated, CR2-bearing T cells may also contribute to the pathogenesis of autoimmunity as they can receive activating/survival signals in the inflamed tissue. Results obtained from mouse experiments however, should be extended to the human system with great care, since there are basic differences between the structure and function of human and murine CR1 and CR2.

Characterization of a trypsin-like serine protease of activated B cells mediating the cleavage of surface proteins.

Activated B cells may cleave their surface receptors due to the proteolytic activity on the cell membrane or in its vicinity. We attempted to isolate and characterize the protease(s) responsible for this cleavage. Zymograms prepared from the supernatant and the plasma membrane fraction of activated human B cells and BL41/95 cell line exhibited a 85-90 kDa doublet band with protease activity, while that of resting B cells did not. Soybean trypsin inhibitor (STI), Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and EDTA treatment abolished the activity of this protease. The excess of Zn(2+) ions in EDTA did not restore the enzymatic activity, while it was completely recovered in the presence of Ca(2+). We affinity-purified a 85-90 kDa protease from the supernatant of BL41/95 cells using STI coupled to Sepharose 4B beads, and measured its kinetic parameters. For the arginyl substrate K(M) was 358+/-59 microM and for the lysyl substrate 582+/-103 microM. TLCK and benzamidine inhibited the protease at micromolar, while STI at nanomolar concentrations. Both the inhibition profile and the substrate specificity suggest that it is a trypsin-like serine protease. We assume that the 85-90 kDa serine protease expressed on and secreted by activated B cells and BL41/95 cell line is responsible for the cleavage of various membrane proteins, including Fcgamma receptors; thus it may play a crucial role in regulating B cell's function.

Developmental differences in B cell receptor-induced signal transduction.

We have compared early signaling events at various stages of B cell differentiation using established mouse cell lines. Clustering of pre-B cell antigen receptor (BCR) or BCR induced the tyrosine phosphorylation of various proteins in all cells, although the phosphorylation pattern differed. In spite of the pre-BCR-induced tyrosine phosphorylation, we could not detect an intracellular Ca(2+) signal in pre-B cells. However, co-clustering of the pre-BCR with CD19 did induce Ca(2+) mobilization. In contrast to the immature and mature B cells, where the B cell linker protein (BLNK) went through inducible tyrosine phosphorylation upon BCR clustering, we observed a constitutive tyrosine phosphorylation of BLNK in pre-B cell lines. Both BLNK and phospholipase C (PLC)gamma were raft associated in unstimulated pre-B cells, and this could not be enhanced by pre-BCR engagement, suggesting a ligand-independent PLC gamma-mediated signaling. Further results indicate that the cell lines representing the immature stage are more sensitive to BCR-, CD19- and type II receptors binding the Fc part of IgG (Fc gamma RIIb)-mediated signals than mature B cells.

Synthesis and receptor binding of IgG1 peptides derived from the IgG Fc region.

The IgG binding Fcgamma receptors (FcgammaRs) play a key role in defence against pathogens by linking humoral and cell-mediated immune responses. Impaired expression and/or function of FcgammaR may result in the development of pathological autoimmunity. Considering the functions of FcgammaRs, they are potential target molecules for drug design to aim at developing novel anti-inflammatory and immunomodulatory therapies. Previous data mostly obtained by X-ray analysis of ligand-receptor complexes indicate the profound role of the CH2 domain in binding to various FcgammaRs. Our aim was to localize linear segments, which are able to bind and also to modulate the function of the low affinity FcgammaRs, like FcgammaRIIb and FcgammaRIIIa. To this end a set of overlapping octapeptides was prepared corresponding to the 231-298 sequence of IgG1 CH2 domain and tested for binding to human recombinant soluble FcgammaRIIb. Based on these results, a second group of peptides was synthesized and their binding properties to recombinant soluble FcgammaRIIb, as well as to FcgammaRs expressed on the cell surface, was investigated. Here we report that peptide representing the Arg(255)-Ser(267) sequence of IgG1 is implicated in the binding to FcgammaRIIb. In addition we found that peptides corresponding to the Arg(255)-Ser(267), Lys(288)-Ser(298) or Pro(230)-Val(240) when presented in a multimeric form conjugated to branched chain polypeptide in uniformly oriented copies induced the release of TNFalpha, a pro-inflammatory cytokine from MonoMac monocyte cell line. These findings indicate that these conjugated peptides are able to cluster the activating FcgammaRs, and mediate FcgammaR dependent function. Peptide Arg(255)-Ser(267) can also be considered as a lead for further functional studies.

Functional mapping of the Fc gamma RII binding site on human IgG1 by synthetic peptides.

Receptors specific for the Fc part of IgG (Fc gamma R) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory Fc gamma R may result in autoimmunity. Thus, the modulation of IgG-Fc gamma R 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 Fc gamma RII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P(234) and S(298) were synthesized and used in binding and functional experiments. Binding of the peptides to Fc gamma R was assayed in vitro and ex vivo, and peptides found to interact were functionally tested. The shortest effective peptide was T(256)-P(271), which bound to soluble recombinant Fc gamma RIIb with K(d)=6 x 10(6) M(-1). The biotinylated peptides R(255)-P(271) and T(256)-P(271) complexed by avidin exhibited functional activity; they induced Fc gamma RIIb-mediated inhibition of the BCR-triggered Ca(2+) response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF-alpha and IL-6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the Fc gamma RII-binding part of IgG1.