The IgG isotype profile of the influenza virus-specific immune response was studied by quantitation of serum antibody (Ab) levels in correlation with the enumeration of antibody-secreting cells (ASC) detected in the lung, spleen, mediastinal lymph nodes (MLN), Peyer's patches and bone marrow (BM). Distinct isotypic patterns for serum Ab and Ab produced by cells present at or close to the site of infection were found after primary or repeated infections. An elevated number of IgM ASC was found after primary challenge in the spleen, lung and MLN. In contrast, the site of IgA and IgG production is restricted to the lung and lymph nodes draining the site of infection. In these organs IgA, IgG2a and IgG1 ASC are found as a result of primary virus infection while viral challenge induces mostly activation of IgA-producing cells and secretion of IgA to the lung lavage. In contrast, the majority (80-90%) of Ab detected in the serum belong to the IgG2a subclass and their serum level is maintained at a high level during the whole period of the response. The relative level of virus-specific serum IgG2a in correlation with the production of IgG2a Ab found predominantly in MLN and lung is highly dependent on the viral dose used for priming or challenge. As IgG2a ASC can be detected at relatively low numbers in the spleen and BM these results suggest that the production of the dominant IgG2a isotype of serum Ab occurs close to the viral challenge site. These data, however, point to distinct isotypic regulation in systemic versus local virus-specific Ab responses.
The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcg receptor (FcgRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domaincontaining molecules to the phosphorylated FcgRIIb. SHP-2, activated upon the binding to FcgRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgRIIb co-aggregated cells.
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.
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