Rheumatoid arthritis is associated with signaling alterations in naturally occurring autoreactive B-lymphocytes.

Immune tolerance established during the development of B lymphocytes can be subverted in mature cells and lead to autoimmunity. This study focuses on the recently discovered subset of CD19(+)CD27(-)IgD(+)IgM(low/-) B cells that recognize self-antigens and have the capacity to produce autoantibodies, but under normal conditions do not generate autoimmune response due to intrinsic signaling inhibition (a condition known as clonal anergy and characterized by impaired antigen receptor signaling). Phosphorylation of intracellular signaling proteins and Ca(2+) responses in anergic B cells were measured by multicolor flow cytometry. Our results demonstrate a distinct phosphorylation pattern for major signal transduction proteins, which distinguishes anergic B cells. Comparison of B cell signaling properties in Rheumatoid Arthritis patients and healthy controls revealed a reversal of pTyr and Ca(2+) anergic signaling features in patients, accompanied by phosphorylation decreases of Blnk, Syk, SHP2, CD19. We identified BCR signaling pathway alterations associated with the loss of anergic B cell tolerance in Rheumatoid Arthritis.

Potentially autoreactive naturally occurring transitional T3 B lymphocytes exhibit a unique signaling profile.

B lymphocytes exhibit phenotypic differences that correlate with their developmental or functional stages and affect humoral immune responses. One recently described subset of naturally occurring immature transitional type 3 (T3) B lymphocytes is believed to consist of potentially autoimmune cells whose signaling properties have not been studied in detail. This study characterizes intracellular signaling in T3 B cells in wildtype C57BL/6 mice. Protein phosphorylation and Ca(2+) responses upon B-cell antigen receptor (BCR) engagement were measured by multicolor flow cytometry. We observed high baseline signaling activity and reduced BCR-mediated responses in T3 cells, which confirmed their anergy - a functional state in which lymphocytes recognize chronically present self-antigens but cannot produce immune response due to intrinsic signaling inhibition. Our results also revealed a previously unknown T3-specific phosphorylation pattern of 24 key signaling molecules involved in BCR signal transduction. These characteristics reflect the balance between stimulatory and inhibitory BCR signaling pathways in anergy. Results obtained in the collagen-induced arthritis model demonstrate the loss of anergy in T3 B cells during the onset of the disease. Our findings provide rationale for further investigating alterations in B-cell signaling patterns as earliest functional biomarkers of changes in the immune tolerance of autoreactive B cells.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Role of initial protein phosphorylation events and localized release-activated calcium influx in B cell antigen receptor signaling.

An increase in intracellular calcium concentration is one of the major initial steps in B cell activation following antigen receptor (BCR) ligation. We show herein that in C57BL/6 murine B lymphocytes and in model cell lines, BCR-mediated calcium ion (Ca(2+)) influx occurs via highly selective Ca(2+) release-activated channels, and stromal interaction molecule 1 (STIM1) plays an important role in this pathway. We also demonstrate the temporal relation between Ca(2+)-dependent signaling events and formation of the immune synapse. Our data indicate that cytoplasmic Ca(2+) levels in areas adjacent to the immune synapse differ from those in the rest of the cytoplasm. Finally, a comparison of phosphorylation patterns of BCR-triggered signaling proteins in the presence or absence of Ca(2+) revealed the unanticipated finding that initial BCR-triggered, Ca(2+)-dependent tyrosine phosphorylation events involve predominantly Ca(2+) released from intracellular stores and that influx-derived Ca(2+) is not essential. This suggests a different role for this phase of Ca(2+) influx.

Intrinsic B cell hypo-responsiveness in mice prematurely expressing human CR2/CD21 during B cell development.

We previously reported that human CR2 (hCR2) prematurely expressed under a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene during the CD43+ CD25- late pro-B cell stage of development results in peripheral B cells with impaired responses to immunization with T-dependent antigens. Herein, we show that hCR2 transgenic (Tg) mice also demonstrate a severe defect in T-independent antigen responses and are substantially protected from clinical arthritis, synovitis and cartilage/bone destruction in a collagen-induced arthritis model. This outcome is found despite the apparently normal development of autoreactive T cells with equivalent cytokine and proliferative responses to antigen when compared to non-Tg control mice. These data suggest the presence of an intrinsic B cell defect in the hCR2 Tg mice. We also show that an hCR2-dependent Ca2+ influx can be generated in both developing and mature Tg B cells, but with different rates of decay as compared to control wild-type (WT) mice. In addition, although analysis of tyrosine-phosphorylated proteins in WT and Tg B cells following B cell receptor (BCR)-induced activation revealed the presence of distinctly different phosphorylation patterns, no differences were identified in several candidate protein targets. Overall, these data suggest that premature hCR2 expression and the consequences thereof during B cell development intrinsically alters the way mature B cells develop and subsequently respond to antigen through the BCR signaling complex.