Chandrasekar Venkataraman

Negative regulation of antigen receptor‐mediated signaling by constitutive asociation of CD5 with the SHP‐1 protein tyrosine phosphatase in B‐1 B cells

CD5, a membrane‐associated glycoprotein, has been shown to negatively regulate antigen receptor‐mediated growth responses in peritoneal B lymphocytes, thymocytes and mature T cells. The CD5‐expressing peritoneal B cells (B‐1) that are normally unresponsive to B cell receptor (BCR)‐mediated growth signals mount a proliferative response to BCR cross‐linking if the CD5 gene is deleted or if the CD5 molecule is sequestered away from the BCR. SHP‐1, a cytosolic protein tyrosine phosphatase, has also been implicated in the negative regulation of antigen receptor‐mediated signaling. The present study shows that SHP‐1 is constitutively associated with the BCR in B‐1 cells. This association is mediated in part by CD5, as it is reduced substantially after antigen receptor ligation in CD5– / – B‐1 cells, and upon sequestration of CD5 from the antigen receptor complexes in wild‐type B‐1 cells. Prior cross‐linking of CD5 also restores a normal calcium mobilization response as well as NF‐κB activation in B‐1 cells. These data support a model whereby CD5 negatively regulates antigen receptor‐mediated growth signals by recruiting SHP‐1 into the BCR complex in B‐1 cells.

CCR8 Is Not Essential for the Development of Inflammation in a Mouse Model of Allergic Airway Disease

Chemokine receptors play an important role in the trafficking of various immune cell types to sites of inflammation. Several chemokine receptors are differentially expressed in Th1 and Th2 effector populations. Th2 cells selectively express CCR3, CCR4, and CCR8, which could direct their trafficking to sites of allergic inflammation. Additionally, increased expression of the CCR8 ligand, TCA-3, has been detected in affected lungs in a mouse model of asthma. In this study, CCR8-deficient mice were generated to address the biological role of CCR8 in a model of allergic airway disease. Using two different protocols of allergen challenge, we demonstrate that absence of CCR8 does not affect the development of pulmonary eosinophilia and Th2 cytokine responses. In addition, administration of anti-TCA-3-neutralizing Ab during allergen sensitization and rechallenge failed to inhibit airway allergic inflammation. These results suggest that CCR8 does not play an essential role in the pathogenesis of inflammation in this mouse model of allergic airway disease.

Cutting Edge: FISP (IL-4-Induced Secreted Protein), a Novel Cytokine-Like Molecule Secreted by Th2 Cells

Th cell subsets, namely Th1 and Th2 cells, play an important role in mounting an immune response against invading pathogens. Several genes are selectively up-regulated during differentiation and effector phases of Th subsets. In this study, we report the identification of a novel cytokine-like molecule designated FISP (IL-4-induced secreted protein), which is selectively expressed and secreted by Th2 cells. Detectable levels of FISP are observed only 3 days after initiation of Th2 differentiation. Expression of FISP in developing Th cells requires at least two signals: TCR signaling involving protein kinase C activation and STAT6-dependent IL-4R signaling.

CD72-MEDIATED B CELL ACTIVATION INVOLVES RECRUITMENT OF CD19 AND ACTIVATION OF PHOSPHATIDYLINOSITOL 3-KINASE

Occupancy of the B cell glycoprotein, CD72 results in syk‐independent activation of phospholipase‐C γ and calcium mobilization. The cytoplasmic tail of CD72 does not contain an immunoreceptor tyrosine‐based activation motif to directly transduce signals into the B lymphocyte. Hence, we investigated whether other coreceptors such as CD19 and its associated phosphatidylinositol 3‐kinase (PI 3‐K) were involved in CD72 signaling. Two specific inhibitors of PI 3‐K inhibited CD72‐stimulated B cell proliferation in a dose‐dependent manner. Activation of B lymphocytes via CD72 resulted in recruitment and activation of PI 3‐K, which was mediated by CD19. Accordingly, CD72 ligation induced CD19 tyrosine phosphorylation. Thus, lipid products generated as a result of PI 3‐K activation may have an important function in CD72‐mediated B lymphocyte activation. The kinetics of CD19 tyrosine phosphorylation induced by CD72 ligation were strikingly different from those seen following B cell antigen receptor (BCR) stimulation. A transient increase in the tyrosine phosphorylation of the complement receptors, CD21 and CD35 was observed in BCR‐ but not CD72‐stimulated cells. Co‐cross‐linking of CD72 and CD19 failed to induce syk tyrosine phosphorylation suggesting that even under these conditions, CD72 signaling was independent of syk activation. A transient and stimulation‐dependent physical association between CD19 and CD72 was observed in CD72‐ligated cells. These observations suggest a mechanism by which CD72 can recruit CD19 and influence activation of CD19‐associated PI 3‐K, which appears to be critical for CD72‐mediated B cell activation.

CpG oligodeoxynucleotides overcome the unresponsiveness of neonatal B cells to stimulation with the thymus-independent stimuli anti-IgM and TNP-Ficoll

Neonates are very vulnerable to pathogenic encapsulated bacteria due to their inability to mount an antibody response to capsular polysaccharides, which are thymus‐independent type 2 (TI‐2) antigens (Ag). Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides induced neonatal B cells to proliferate to anti‐IgM, a TI‐2 stimulus. CpG ODN inhibited the spontaneous and B cell receptor‐mediated apoptosis of neonatal B cells and reduced the amount of the pro‐apoptotic Bcl‐xS , strongly correlated with anti‐IgM‐induced apoptosis of neonatal B cells. CpG ODN protected neonatal B cells from apoptosis by down‐regulation of the Bcl‐xS protein. Neonatal B cells underwent polyclonal differentiation upon stimulation with CpG ODN, but unlike in adult B cells, this was not preceded by IL‐6 secretion. CpG ODN stimulated neonatal B cells to mount an Ag‐specific antibody response to TNP‐Ficoll, another TI‐2 Ag. Thus CpG ODN could provide a novel approach to induce the immune system in neonates to respond to harmful encapsulated bacteria.

Bacterial lipopolysaccharide induced B cell activation is mediated via a phosphatidylinositol 3-kinase dependent signaling pathway.

Bacterial lipopolysaccharide (LPS) is a potent stimulant of B cells and macrophages. LPS induces B cell proliferation and differentiation into antibody secreting cells. In addition, LPS also stimulates IL-6 secretion in mature B cells and in immature B cell lines such as WEHI-231. Although sufficient literature is available on LPS induced signaling events in monocytes and macrophages, the mechanisms involved in LPS induced B cell activation are not well understood. In this report, it is shown that both LPS mediated B cell proliferation and IL-6 secretion are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways. The B cell specific co-receptor, CD19 is not tyrosine phosphorylated in LPS stimulated B cells. Thus, in contrast to B cell antigen receptor (BCR) signaling, the activation of PI 3-kinase appears not to be related to the recruitment of PI 3-kinase to tyrosine phosphorylated CD19. This is the first demonstration of the importance of PI 3-kinase signaling pathway in LPS mediated B lymphocyte activation.

Positive Signaling Through CD72 Induces Mitogen-Activated Protein Kinase Activation and Synergizes with B Cell Receptor Signals to Induce X-Linked Immunodeficiency B Cell Proliferation

CD72 is a 45-kDa B cell transmembrane glycoprotein that has been shown to be important for B cell activation. However, whether CD72 ligation induces B cell activation by delivering positive signals or sequestering negative signals away from B cell receptor (BCR) signals remains unclear. Here, by comparing the late signaling events associated with the mitogen-activated protein kinase pathway, we identified many similarities and some differenes between CD72 and BCR signaling. Thus, CD72 and BCR activated the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase. Both CD72- and BCR-mediated ERK and JNK activation required protein kinase C activity, which was equally important for CD72- and BCR-induced B cell proliferation. However, CD72 induced stronger JNK activation compared with BCR. Surprisingly, the JNK activation induced by both BCR and CD72 is Btk independent. Although both CD72 and BCR induced Btk-dependent ERK activation, CD72-mediated proliferation is more resistent to blocking of ERK activity than that of BCR, as shown by the proliferation response of B cells treated with PD98059 and dibutyryl cAMP, agents that inhibit ERK activity. Most importantly, CD72 signaling compensated for defective BCR signaling in X-linked immunodeficiency B cells and partially restored the proliferation response of X-linked immunodeficiency B cells to anti-IgM ligation. These results suggest that CD72 signals B cells by inducing BCR-independent positive signaling pathways.

Cutting edge: Chandra, a novel four-transmembrane domain protein differentially expressed in helper type 1 lymphocytes.

Development of naive Th cells into Th1 and Th2 effector populations requires coordinated expression of a complex set of genes. In this study, we have identified a novel four-transmembrane domain protein, Chandra, that is differentially expressed in Th1 cells. Chandra expression is observed in STAT4- and IFN-γ-deficient mice, indicating that Chandra is not an IL-12- or IFN-γ-responsive gene. Interestingly, Chandra mRNA is detected in anti-CD3-activated T cells from STAT6-deficient mice in the absence of any differentiation conditions. Furthermore, neutralization of IL-4 signaling is sufficient to induce transcription of Chandra in anti-CD3-activated T cells from wild-type mice, demonstrating that STAT6 signaling is required to repress Chandra expression in activated T cells and Th2 subsets. This is the first demonstration of a differentially expressed four-transmembrane domain protein in Th1 cells.

Defective CD19-dependent signaling in B-1a and B-1b B lymphocyte subpopulations

Peritoneal and pleural cavities in mice and humans contain a unique population of B-lymphocytes called B-1 cells that are defective in B cell antigen receptor (BCR) signaling but have an increased propensity to produce autoantibodies. Several molecules such as Btk, Vav, and CD19 known to be important for BCR signaling have been shown to be critical for the development of B-1 cells from undefined precursors. Here we demonstrate that B-1 cell unresponsiveness to BCR cross-linking is in part due to defective signaling through CD19, a molecule known to modulate signaling thresholds in B cells. The defective CD19 signaling is manifested in reduced synergy between mIgM and CD19 to stimulate calcium mobilization in B-1 cells. BCR induced tyrosine phosphorylation of CD19 was transient in B-1 cells while it was prolonged in splenic B-2 cells. In both B-1 and B-2 cells BCR cross-linking induced a modest increase of CD19 associated Lyn, a Src family protein tyrosine kinase (PTK) thought to be important for CD19 phosphorylation. However, the tyrosine phosphorylated CD19 in B-1 cells binds less phosphatidylinositol 3-kinase (PI3-K) compared to B-2 cells. Most interestingly, we find that Vav-1 and Vav-2, proteins thought to be critical for CD19 signal transduction, are severely reduced in B-1 cells resulting in a complete absence of any CD19 associated Vav. Also we showed that both B-1a and B-1b B cells failed to proliferate in response to BCR cross-linking which in part appears to be due to defects in CD19 mediated amplification of BCR induced calcium mobilization.

Interleukin-4 overcomes the negative influence of cyclic AMP accumulation on antigen receptor stimulated B lymphocytes.

Activation of protein kinase A (PKA) in B lymphocytes prior to the ligation of the B cell antigen receptor (BCR) results in a profound inhibition of BCR induced proliferation. The major effect of increased PKA activity in B lymphocytes was the induction of apoptosis leading to a reduced BCR induced growth response. The growth promoting cytokine IL-4 rescued B lymphocytes from PKA mediated negative effects. IL-4 protected BCR stimulated cells from PKA mediated inhibition primarily by preventing apoptosis and growth arrest. PKA-activation caused a downregulation of anti-IgM induced expression of Bcl-xL protein, that was restored by IL-4. Previous studies have shown that PKA-activation blocks BCR induced phospholipase Cgamma-activation and calcium mobilization. IL-4 was unable to overcome the block in anti-IgM mediated calcium mobilization due to PKA-activation. B cell apoptosis induced by PKA-activation was also seen in CD72 stimulated cells, although CD72 mediated B-lymphocyte proliferation was not affected. PKA mediated block in phospholipase gamma-activation and calcium mobilization were not due to alterations in the activation of tyrosine kinases lyn, blk and syk. Moreover, BCR mediated tyrosine phosphorylation of PLC gamma2 and CD19 were also unaffected by cAMP accumulation. These observations are in contrast to the ability of PKA to drastically reduce the activity of ZAP-70 and syk in T lymphocytes and neutrophils, respectively. The IL-4 mediated protection appears to be due to a change in late events in BCR signaling, which are important for Bcl-xL expression.