A filarial nematode-secreted phosphorylcholine-containing glycoprotein uncouples the B cell antigen receptor from extracellular signal-regulated kinase-mitogen-activated protein kinase by promoting the surface Ig-mediated recruitment of Src homology 2 domain-containing tyrosine phosphatase-1 and Pac-1 mitogen-activated kinase-phosphatase.

Unraveling the molecular mechanisms by which filarial nematodes, major human pathogens in the tropics, evade the host immune system remains an elusive goal. We have previously shown that excretory-secretory product-62 (ES-62), a homologue of phosphorylcholine-containing molecules that are secreted by human parasites and which is active in rodent models of filarial infection, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal transduction elements in B lymphocytes. Such activation mediates desensitization of subsequent B cell Ag receptor (BCR) ligation-induced activation of extracellular signal-regulated kinase-mitogen-activated protein (ErkMAP) kinase and ultimately B cell proliferation. We now show that the desensitization is due to ES-62 targeting two major regulatory sites of B cell activation. Firstly, pre-exposure to ES-62 primes subsequent BCR-mediated recruitment of SHP-1 tyrosine phosphatase to abolish recruitment of the RasErkMAP kinase cascade via the Igalphabeta-ShcGrb2Sos adaptor complex interactions. Secondly, any ongoing ErkMAP kinase signaling in ES-62-primed B cells is terminated by the MAP kinase phosphatase, Pac-1 that is activated consequently to challenge via the BCR.

B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells.

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A(2) activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A(2) expression, induction of mitochondrial phospholipase A(2) activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Delta psi(m)), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Delta psi(m), ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Delta psi(m) stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-x(L) cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Delta psi(m) and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.

Immunomodulatory properties of a phosphorylcholine-containing secreted filarial glycoprotein.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein which is secreted by the rodent filarial nematode Acanthocheilonema viteae. A homologue exists in the human filarial nematode Brugia malayi and indeed PC is found attached to glycoproteins of many, if not all, filarial species. At concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans, ES-62 is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B and T lymphocytes following in-vitro exposure. Although this interaction is insufficient to cause lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways and hence also to proliferation via the antigen receptors. The active component of ES-62 appears to be PC, as the results obtained with ES-62 are broadly mimicked by PC conjugated to BSA or PC alone. Although PC can also be shown to desensitize B cells following in-vivo administration, not all cells are affected, as it is still possible to generate an antibody response. Dissection of this response indicates that it is of the Th2 type.

MAPkinase: a second site of G-protein regulation of B-cell activation via the antigen receptors.

Ligation of the antigen receptors on B cells transduces transmembrane signals leading to the induction of DNA synthesis. We now show that a pertussis toxin-sensitive heterotrimeric G-protein(s) of the Gi class plays a key role in the regulation of surface immunoglobulin (sIg)-mediated DNA synthesis in B cells. This site of G-protein regulation is distinct from that we have previously reported to govern the coupling of the antigen receptors on B cells to the phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. We have, moreover, identified a candidate target for this new G-protein regulation by showing that mitogen-activating protein kinase (MAPkinase) activity, which plays a key role in the transduction of sIg-mediated proliferative signals in B cells, is abrogated by pre-exposure to pertussis toxin that covalently modifies and inactivates heterotrimeric G-proteins of the Gi class. Furthermore, our data suggest that this pertussis toxin-sensitive G-protein couples the antigen receptors to MAPkinase activation, at least in part, by regulating sIg-coupling to Lyn, Syk and perhaps Blk and Fyn activity, results consistent with studies in other systems which show that classical G-protein-coupled receptors recruit such protein tyrosine kinases to tranduce MAPkinase activation. Interestingly, however, this G-protein plays no apparent role in the control of up-regulation of major histocompatibility complex class II expression on B cells, suggesting that such G-protein-regulated-tyrosine kinase and MAPkinase activation is not required for the induction of this biological response following antigen receptor ligation.

A phosphorylcholine-containing filarial nematode-secreted product disrupts B lymphocyte activation by targeting key proliferative signaling pathways.

Filarial nematodes infect more than 100 million people in the tropics, causing elephantiasis, chronic skin lesions, and blindness. The parasites are long-lived as a consequence of being able to evade the host immune system, but an understanding of the molecular mechanisms underlying this evasion remains elusive. In this study, we demonstrate that ES-62 (2 microg/ml), a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B lymphocytes. Although this interaction is insufficient to cause B lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase and Ras mitogen-activating protein kinase pathways, and hence also to proliferation, via the Ag receptor. The active component of ES-62 appears to be PC, a molecule recently shown to act as an intracellular signal transducer, as the results obtained with ES-62 are broadly mimicked by PC alone. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES, by interfering with B cell function, could play a role in prolonging filarial infection in parasitized individuals.

Induction of signalling anergy via the T-cell receptor in cultured Jurkat T cells by pre-exposure to a filarial nematode secreted product.

Filarial nematodes constitute major causes of morbidity in the Tropics. The worms have a life-span exceeding five years, a longevity which is considered to reflect at least in part, their ability to interfere with host lymphocyte responsiveness. To date the molecular mechanisms underlying this ability have not been defined but we now demonstrate that ES-62, a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to render Jurkat T cells anergic to intracellular signalling via the antigen receptor (TCR). In particular, ES-62 acts by modulating activation of the tyrosine kinases Fyn, Lck and ZAP-70 leading to selective disruption of TCR coupling to the phospholipase D, protein kinase C, phosphoinositide-3-kinase and RasMAPkinase signalling cascades. These cascades are key elements in the transduction of transcriptional and proliferative signals following ligation of TCR. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES may play a role in the induction of T lymphocyte hyporesponsiveness observed during filarial infections.

A filarial nematode secreted product differentially modulates expression and activation of protein kinase C isoforms in B lymphocytes.

Filarial nematodes, parasitic worms that cause elephantiasis, chronic skin lesions, and blindness in the tropics, release a number of molecules, some of which appear to be immunomodulatory/suppressive, into the host environment. Here we demonstrate that ES-62, a phosphorylcholine-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, interferes with activation of B lymphocytes by differential modulation of protein kinase C isoform expression. Indeed, while ES-62 selectively down-regulates expression of the alpha, beta, iota/lambda, delta, and zeta isoforms of PKC, it up-regulates expression of PKC-gamma and -epsilon in B cells. Inhibitor studies suggest that ES-62 appears to promote down-regulation of PKC isoforms mainly by stimulating proteolytic degradation. ES-62 also disrupts the normal activation and nuclear translocation patterns of the alpha and iota/lambda isoforms of PKC following ligation of the Ag receptor. The effects of ES-62 on certain PKC isoforms were found to be modified by coculture with IL-4. Of particular interest was the observation that IL-4 prevented down-regulation of PKC alpha and iota/lambda, isotypes considered to be active in transducing mitogenic signals. Phosphorylcholine-containing secreted products (phosphorylcholine-ES) are also released by human filarial parasites; hence we discuss how these findings may relate to the nature of the human B cell response during filarial infections.

Mechanical stretch increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells.

OBJECTIVE: To determine whether changes in haemodynamic load, simulated in vitro by mechanically stretching cultured vascular smooth muscle cells, could be transduced into biochemical signals similar to those produced by growth factors. DESIGN: A system was developed which was capable of stretching cultured vascular smooth muscle cells from 0 to 20%. The effect of stretching quiescent vascular smooth muscle cells on both c-fos messenger RNA (mRNA) expression and release of total inositol phosphates was determined over a time interval of 0-360 min. METHODS: Rat mesenteric artery vascular smooth muscle cells were grown using standard cell culture methods. Induction of the proto-oncogene, c-fos, was determined by Northern blotting. Phosphoinositide breakdown was assessed by measuring [3H]-inositol phosphates released from prelabelled cells. RESULTS: A 20% fixed stretch resulted in a rapid induction of c-fos mRNA which reached maximal levels by 15 min. The amount of c-fos mRNA detected was dependent on the degree of stretch, with maximum induction obtained for 15 and 20% stretch. The effects of mechanical stretch were also assessed on phosphoinositide turnover by measuring [3H]-inositol phosphates released from prelabelled cells. A 20% fixed stretch of vascular smooth muscle cells for 20 min resulted in a 3.2-fold increase in total [3H]-inositol phosphates released compared with unstretched cells. CONCLUSIONS: Our results show that mechanical stretch increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells in vitro. These observations suggest that mechanical stretch and growth factors share common signal transduction pathways which may be important in the development of vascular hypertrophy.