Differentiation of B lymphocytes in C3H/HeJ mice: the induction of Ia antigens by lipopolysaccharide.

The lipid A moiety of bacterial lipopolysaccharide (LPS) elicits several types of responses in murine B lymphocytes. First, lipid A induces the nonproliferative expression of cell surface antigens in more immature cell types. Second, lipid A induces a mitogenic response in more mature B cell types. Lipid A induces the expression of Ia antigens on bone marrow cells from C3H/DiSn but not C3H/HeJ mice. The Ia-inducible cells possess surface immunoglobulin. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) induce the appearance of Ia antigens on B lymphocytes from both C3H/HeJ and C3H/DiSn mice, suggesting that lipid A exerts its inductive effects by increasing cyclic AMP levels in cells. In contrast to what is observed by using other strains of mice, mature B lymphocytes from C3H/HeJ mice do not support a mitogenic response to lipid A. The subpopulation of B lymphocytes in C3H/HeJ mice that normally respond mitogenically to LPS not only appear to lack an LPS-response mechanism utilized in the mitogenic pathway, but they lack the LPS-response pathway of the immature B cell types. A lipid A-bound protein (LAP) induces both the expression of Ia and a mitogenic response in the different subpopulations of B lymphocytes from C3H/HeJ and C3H/DiSn mice. The genetic defect in C3H/HeJ mice that limits responses to lipid A may be associated with a receptor that is normally expressed on many different cell types.     

Inhibitors of protein kinase C block activation of B lymphocytes by bacterial lipopolysaccharide

Activation of murine splenic B lymphocytes (B cells) by bacterial lipopolysaccharide (LPS) was found to be markedly inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), two potent inhibitors of protein kinases. The higher sensitivity of DNA synthesis, RNA synthesis and protein N-glycosylation activity to H-7, relative to H-8, strongly supports the proposal that protein kinase C plays a critical role in the activation of B cells. A kinetic study on the time of addition of H-7 indicated that protein kinase C promoted the activation process continuously after the addition of LPS.     

Glucocorticoid effects on membrane lipid mobility during differentiation of murine B lymphocytes

The lateral motion of membrane lipids on lipopolysaccharide-stimulated murine B lymphocytes was measured using photobleaching recovery techniques. The mobility of the phospholipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured at 37 degrees C on B lymphocytes 48 h after stimulation by various concentrations of lipopolysaccharide. DiI mobility on lymphoblasts from cultures stimulated with 10 micrograms/ml lipopolysaccharide was reduced 50% compared with unstimulated, small B cells. However, both lower and higher lipopolysaccharide concentrations caused some decrease in lipid mobility. Lipid mobility was measured on B cells stimulated with 10 micrograms/ml lipopolysaccharide at zero time, on lymphoblasts at 18, 24, 48 and 72 h, and on immunoglobulin (Ig) -secreting lymphocytes at 96 h. The diffusion coefficient of DiI on both control and lipopolysaccharide-treated cells at zero time is 6.3 X 10(-9) cm2 X s-1. This value remains unchanged for unstimulated cells over 72 h. Lipid mobility of lipopolysaccharide-activated lymphoblasts decreased during incubation with lipopolysaccharide to 5.0, 3.4, 2.8 and 2.4 X 10(-9) cm2 X s-1 after 18, 24, 48 and 72 h, respectively. DiI mobility on immunoglobulin (Ig) -secreting lymphocytes identified at the foci of Protein A-coated sheep red blood cells plaques is 8.6 X 10(-9) cm2 X s-1, a value similar to that of unstimulated B cells. The effect of introducing various concentrations of a synthetic glucocorticoid, triamcinolone acetonide (TA), to 48 h lipopolysaccharide-stimulated cells for 6 h was examined. Maximal TA effect was observed at a concentration of 10(-7) M, which caused an increase in lipid mobility to 7.5 X 10(-9) cm2 X s-1. Exposing resting B cells (t = 0) or lymphoblasts (t = 24, 48 or 72 h) to TA for 3 h had no effect on lipid mobility. Treatment for 6 h with 10(-7) MTA increased DiI diffusion to 12.6, 9.9, 7.5 and 6.8 X 10(-9) cm2 X s-1 on control cells and on 24, 48 and 72 h lipopolysaccharide-activated lymphoblasts, respectively. A longer incubation of 12 h with 10(-7) MTA caused no further change in lipid lateral diffusion. The response was glucocorticoid-specific. In lymphoblasts (48 h) incubated an additional 6 h with 10(-7) MTA and a 100-fold excess of cortexolone or progesterone, the increase in lipid mobility was substantively blocked; estradiol and testosterone had no effect on lipid lateral diffusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased rate of phosphorylation-dephosphorylation of the translational initiation factor eIF-4E correlates with the induction of protein and glycoprotein biosynthesis in activated B lymphocytes

A 10-50-fold, biphasic increase in the rate of 32Pi labeling of eIF-4E was closely correlated with the induction of protein and glycoprotein biosynthesis when resting murine splenic B lymphocytes (B cells) were activated by bacterial lipopolysaccharide or the combination of phorbol 12-myristate 13-acetate and ionomycin. The fraction of eIF-4E which was phosphorylated only increased from 46% in resting cells to 83% in lipopolysaccharide-activated cells. This discrepancy between the increase in the fraction of phosphorylated eIF-4E and the increase in 32Pi labeling suggested that the phosphoryl group of eIF-4E turns over slowly in resting B cells compared with activated cells. The turnover rate for the eIF-4E phosphate moiety in lipopolysaccharide-activated cells was rapid (t1/2 = 2 h) in comparison to the eIF-4E polypeptide chain, which did not turn over detectably in 6 h. Neither protein kinase C nor a cyclic nucleotide-dependent protein kinase appeared to be involved in eIF-4E phosphorylation in B cells, based on the observations that the metabolic labeling of eIF-4E by 32Pi was insensitive to the protein kinase inhibitors H-7 and HA1004, and that maximal labeling occurred after protein kinase C activity was "down-regulated" to very low levels in phorbol 12-myristate 13-acetate/ionomycin-activated cells. Dephosphorylation in vivo was blocked by okadaic acid (IC50 = 200 nM). These results indicate that a rapid phosphorylation-dephosphorylation of eIF-4E is associated with high translation rates during the activation of B cells, and implicate protein phosphatase-1 (or possibly-2A) in the dephosphorylation of the initiation factor.     

Synergy between Sphingosine 1-Phosphate and Lipopolysaccharide Signaling Promotes an Inflammatory,Angiogenic and Osteogenic Response in Human Aortic Valve Interstitial Cells

Given that the bioactive lipid sphingosine 1-phosphate is involved in cardiovascular pathophysiology, and since lipid accumulation and inflammation are hallmarks of calcific aortic stenosis, the role of sphingosine 1-phosphate on the pro-inflammatory/pro-osteogenic pathways in human interstitial cells from aortic and pulmonary valves was investigated. Real-time PCR showed sphingosine 1-phosphate receptor expression in aortic valve interstitial cells. Exposure of cells to sphingosine 1-phosphate induced pro-inflammatory responses characterized by interleukin-6, interleukin-8, and cyclooxygenase-2 up-regulations, as observed by ELISA and Western blot. Strikingly, cell treatment with sphingosine 1-phosphate plus lipopolysaccharide resulted in the synergistic induction of cyclooxygenase-2, and intercellular adhesion molecule 1, as well as the secretion of prostaglandin E₂, the soluble form of the intercellular adhesion molecule 1, and the pro-angiogenic factor vascular endothelial growth factor-A. Remarkably, the synergistic effect was significantly higher in aortic valve interstitial cells from stenotic than control valves, and was drastically lower in cells from pulmonary valves, which rarely undergo stenosis. siRNA and pharmacological analysis revealed the involvement of sphingosine 1-phosphate receptors 1/3 and Toll-like receptor-4, and downstream signaling through p38/MAPK, protein kinase C, and NF-κB. As regards pro-osteogenic pathways, sphingosine 1-phosphate induced calcium deposition and the expression of the calcification markers bone morphogenetic protein-2 and alkaline phosphatase, and enhanced the effect of lipopolysaccharide, an effect that was partially blocked by inhibition of sphingosine 1-phosphate receptors 3/2 signaling. In conclusion, the interplay between sphingosine 1-phosphate receptors and Toll-like receptor 4 signaling leads to a cooperative up-regulation of inflammatory, angiogenic, and osteogenic pathways in aortic valve interstitial cells that seems relevant to the pathogenesis of aortic stenosis and may allow the inception of new therapeutic approaches.     

Protein kinase C modulates actin conformation in human T lymphocytes

We studied the effect of activators and inhibitors of protein kinase C on actin conformation in human blood lymphocytes by flow cytometry and gel electrophoresis. PMA, 1-oleyl-2-acetyl-glycerol, and mezerein, activators of protein kinase C, caused an increase in lymphocyte F-actin within 2 to 5 min. After stimulation with PMA, lymphocytes formed pseudopods containing an increased concentration of F-actin and had an increase of actin in the Triton-insoluble cytoskeletal fraction. Sphingosine and H-7, inhibitors of protein kinase C activation, inhibited the increase in F-actin induced by PMA. The increase in F-actin in response to PMA was striking in Th and Ts lymphocytes (2- to 3-fold increase), but B lymphocytes had only a slight increase (1.15-fold). Thus, activation of protein kinase C modulates actin conformation specifically in T lymphocytes.     

Activation of protein kinases A and C increase lymphocyte penetration through endothelial monolayers

A brief incubation of lymphocytes with either PMA, stimulating protein kinase C, or with dibutyryl-cAMP, leading to protein kinase A activation, led to increased lymphocyte penetration through intact endothelial monolayers in vitro. The PMA-induced penetration could be dose-dependently down-regulated with a protein kinase C inhibitor, H7. Similarly HA 1004, being mainly a protein kinase A inhibitor, decreased the dibutyryl-cAMP induced penetration. Treatment of lymphocytes with PMA and cAMP did not alter the expression of CD44 homing receptors on lymphocytes. Stimulation of lymphocytes with dibutyryl-cGMP or calcium ionophore had no effect on lymphocyte penetration. These results suggest that activation of both protein kinases A and C is important in the lymphocyte binding to endothelium.     

Effects of bacterial lipopolysaccharide on the binding of lymphocytes to endothelial cell monolayers

Preincubation of human umbilical vein endothelial cell (EC) monolayers with 1 ng to 10 micrograms/ml lipopolysaccharide (LPS) increased the binding of T lymphocytes to EC. The effect was maximal at LPS concentrations of 0.1 to 10 micrograms/ml, and occurred with LPS derived from Escherichia coli (serotypes 0111:B4 and 0127:B8), Shigella flexneri (serotype 2a), Serratia marcescens (serotype 0:3), and Yersinia entercolitica (serotype 0:3). The increased binding appeared to be mediated primarily through an action on EC; preincubation of T cells rather than EC with LPS did not lead to enhanced binding. The onset of enhanced binding was very rapid, being observed after 2 to 3 min of preincubation and becoming maximal after 1 hr. EC were unresponsive to LPS after fixation with 2% paraformaldehyde-L-lysine-periodate and also when the LPS was incubated with EC at 4 degrees C. Enhanced binding was seen with lipid A and with LPS from Salmonella minnesota Re 595 (mainly lipid A) and was abolished by conjugation with polymyxin B. The observed increase in the binding of lymphocytes to EC exposed to LPS suggests that the lymphocytopenia induced by endotoxemia may result from augmentation of the adherence of lymphocytes to altered endothelium.     

Calcium- and lipid-independent protein kinase C autophosphorylation. Activation by low pH

The activity of protein kinase C is dependent on communication between a catalytic domain and a Ca2+- and lipid-binding regulatory domain in the kinase molecule. It is shown here that acidic reaction conditions can bypass the calcium and lipid requirement in the autophosphorylation of protein kinase C. Acidic pH does not entirely deregulate the kinase, though, since only autophosphorylation is favored between pH 4 and 6 and not the phosphorylation of alternative substrate proteins. Interestingly, low pH stably activated protein kinase C: when restored to neutral pH, the autophosphorylation reaction remained independent of Ca2+ and lipid. These observations suggest that protonation of functional groups in the protein kinase C molecule, with their pKa suggestive of histidine imidazole, can produce a stable conformation where regulatory constraints on enzyme activity have been removed.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.     

TLR4-dependent lipopolysaccharide-induced shedding of tumor necrosis factor receptors in mouse bone marrow granulocytes

We reported previously that bone marrow granulocytes respond to small amounts of enterobacterial lipopolysaccharide (LPS) via a CD14-independent and TLR4-mediated mechanism by de novo expression of an inducible receptor (CD14) and by down-modulation of a constitutive receptor (L-selectin). In this report we address another effect of LPS: the down-regulation of receptors for tumor necrosis factor-alpha. In mouse bone marrow cells (BMC), this down-regulation is detectable soon (20 min) after exposure of the cells to low levels (0.5 ng/ml) of LPS. This temperature-dependent effect is rather selective for LPS and requires the presence of a conventional lipid A structure in the LPS molecule and a functional TLR4 molecule in the cells. The down-modulation, due to a shedding of the receptors, is blocked by p38 MAPK inhibitors, by a furin inhibitor, and by three metalloproteinase inhibitors (BB-3103, TIMP-2, and TIMP-3). In contrast, inhibitors of MEK, protein kinase C, cAMP-dependent protein kinase, and kinases of the Src family do not block the shedding. Analysis of BMC from mice lacking tumor necrosis factor receptor-1 (CD120a-/-) or tumor necrosis factor receptor-2 (CD120b-/-) indicates that the LPS-induced shedding is specific for CD120b. Thus, exposure of BMC to LPS triggers a rapid shedding of CD120b via a protein kinase C- and Src-independent pathway mediated by p38 MAPK, furin, and metalloproteinase. The additive effects of furin and metalloproteinase inhibitors suggest that these enzymes are involved in parallel shedding pathways.     

Interaction between protein kinase C and sphingomyelin/cholesterol

Physical characteristics of binding of protein kinase C with sphingomyelin/cholesterol lipid bilayers were analysed using three complementary approaches: acrylodan fluorescence, fluorescence energy transfer and quenching of tryptophan fluorescence. It was demonstrated that sphingomyelin/cholesterol lipid membranes were available for protein kinase C binding. The intensity of the binding was dependent on the sphingomyelin content. The results of quenching of intrinsic tryptophan fluorescence showed that the enzyme molecule penetrated the sphingomyelin/cholesterol lipid bilayer to the C-16 position of labeled fatty acid probes. Our results also showed sphingomyelin itself restrains protein kinase C activity. A possible explanation for our results is that caveolae function as signaling storage devices.     

Evidence that guanine-nucleotide binding regulatory proteins couple cell-surface receptors to the breakdown of inositol-containing lipids during T-lymphocyte mitogenesis

We have studied the role of guanine-nucleotide binding regulatory proteins (G proteins) in the stimulation of inositol lipid breakdown during mitogenic activation of normal human T lymphocytes. The effect of the mitogen phytohemagglutinin (PHA) was compared with the action of two G-protein activators, fluoroaluminate (AlF4-) and guanosine-5'-O-thiotriphosphate (GTP gamma S). PHA and AlF4- stimulated the breakdown of inositol lipids via both the phospholipase A and C pathways when added to intact lymphocytes. PHA, AlF4- and GTP gamma S also triggered both these pathways when added to permeable lymphocytes. The magnitude of the response obtained with AlF4- and GTP gamma S was about four-fold less than with PHA. This difference was attributable to increases in cAMP elicited by AlF4- and GTP gamma S which inhibited the phospholipase pathways. AlF4-, GTP gamma S, and PHA all stimulated the phosphorylation of a 42 kDa protein on tyrosine residues. We propose a model for the early steps following mitogen binding, including sequential activation of a G protein, phospholipase C, protein kinase C and a tyrosine protein kinase. A parallel pathway involving G protein mediated activation of phospholipase A is also implicated.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.     

Comparison of murine B cell clonal expansions by synthetic lipid A and muramyl dipeptide analogs

Direct stimulations of murine B lymphocytes with synthetic lipid A analogs and synthetic muramyl dipeptide (MDP) derivatives were studied using a limiting dilution assay system. Synthetic lipid A analogs, GLA-27 and GLA-40, when conjugated with bovine serum albumin (BSA) had the ability to induce B cell clonal expansion of a single B cell from the spleen or bone-marrow. Their activities were almost the same as those of naturally obtained lipid A, but were lower than that of bacterial lipopolysaccharide (LPS). Addition of dextran sulfate (DXS) enhanced the effect of lipid A analogs. In contrast, synthetic MDP and its derivatives, although they had many biological and immunological activities in experimental animals, could not stimulate a single B cell to induce clonal expansion regardless of the presence or absence of DXS. These results suggested that lipid A analogs can directly cause the proliferation of B cells, but MDPs can not.     

Enhancement of B-cell stimulation by muramyl dipeptide through a mechanism not involving interleukin 1 or increased Ca2+ mobilization or protein kinase C activation

Muramyl dipeptide (MDP) enhanced mitogenic stimulation of mouse lymphocytes by polyclonal B cell activators (peptidoglycan, lipopolysaccharide, Staphylococcus aureus Cowan I cells, and pokeweed mitogen), but not by T-cell mitogens (phytohemagglutinin and concanavalin A). Only adjuvant-active MDP analogs were effective, whereas adjuvant-inactive MDP analogs, muramic acid, peptidoglycan pentapeptide, and low Mr digests of peptidoglycan were not. The half-maximal enhancement was seen at 5-10 microM MDP and occurred at both optimal and suboptimal concentrations of B cell mitogens. The enhancing effect of MDP was exerted on the B cells, since it was T cell- and macrophage-independent and was not mediated by IL-1. MDP was effective during the first 12 hrs of culture, and most strongly enhanced the mitogen-induced DNA synthesis, although significant enhancement of RNA synthesis and B cell differentiation into antibody-secreting cells was also observed. The enhancement of mitogenic response was not due to changed requirements for extracellular or intracellular Ca2+ or to increased activation of protein kinase C. These results demonstrate a novel immunoenhancing effect of MDP that should be useful in the studies on the mechanism of B cell activation.     

B cell lipid rafts regulate both peptide-dependent and peptide-independent APC-T cell interaction

Formation of an immunological synapse (IS) between APCs and T CD4(+) lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.     

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.