Marc Fontaine

Regulation by complement C3a and C5a anaphylatoxins of cytokine production in human umbilical vein endothelial cells

C3a and C5a anaphylatoxins are cytokine‐like polypeptides generated during complement (C) system activation and released at the inflammatory site. They exert several biological activities through binding to the G‐protein‐coupled receptors C3aR and C5aR, respectively. Cloning and Northern blot experiments have indicated that both receptors are expressed by myeloid as well as nonmyeloid cells (e.g., endothelial and epithelial cells). To better understand the roles of C anaphylatoxins during inflammation, we investigated their effects on the expression of cytokine and chemokine genes by cultured human umbilical cord endothelial cells (HUVEC). HUVEC constitutively expressed both anaphylatoxin receptors, and addition of physiological concentrations of C3a or C5a (nM range) caused a strong up‐regulation of IL‐8, IL‐1β, and RANTES mRNA in a time‐ and dose‐dependent manner. Conversely, a decrease in IL‐6 mRNA was observed, but only with C5a stimulation. These variations in mRNA levels were inhibited by pretreatment with anti‐C5aR and anti‐C3aR antibodies as well as pertussis toxin, indicating that G‐proteins are involved in anaphylatoxin‐activated signal transduction pathways. Finally, we showed that C3a and C5a both strongly activate downstream MAP kinase signaling pathways (p44 and p42 Erk kinases).—Monsinjon, T., Gasque, P., Chan, P., Ischenko, A., Brady, J.J., Fontaine, M. Regulation by complement C3a and C5a anaphylatoxins of cytokine production in human umbilical vein endothelial cells. FASEB J. 17, 1003–1014 (2003)

Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoked apoptotic cell death. Incubation of granule cells with PACAP prevented ethanol-induced apoptosis, and this effect was not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induced a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provoked a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevented ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome.

Expression of Receptors for Complement Anaphylatoxins C3a and C5a Following Permanent Focal Cerebral Ischemia in the Mouse

In the present study, we have examined the expression of anaphylatoxin C3a and C5a receptors (C3aR and C5aR) at the mRNA and protein levels in ischemic brain tissues following permanent middle cerebral artery (MCA) occlusion in the mouse. C3aR and C5aR mRNAs were both detected by semiquantitative reverse transcription and polymerase chain reaction (RT-PCR) and the cellular distribution of each receptor was analyzed by immunohistochemistry. Significant increases in the expression of C3aR and C5aR mRNAs in the ischemic cortex were observed; the expression of both reached a peak at 2 days after MCA occlusion (4.3- and 3.4-fold increases, respectively, compared with nonoperated control cortical samples; P < 0.00625 with Bonferronis correction, n = 3). C3aR and C5aR stainings were found constitutively on neurons and astrocytes. In ischemic tissues, we observed that C3aR and C5aR were expressed de novo on endothelial cells of blood vessels, at 6 h and 2 days after MCA occlusion, respectively. C3aR and C5aR immunostaining was increased in macrophage-like cells and reactive astrocytes 7 days postocclusion. C3a and C5a may play an important role in promoting inflammatory and/or repair processes in the ischemic brain by regulating glial cell activation and chemotaxis.

Evidence for alpha-1-acid glycoprotein populations of different pI values after concanavalin a affinity chromatography Study of their evolution during inflammation in man

Isoelectric focusing in polyacrylamide gel was used to reveal the two alpha 1-acid glycoprotein populations separated by affinity chromatography on Con A-Sepharose from human purified alpha 1-acid glycoprotein, whole normal serum and sera from patients undergoing an acute inflammatory process. The concanavalin A non-reactive peak had a lower isoelectric point than did the concanavalin A reactive peak. Moreover, crossed immunoaffinity electrophoresis with free concanavalin A revealed three alpha 1-acid glycoprotein populations (concanavalin A reactive, weakly and non-reactive). A significant difference was observed between the patterns of normal and of inflammatory sera, since the concanavalin A reactive fraction was enhanced during the inflammatory process. These results suggest a variation in relative ratios of the different types of peripheral oligosaccharide branches in the alpha 1-acid glycoprotein populations.

Pituitary adenylate cyclase-activating polypeptide prevents C2-ceramide-induced apoptosis of cerebellar granule cells

The sphingolipid metabolites, ceramides, are critical mediators of the cellular stress response and play an important role in the control of programmed cell death. In particular, ceramides have been shown to induce apoptosis of cerebellar granule cells. We show that pituitary adenylate cyclase‐activating polypeptide (PACAP) prevents C2‐ceramide‐induced apoptosis. The neuroprotective effect of PACAP was dose‐dependent and blocked by its antagonist, PACAP6‐38, whereas the PACAP‐related peptide VIP was inactive. The effect of PACAP on cell survival was mimicked by dibutyryl‐cAMP (dbcAMP) and forskolin and prevented by the MEK inhibitor U0126, indicating that both the adenylyl‐cyclase and MAP‐kinase pathways contribute to the neuroprotective action of the peptide. C2‐ceramide and PACAP induced opposite effects on phosphorylated forms of ERK and JNK without affecting the total amounts of ERK and JNK, suggesting that a balance between these two MAP‐kinases is critical for the cell survival/death decision. The effect of PACAP on ERK phosphorylation was blocked by U0126, but was not affected by H89 or chelerythrine indicating that PACAP activates ERK through a PKA‐ and PKC‐independent mechanism. C2‐ceramide induced a time‐dependent activation of caspase‐3, enhanced the amount of cleaved caspase‐3 and stimulated the DNA fragmentation process, while PACAP strongly inhibited the C2‐ceramide‐induced activation of caspase‐3, reduced the expression of cleaved caspase‐3 and blocked DNA fragmentation. Taken together, the present results show that C2‐ceramide induces apoptosis of cerebellar granule cells through a mechanism involving activation of caspase‐3. Our data also demonstrate that PACAP is a potent inhibitor of C2‐ceramide‐induced apoptosis.

Complement anaphylatoxin C3a is selectively protective against NMDA-induced neuronal cell death.

The anaphylatoxin C3a is a potent inflammatory polypeptide released at sites of complement activation. To test whether C3a might alter neuronal outcome following an ischemic insult, we determined the effects of purified human C3a on murine primary cortical cell cultures exposed to apoptotic or excitotoxic paradigms. C3a prevented neither serum deprivation-induced apoptotic neuronal death, nor AMPA/kainate-mediated excitotoxicity. However, in mixed cultures of neurons and astrocytes, C3a dose-dependently protected neurons against NMDA toxicity (47% neuroprotection using 100 nM C3a, p < 0.01, n = 12). The neuroprotective effect of C3a was observable only in the presence of astrocytes. These observations suggest that C3a is involved in excitotoxicity-mediated neuronal death through astrocyte stimulation and extend its role beyond immune functions.

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells

The sphingomyelin‐derived messenger ceramides provoke neuronal apoptosis through caspase‐3 activation, while the neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase‐3 activity. However, the mechanisms leading to the opposite regulation of caspase‐3 by C2‐ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2‐ceramide‐induced inhibition of mitochondrial potential and C2‐ceramide‐evoked cytochrome c release. C2‐ceramide stimulated Bax expression, but had no effect on Bcl‐2, while PACAP abrogated the action of C2‐ceramide on Bax and stimulated Bcl‐2 expression. The effects of C2‐ceramide and PACAP on Bax and Bcl‐2 were blocked, respectively, by the JNK inhibitor L‐JNKI1 and the MEK inhibitor U0126. L‐JNKI1 prevented the alteration of mitochondria induced by C2‐ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2‐ceramide on mitochondrial potential. Moreover, L‐JNKI1 inhibited the stimulatory effect of C2‐ceramide on caspase‐9 and ‐3 and prevented C2‐ceramide‐induced cell death. U0126 blocked PACAP‐induced Bcl‐2 expression, abrogated the inhibitory effect of PACAP on ceramide‐induced caspase‐9 activity, and promoted granule cell death. The present study reveals that C2‐ceramide and PACAP exert opposite effects on Bax and Bcl‐2 through, respectively, JNK‐ and ERK‐dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro‐ and anti‐apoptotic effects of C2‐ceramide and PACAP.

Glial responses, clusterin, and complement in permanent focal cerebral ischemia in the mouse

There is considerable evidence that complement activation occurs within the CNS in inflammatory and degenerative disorders, but little is known about its involvement in the pathophysiology of cerebral ischemia. Our study sought to characterize the glial response and the expression of complement factors after permanent focal cerebral ischemia in the mouse, using semiquantitative reverse transcription‐polymerase chain reaction (RT‐PCR), in situ hybridization, and immunohistochemistry. mRNA expression of glial fibrillary acidic protein (GFAP) increased at day 1 and peaked 3 days after middle cerebral artery (MCA) occlusion in the perifocal area. Immunohistochemical staining for GFAP indicated that astroglia were activated the day after MCA occlusion. Microglial activation, as assessed by lectin‐binding experiments, increased by 1 day after MCA occlusion in the perifocal area and peaked at 3 days postocclusion. RT‐PCR experiments demonstrated an increased expression of clusterin, C1qB, and C4 mRNA in the ischemic cortex, with a peak level at 3 days after MCA occlusion. Clusterin, C1qB, and C4 mRNA were located in the perifocal area, as assessed by in situ hybridization. Reactive astrocytes within the cortex medial to the ischemic lesion were found to be strongly immunoreactive for clusterin. In addition, we observed C1q‐positive macrophage‐like cells within the infarcted core at 3 days postocclusion. At 7 days after the onset of ischemia, increased C4 immunostaining was restricted to perifocal neurons. We conclude that local expression of complement components may contribute to the inflammation observed in this model, thereby representing an important process in secondary injury mechanisms after focal cerebral ischemia. GLIA 31:39–50, 2000.

Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins

Two human neuroblastoma cell lines activated the classical pathway of complement in serum. Activation caused the opsonisation of these cells with complement fragments but with moderate cell killing. Neuroblastoma expressed regulators MCP and CD59 but did not express DAF or CR1. Neutralisation of CD59 rendered the cells susceptible to killing. Neuroblastoma also expressed C1-inhibitor, factor H, clusterin and S-protein. Expression of several regulators was enhanced by incubation with cytokines. Complement inhibition using soluble CRI markedly reduced opsonisation and killing of neuroblastoma. Our results suggest that complement might play a role in neuronal loss and that treatment with complement inhibitors might be of therapeutic value.

Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression.

Abstract: C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement activation that act through distinct Gi protein‐coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐α, and transforming growth factor‐β mRNA expression was studied by quantitative RT‐PCR. Whereas IL‐1β, tumor necrosis factor‐α, and transforming growth factor‐β mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL‐6 mRNA level. The amount of IL‐6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti‐C3aR or anti‐C5aR antibodies completely blocked the IL‐6 mRNA increase. The IL‐6 response was also investigated at the protein level, but IL‐6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin‐mediated transcriptional activation of IL‐6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.