Philippe Chan

Interleukin-1β and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes

C3a and C5a anaphylatoxins are proinflammatory polypeptides released during complement activation. They exert their biological activities through interaction with two G protein-coupled receptors named C3aR and C5aR, respectively. In the brain, these receptors are expressed on glial cells, and some recent data have suggested that anaphylatoxins could mediate neuroprotection. In this study, we used RT-PCR and ribonuclease protection assays (RPA) to investigate the role of anaphylatoxins on neurotrophin expression by the human glioblastoma cell line T98G and by rat astrocytes. Our data show that for both cell types, anaphylatoxins upregulate expression of NGF mRNA. This response depended on a G protein-coupled pathway since pre-treatment of cells with pertussis toxin (PTX) completely blocked NGF mRNA increases. This effect was anaphylatoxin-specific since pre-incubation with anti-C3a or anti-C5aR antibodies abolished the effects of C3a and C5a, respectively. The regulation of NGF mRNA by anaphylatoxins was not accompanied by translation into protein expression, but there was a significant synergic effect of anaphylatoxins/IL-1b costimulation. Our demonstration of involvement of anaphylatoxins in the NGF release process by astrocytes suggests that C3a and C5a could modulate neuronal survival in the CNS.

Complement component anaphylatoxins upregulate chemokine expression by human astrocytes

The complement (C) system, a major component of the innate immune system, has been described as a factor implicated in some brain disorders. C activation leads to the release of anaphylatoxins, two proinflammatory polypeptides acting through specific receptors that have been detected on brain cells. Here, we examined the effect of anaphylatoxins on chemokine expression by human astrocytes. We showed that anaphylatoxins significantly increase chemokine mRNA expression. However, anaphylatoxin‐induced chemokine secretion (interleukin‐8) was observed only in the presence of interleukin‐1β. Thus, anaphylatoxins could initiate a chemokine cascade and, at least in part, be involved in pathogenesis of the brain.

Characterization of rat C5a anaphylatoxin receptor (C5aR): cloning of rat C5aR cDNA and study of C5aR expression by rat astrocytes

Complement system activation within the central nervous system (CNS) is involved in demyelinating and neurodegenerative disorders, but the role of complement in the pathogenic process or in the repair remains unclear. Besides the direct lytic effects of complement on target cells (oligodendrocytes or neurons), complement can exert other functions through interaction of complement fragments with specific receptors. The C5a anaphylatoxin, an inflammatory peptide which is formed during complement activation, might play a role in the CNS pathogenesis, and activation and recruitment of glial cells by binding to its receptor (C5aR) on CNS cells. Using degenerate primers corresponding to homologous regions between human and mouse C5aR cDNAs, we have cloned a rat C5aR cDNA probe from rat monocytes RNAs after RT-PCR experiment. The rat C5aR probe isolated by this procedure allowed us to clone the rat C5aR cDNA-coding sequence using a library screening cloning strategy. This probe was also used to study the expression of the C5aR mRNA in the rat CNS. Northern blotting and RT-PCR experiments demonstrated the constitutive expression of C5aR mRNA in brain, spleen, kidney and lung. This transcript was also observed in primary culture of rat astrocytes. Microfluorimetry experiments demonstrated that C5aR expressed by astrocytes in culture is functional since the addition of C5a induced a dose-dependent increase of intracellular calcium concentration. The expression of the C5aR by astrocytes suggests new roles for the C5a anaphylatoxin in reactive astrogliosis to CNS injuries.

Mouse α-1-microglobulin/bikunin precursor: cDNA analysis, gene evolution and physical assignment of the gene next to the orosomucoid locus

alpha-1-Microglobulin (A1m) is a member of the lipocalin superfamily whereas bikunin is a Kunitz-type proteinase inhibitor. A1m and bikunin originate from a shared precursor. A comparison of mammalian cDNAs for the precursor indicates a highly conserved amino acid sequence along with muridae-specific deletions in both A1m and bikunin. In rodents, the gene for this precursor is less than 300 kb apart from the orosomucoid gene, another lipocalin gene. This precursor likely results from the assembly of two lipocalin and Kunitz-type genes, between 270 and 80 million years ago.

Expression of clusterin and C4 mRNA during rat peripheral nerve regeneration

The complement system (C) is a major piece of the inflammatory processes triggered after tissue injury. Since implication of the C has been demonstrated during neurodegeneration and Wallerian degeneration, without being clearly explained, we investigated the expression of C4 and clusterin mRNA, at the lesion site, after rat sciatic nerve crush injury. This pilot study was then realized over 28 days during peripheral nerve regeneration. We determined mRNA expression levels in naive control animals (N) and 2, 7, 14 and 28 days (D) after crush experiment, by using semi-quantitative RT-PCR. We observed a basic constitutive expression of both mRNAs in group N. Clusterin mRNA level increased between D2 and D7 to reach 2.5-fold the basic level of expression (N) at D7 and D14, and slightly decreased until D28. C4 mRNA underwent a rapid and marked increase and represented 2-3-fold the N level from D2 to D14, then it decreased until D28 to return to the basic level of expression (N). These preliminary data exhibit very interesting individual variations in mRNA expression and show that a peripheral nerve trauma can stimulate the expression of C4 and clusterin mRNA at the lesion site.

C3d Binding to the Myelin Oligodendrocyte Glycoprotein Results in an Exacerbated Experimental Autoimmune Encephalomyelitis

The complement system is known to contribute to demyelination in multiple sclerosis and experimental autoimmune encephalomyelitis. However, there are few data concerning the natural adjuvant effect of C3d on the humoral response when it binds to myelin Ags. This study addresses the effect of C3d binding to the myelin oligodendrocyte glycoprotein (MOG) in the induction of experimental autoimmune encephalomyelitis in C57BL/6J mice. Immunization with human MOG coupled to C3d was found to accelerate the appearance of clinical signs of the disease and to enhance its severity compared with MOG-immunized mice. This finding was correlated with an increased infiltration of leukocytes into the central nervous system accompanied by increased complement activation and associated with areas of demyelination and axonal loss. Furthermore, B cell participation in the pathogenesis of the disease was determined by their increased capacity to act as APCs and to form germinal centers. Consistent with this, the production of MOG-specific Abs was found to be enhanced following MOG/C3d immunization. These results suggest that binding of C3d to self-Ags could increase the severity of an autoimmune disease by enhancing the adaptive autoimmune response.