Caratterizzazione molecolare e funzionale degli amebociti di Ciona intestinalis nella risposta immune mediata dal sistema del complemento

Dottorato di Ricerca in Biologia Animale, XXII Ciclo,a.a.2008-2009Research over the past ten years indicates that the Complement System, the major effector arm of the vertebrate innate immunity, emerged at least 1,300 million years ago, long before the appearance of the adaptive immune response. These studies also pointed to the ascidian Ciona intestinalis (Urochordata), which occupies a key phylogenetic position in chordate evolution, as a suitable animal model for investigating the immune system evolution. In this context, two C3-like molecules, CiC3-1 and CiC3-2, exhibiting all the characteristic features of the mammalian C3s, have been previously identified, sequenced, and characterized in C. intestinalis. Furthermore, it has been found that the activation of CiC3-1 results in the release of the bioactive fragment C3-1a, a potent mediator of inflammatory reactions. The C3-1a fragment exerts its functional activity through the specific binding to a cell surface G protein-coupled seven-transmembrane receptor (CiC3aR), which has been more recently identified and characterized. Both C3-1a and its receptor are expressed only by granular and hyaline amoebocytes, two renewing blood cell types. In Ciona, blood cell proliferation occurs in circulating blood and in lymph nodules, mainly localized in the pharynx. In the present study I have first of all assessed whether Ciona C3 could represent an early molecular marker of amoebocyte proliferation in pharyngeal limph nodules. Using real-time PCR analysis I have found that CiC3-1 is constitutively expressed in the pharynx and that its expression is up-regulated by the treatment of the animals with lipopolysaccharides (LPS), a well-known inflammatory agent. By using two specific anti-C3 and anti-C3a polyclonal antibodies in immunohistological staining of pharynx sections, I have found that also the gene product is present in the pharynx, localized in areas identified as sites of hematogenic activity, and in amoebocytes occupying the pharyngeal bars. The same antibodies used in Western blot analysis indicated that CiC3-1 and its activation products, namely CiC3-1b and CiC3-1a, are present in homogenates of pharynx. The finding that the amount of the bioactive fragment CiC3-1a increases in the pharynx of LPS treated animals, clearly indicates that CiC3-1, present in the pharynx and labeling the maturing amoebocytes, is functionally active. The identification in C. intestinalis of both the partners participating in the complement-mediated inflammatory response gave me the opportunity for further investigating the C3a-C3aR interaction at both the molecular and cellular levels. In another series of experiments, I have focused my attention on cellular mechanisms acknowledged to be fundamental in ligand-receptor interaction. I have analyzed by confocal microscopy the dynamic process of internalization of the CiC3aR, induced by the binding of CiC3-1a in the form of the eighteen amino acid C-terminal peptide CiC3-1a59-76 of CiC3-1a fragment. These experiments have been performed on amoebocytes of circulating blood, which constitutively express both CiC3-1 and CiC3aR. Results of these experiments indicate that CiC3aR, following the agonist stimulus, is internalized in few minutes, and recycled to the cell surface within 30 min, made again available for CiC3-1a binding. My observations also revealed that in response to CiC3aR activation, Ciona arrestin (CiArr) is translocated to the plasma membrane in 30 sec. This localization profile is consistent with the participation of CiArr in CiC3aR internalization, and suggests, for the first time in Ciona, a role for CiArr as the agonist driven switch initiating receptor endocytosis.Università della Calabri