Browsing by Author "Pinto, Maria Rosaria"

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Caratterizzazione molecolare e funzionale degli amebociti di Ciona intestinalis nella risposta immune mediata dal sistema del complemento
(2014-03-25) Giacomelli, Stefano; Canonaco, Marcello; De Santis, Rosaria; Pinto, Maria Rosaria
Research 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.
Pathway infiammatorio del Sistema del Complemento nelle ascidie: sequenziamento e caratterizzazione funzionale del recettore dell’anafilatossina C3a di Ciona intestinalis
(2014-03-26) Melillo, Daniela; Tota, Bruno; Panno, Maria Luisa; Pinto, Maria Rosaria
In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, a C3a-mediated chemotaxis of hemocytes has been demonstrated in the deuterostome invertebrate Ciona intestinalis and an important role for this molecule in inflammatory processes has been suggested. In this study, we have cloned and characterized the CiC3aR molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence of CiC3aR, encoding a 95,394 Da seventransmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent, in the ovaries, hemocytes, and larvae. Three polyclonal antibodies raised against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop, react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific antibodies revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the antibodies against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis.expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process