Main results

 

The Laboratory of Immunopharmacology is essentially focused on the characterization of the long pentraxin PTX3, cloned by this group in the early 80’s. PTX3 is a multimeric protein belonging to the pentraxin family and first member of the long pentraxin subfamily; it shares homology in its C terminal portion to the prototypic pentraxin C reactive protein (CRP), but it is distinct from CRP for the presence of a long N-terminal domain. In addition PTX3 differs from CRP for gene organization, cellular source and ligand–binding properties. Microbial recognition and inflammatory signals (IL-1 and TNF) induce PTX3 production by different cell types (i.e. monocytes, dendritic cells, fibroblasts, epithelial and endothelial cells). Structural analysis and gene-modified models have provided new insight on the biological role of PTX3. Data collected so far indicate that PTX3 is a multifunctional protein at the crossroad between immunity, inflammation, extracellular matrices (ECM) construction and female fertility.

The Laboratory of Immunopharmacology has participated to the development of original tools (recombinant protein and its domains; monoclonal and polyclonal antibodies) and since the identification of the protein it has been working on the characterization of the ligand binding properties of PTX3 and on the biological significance of such interactions. It participates to the identification of different PTX3 ligands, including microbial components (Jeannin et al., Immunity 2005, 22, 55), complement components (Bottazzi et al., JBC 1997, 272, 32817; Deban et al., J. Immunol. 2008, 181, 8423), ECM components (Salustri et al., Development 2004, 131, 1577; Scarchilli et al., JBC 2007, 282, 30161) and angiogenic factors (Rusnati et al., Blood 2004, 104, 92; Camozzi et al., JBC 2006, 281, 22605). In strict collaboration with the Laboratory directed by Cecilia Garlanda, the group has contributed to define the role of PTX3 in the context of innate immune response to pathogens (Garlanda et al., Nature 2002, 420, 182; Reading et al., J. Immunol. 2008, 180, 3391; Jeannin et al., Immunity 2005, 22, 55) In addition, in collaboration with industrial and academic partners, the Laboratory is involved in the characterization of the structural features of PTX3 (Inforzato et al., JBC 2008,283, 10147). Finally efforts are conducted to validate the use of PTX3 as diagnostic and prognostic marker in human pathology.

 

The most recent results obtained by the Laboratory include in particular:

1.  Interaction of PTX3 with microbes: in collaboration with Patrick Reading (Melbourne – Australia) we have investigated the role of PTX3 in Influenza Virus (IV) infection. We observed that PTX3 binds viral hemoagglutinin through sialylated ligands present on PTX3 molecule. Thanks to this interaction PTX3 acts as a potent  inhibitor of IV, neutralizing virus infectivity in vitro and protecting models in vivo (Reading et al., J. Immunol. 2008, 180, 3391).

2.   Role of PTX3 on complement activation: it has been already shown that PTX3 could affect the classical pathway of complement activation through interaction with C1q. Moreover we recently identify Factor H (FH), the most important inhibitor of the alternative pathway of complement activation, as a new ligand of PTX3. The data obtained so far suggest that surface-bound PTX3 can localize FH modulating the alternative pathway of complement activation and preventing excessive inflammatory response to tissue injury (Deban et al., J. Immunol. 2008, 181, 8423 ).

 

Main objectives and research lines

 

The Laboratory is involved in the characterization of the prototypic long pentraxin PTX3, in strict collaboration with the Laboratory of Cecilia Garlanda and with external collaborators. In general, by studying PTX3 we expect to learn lessons on the function of humoral innate immunity and its interplay with the cellular arm of innate immunity. The laboratory will focus on the following aspects of the biology of this protein:

 

Define the repertoire of PTX3 ligands : a common feature of all pentraxins is the capacity to interact with several ligands. The spectrum of ligands recognized by PTX3 will be expanded, focusing in particular on microbial moieties. Prompted by encouraging preliminary data we will characterize components of meningococcus type B (Men B) recognized by PTX3, taking advantage of the collaboration with Novartis-Vaccine.

 

Identify PTX3 receptor(s) on myeloid cells: cells of the myeloid lineage present a binding site for PTX3. Several evidences indicate Fcgamma as putative pentraxin receptors, however they do not fully account for cellular binding sites. Genetic approach (Fcgamma receptor deficient models) will be used to evaluate the involvement of Fcgamma as PTX3 receptors. To identify other possible candidates different strategies will be followed, including screening of a cDNA library from a experimental model's macrophage cell line, generation of blocking antibodies and proteomic approaches.

 

Define the role of PTX3 in inflammation : Pentraxins have long been known to regulate inflammatory reactions, however the molecular basis for this effect are not completely defined. We unexpectedly found that PTX3 binds to P-selectin. The interaction of PTX3 with P-selectin will be characterized in terms of mapping of domains involved and biochemical requirements. The role of this interaction in the modulation of cell extravasation will be evaluated in in vivo models of inflammation.

 

Define the interplay with complement: PTX3 binds C1q and activate the classical pathway of complement activation. In addition it interacts with Factor H, the most important inhibitor of the alternative pathway. Finally, in a first set of “fishing” experiments performed on human serum, we have surprisingly found that PTX3 can interact with members of the Ficolin family, possibly affecting the lectin pathway of complement activation. Thus PTX3 could exert a unique regulatory function on complement activation by affecting the classic, alternative and lectin pathways and further experiments will be performed to better define the function of this protein in the modulation of complement activation

 

Define the relative importance of different cellular sources : it was unexpectedly observed that lymphatic endothelial cells (LEC) constitutively express PTX3 and that PTX3 accumulates selectively around lymphatic vessels. Recent results demonstrate that the perilymphatic ECM likely plays a key role in the function of these vessels. Given the role of PTX3 as integral component of ECM, we will evaluate whether PTX3 could confer viscoelastic properties to the perilymphatic ECM, hence playing a role in fluid and cell traffic at sites of inflammation.

 

PTX3 as marker of pathology : the development of specific reagents for the identification of PTX3 in biological fluids allows the measurement of PTX3 levels in humans. PTX3 levels are increased in different pathological conditions with an inflammatory or infectious origin and in some context correlate with severity of disease. In collaboration with different clinical groups from Istituto Clinico Humanitas and from other hospital, efforts are conducted to validate the use of PTX3 as early marker and prognostic factor for different human pathologies.

 

Provisional of reagents : evaluation of PTX3 levels in biological fluids are made possible by an ELISA assay developed on the basis of our original reagents. The Laboratory is in charge for the purification of human and models' recombinant PTX3, human N- and C- terminal PTX3 domains, polyclonal and monoclonal antibodies against both human and models' protein. Purified reagents are used in our day-to-day activity, both in in vitro and in vivo assays,  and are distributed on a collaborative basis to various external groups.