Inhibitory Functions of PECAM-1: Regulation by Sequential ITIM Phosphorylation and Consequences for Recovery from Infectious Disease

Platelet endothelial cell adhesion molecule (PECAM-1) is a type I transmembrane glycoprotein constitutively expressed on the surfaces of endothelial cells and the majority of hematopoietic cells. The goal of this dissertation was to determine whether the inhibitory functions of PECAM-1 play a role in protection from acute infections and to better understand the mechanism that regulates initiation of PECAM-1 inhibitory signaling during thrombosis and inflammation.;Specific Aim 1: Determine the effect of PECAM-1 expression on the lethality associated with acute infection. We tested whether the lessened immune response of PECAM-1 expressing mice would be beneficial or detrimental when challenged with an infectious stimulus. To evaluate PECAM-1’s role in resolution of acute inflammation due to infection, WT and PECAM-1 deficient mice were subjected to two different types of bacterial infections or to Sendai virus (SeV), which primarily elicit innate and adaptive immune responses, respectively. Following challenge with an intrapertioneal injection of E. coli, WT mice exhibited increased survival compared to their PECAM-1 deficient counterparts similar to results obtained with non-infectious inflammatory models. However, PECAM-1 deficiency had no effect on the mortality or recovery of mice that were challenged with either SeV or colon ascendens stent peritonitis (CASP). These data show that PECAM-1 is not protective against the lethal effects of all acute infections; rather, PECAM-1 plays a pathogen-specific role in protecting mice from the lethal effects of E. coli infection.;Specific Aim 2: Determine the mechanism that regulates initiation of PECAM-1 signaling. The objective of this aim was to test the hypothesis that PECAM-1 ITIM phosphorylation occurs in a two-step process, wherein initial phosphorylation of Y686 by a SFK creates a docking site for another SH2 domain-containing non-receptor tyrosine kinase (NRTK) that, once docked, is maintained in a position to allow for phosphorylation of Y663. To test this hypothesis, we evaluated the ability of three enzymes known to participate in platelet activation by a PECAM-1-regulated ITAM-coupled receptor to phosphorylate the two PECAM-1 ITIMs. Enzymes that were evaluated included the SFK Lyn, Bruton’s tyrosine kinase (Btk), and C-terminal Src kinase (Csk). In this study we found that only the SFK Lyn was able to efficiently phosphorylate the C-terminal ITIM of PECAM-1 (Y686) in vitro. Furthermore, we found that pY686 enhanced the ability of Lyn, Btk, and Csk to phosphorylate Y663 in vitro, but only when both ITIMs were linked within a contiguous polypeptide. However, Btk and Csk were more efficient than Lyn at this enhanced phosphorylation. In addition, the disruption of SH2 domain-dependent binding of Csk to pY 686 significantly reduced its capacity to phosphorylate Y663. These data support a two-step, two-enzyme mechanism for PECAM-1 ITIM phosphorylation in which initial phosphorylation of Y686 by Lyn facilitates recruitment and activation of Csk via its SH2 domain and Btk via an unidentified mechanism, allowing them to phosphorylate Y663 and initiate inhibitory signaling.;Previous non-infectious inflammatory model studies ordained PECAM-1 as an important regulator of inflammation. The studies performed in Aim 1 of this thesis determined that, in the context of acute infectious disease, PECAM-1 is a pathogenspecific rather than a global regulator of inflammation in that it regulates responses to infection with the Gram-negative bacteria E. coli, but not to infection with a virus or with multiple bacteria as is the case with CASP. Furthermore, the anti-inflammatory effects of PECAM-1 do not impair the ability of an infected host to clear acute infections. These results suggest that a therapy designed to augment PECAM-1’s functions could be used to dampen responses to non-infectious chronic inflammatory stimuli without making the host more susceptible to the lethal effects of infectious agents. Aim 2 of this thesis provided a novel mechanistic insight into the phosphorylation of PECAM-1 ITIMs. In contrast to the current paradigm of ITIM phosphorylation in which SFKs phosphorylate both ITIMs, these studies have demonstrated that the ITIMs of PECAM-1 are phosphorylated by multiple kinases in a sequential manner. (Abstract shortened by UMI.).