CD31 mediates homophilic interactions between leukocytes and macrophages during inflammation, apoptotic cells remain attached and are engulfed whereas viable cells actively detach. We hypothesised that differential recruitment of signalling and adapter molecules to the cytoplasmic domain were responsible for the disengagement of the viable leukocyte from macrophages. Investigation with a static attachment assay using THP-1 as a macrophage model showed the ITIM of CD31 on the leukocyte was important for viable cell detachment. Our data also implicated a role for the recruitment of SHP-2 which we attempted to knock-down by siRNA delivered by lentivirus. SHP-2, in addition to its phosphatase activity, also acts as a docking protein. To examine for potential interacting partners we fused the cytoplasmic domain of CD31 to GST which we used in pulldown assays from lysates of viable and apoptotic leukocytes. We demonstrated that the recruitment of SHP-1 and SHP-2 were dependent on an intact ITIM (immunoreceptor tyrosine-based inhibitory motif). Interestingly, apoptotic cell lysates promoted dephosphorylation of the in vitro phosphorylated GST-CD31, suggesting an increase in phosphatase activity in aged neutrophils. We were unable to demonstrate an interaction between the cytoplasmic domain of CD31 with putative binding partners β-catenin, src, RasGAPp120, RhoGAPp190, talin or calmodulin. A proteomic approach by MALDI-TOF and MS/MS identified Hsp90 as a novel binding partner of CD31 irrespective of the phosphorylation state. In contrast, 14-3-3ε bound to phosphorylated CD31, whereas eIF3 specifically bound to an ITIM double tyrosine mutant. The binding of Hsp90 to CD31 was proposed to occur via a TPR motif within the cytoplasmic domain of CD31 which comprises a surface fold of basic amino acids complexing a highly acidic carboxy tail of Hsp90. Truncation and site directed mutagenesis of the cytoplasmic domain revealed multiple binding sites for Hsp90; specifically two regions containing the sequences (KAFYLRKAKAK), previously shown to be the calmodulin binding region, and a novel area (SNNEKMSDMEANSHY) which has significant homology with other TPR-containing proteins. Systematic mutagenesis of the putative basic charged amino acids within the cytoplasmic domain of CD31 which may mediate the interaction with Hsp90 also supports the presence of a TPR motif. The importance of Hsp90, 14-3-3ε and eIF3 is currently unknown, although it is interesting to note that CD31 was recently found within this laboratory to associate with the voltage-gated potassium channel HERG which also binds 14-3-3ε and Hsp90. eIF3 is an RNA helicase that may link CD31 and leukocyte motility to spreading initiation centres where motility can be viewed as a rapid turnover of focal adhesion complexes. Together, these studies have identified novel binding partners of CD31 may form a macromolecular complex to promote CD31-dependent leukocyte motility and detachment from macrophages.

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system initiates tissue repair following injury. In response to sterile tissue injury, neutrophils infiltrate the tissue to remove tissue debris and subsequently undergo apoptosis. Proper clearance of apoptotic neutrophils in the tissue by recruited macrophages, in a process termed efferocytosis, is critical to facilitate the resolution of inflammation and tissue repair. However, the events leading to suppression of sterile inflammation following efferocytosis, and the contribution of other innate cell types are not clearly defined in an in vivo setting. Using a sterile mouse peritonitis model, we identified IL-4 production from efferocytosing macrophages in the peritoneum that activate invariant NKT cells to produce cytokines including IL-4 and IL-13. Importantly, IL-4 from macrophages functions in autocrine and paracrine circuits to promote alternative activation of peritoneal exudate macrophages and augment type-2 cytokine production from NKT cells to suppress inflammation. The increased peritonitis in mice deficient in IL-4, NKT cells, or IL-4Ra expression on myeloid cells suggested that each is a key component for resolution of sterile inflammation. The phagocyte NADPH oxidase, a multi-subunit enzyme complex we demonstrated to require a physical interaction between the Rac GTPase and the oxidase subunit gp91phox for generation of reactive oxygen species (ROS), is required for production of ROS within macrophage phagosomes containing ingested apoptotic cells. In mice with X-linked chronic granulomatous disease (X-CGD) that lack gp91phox, efferocytosing macrophages were unable to produce ROS and were defective in activating iNKT during sterile peritonitis, resulting in enhanced and prolonged inflammation. Thus, efferocytosis-induced IL-4 production and activation of IL-4-producing iNKT cells by macrophages are immunomodulatory events in an innate immune circuit required to resolve sterile inflammation and promote tissue repair.

Indiana University-Purdue University Indianapolis (IUPUI) / More than 300 million people world-wide suffer from breathlessness, wheezing, chest tightness, and coughing characteristic of chronic bronchial asthma, the global incidence of which is on the rise. Allergen-sensitization and challenge elicits pulmonary expression of chemoattractants that promote a chronic eosinophil-rich infiltrate. Eosinophils are increasingly recognized as important myeloid effectors in chronic inflammation characteristic of asthma, although few eosinophil molecular signaling pathways have successfully been targeted in asthma therapy. p21 activated kinases (PAKs), members of the Ste-20 family of serine/threonine kinases, act as molecular switches in cytoskeletal-dependent processes involved in cellular motility. We hypothesized that PAK1 modulated eosinophil infiltration in an allergic airway disease (AAD) murine model. In this model, Pak1 deficient mice developed reduced inflammatory AAD responses in vivo with notable decreases in eosinophil infiltration in the lungs and broncho-alveolar lavage fluids (BALF). To test the importance of PAK1 in hematopoietic cells in AAD we used complementary bone marrow transplant experiments that demonstrated decreased eosinophil inflammation in hosts transplanted with Pak1 deficient bone marrow. In in vitro studies, we show that eotaxin-signaling through PAK1 facilitated eotaxin-mediated eosinophil migration. Ablating PAK1 expression by genetic deletion in hematopoietic progenitors or siRNA treatment in derived human eosinophils impaired eotaxin-mediated eosinophil migration, while ectopic PAK1 expression promoted this migration. Together these data suggest a key role for PAK1 in the development of atopic eosinophil inflammation and eotaxin-mediated eosinophil migration.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic inflammatory disorders are characterized by an increase in excitability of small diameter sensory neurons located in dorsal root ganglia (DRGs). This sensitization of neurons is a mechanism for chronic inflammatory pain and available therapies have poor efficacy and severe adverse effects when used chronically. Prostaglandin E₂ (PGE₂) is an inflammatory mediator that plays an important role in sensitization by activating G-protein coupled receptors (GPCRs) known as E-series prostaglandin receptors (EPs) coupled to the protein kinase A (PKA) pathway. EPs are known to downregulate upon prolonged exposure to PGE₂ or in chronic inflammation, however, sensitization persists and the mechanism for this is unknown. I hypothesized that persistence of PGE₂-induced hypersensitivity is associated with a switch in signaling caused by prolonged exposure to PGE₂ or the neurotrophin nerve growth factor (NGF), also a crucial inflammatory mediator. DRG cultures grown in the presence or absence of either PGE₂ or NGF were used to study whether re-exposure to the eicosanoid is able to cause sensitization and activate PKA. When cultures were grown in the presence of NGF, PGE₂-induced sensitization was not attenuated by inhibitors of PKA. Activation of PKA by PGE₂ was similar in DRG cultures grown in the presence or absence of NGF when phosphatase inhibitors were added to the lysis and assay buffers, but significantly less in cultures grown in the presence of NGF when phosphatase inhibitors were not added. In DRG cultures exposed to PGE₂ for 12 hours-5 days, sensitization after re-exposure to PGE₂ is maintained and resistant to PKA inhibition. Prolonged exposure to the eicosanoid caused complete loss of PKA activation after PGE₂ re-exposure. This desensitization was homologous, time dependent, reversible, and insurmountable by a higher concentration of PGE₂. Desensitization was attenuated by reduction of expression of G-protein receptor kinase 2 and was not mediated by PKA or protein kinase C. The presented work provides evidence for persistence of sensitization by PGE₂ as well as switch from the signaling pathway mediating this sensitization after long-term exposure to NFG or PGE₂.