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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.

Glucocorticoids profoundly influence the immune system and pharmacological doses exert potent anti-inflammatory actions. During inflammation, glucocorticoids limit oedema and influence cell trafficking, differentiation programmes and gene transcription in glucocorticoid-sensitive leukocytes. Within cells, glucocorticoid action is modulated by a pre-receptor mechanism; glucocorticoid metabolism by the enzyme 11β- hydroxysteroid dehydrogenase (11β-HSD). Two 11β-HSD isozymes exist: 11β-HSD1, which catalyses amplification of glucocorticoid levels in intact cells by oxo-reduction of intrinsically inert cortisone (11-dehydrocorticosterone in rodents) into active cortisol (corticosterone in rodents) and 11β-HSD2, which performs the opposite reaction. Thus, amplification of intracellular glucocorticoid levels by 11β-HSD1 may represent an endogenous anti-inflammatory mechanism. This hypothesis has been tested in Hsd11b1-/- mice (homozygous for a targeted disruption in the Hsd11b1 gene, encoding 11β-HSD1), using carageenan-induced pleurisy and experimental model of arthritis induced by injection of arthritogenic antibodies. In both models, Hsd11b1-/- mice showed more severe acute inflammation than control mice. During carrageenan-induced pleurisy, Hsd11b1-/- mice recruited more inflammatory cells to the pleural cavity than congenic controls, with a greater proportion of viable cells, at the onset and peak of pleurisy, suggesting a worse inflammatory response. Histological examination suggested impaired resolution of inflammation in Hsd11b1-/- mice with persistence of inflammation in the visceral pleura, activation of lymphoid aggregates, and uniquely in Hsd11b1-/- mice, formation of fibrous adhesions between lung lobes 48h after initiation of pleurisy. During experimental arthritis induced by injection of serum from arthritic K/BxN mice, clinical signs of inflammation occurred earlier in Hsd11b1-/- mice and were slower to resolve than in control mice. Histological assessment of the acute phase (2d) of arthritis showed no difference in joint pathology between genotypes, despite greater oedema and higher clinical scores in the Hsd11b1-/- mice. However, when the inflammation had resolved (21d following injection of serum), compared to control mice, Hsd11b1-/- mice showed more severe exostosis, intense periarticular inflammation, more collagen deposition and uniquely, ganglion cyst formation. At 21d, whereas basal (morning) plasma corticosterone levels were normal in control mice, they remained elevated in Hsd11b1-/- mice, suggesting ongoing inflammation and persistent activation of the hypothalamic-pituitary-adrenal axis. Mast cells are critical in the initiation of an inflammatory response and are essential in this model of arthritis. Mast cells expressed 11β-HSD1 (but not 11β-HSD2) mRNA and activity. Although mast cell number did not differ in joints or peritoneum of Hsd11b1-/- mice, 11-HSD1-deficient mast cells had a lower threshold for degranulation induced by K/BxN arthritogenic serum. As well as implicating a role for mast cell 11β-HSD1 in limiting initial inflammation in arthritis, these findings also have implications for infection, allergy and tolerance. Collectively, these data suggest that 11β-HSD1 deficiency worsens acute inflammation and results in slower resolution. Therefore, amplification of intracellular glucocorticoids levels, by 11β-HSD1, may represent an important mechanism to limit the acute inflammatory response and programme its subsequent resolution. Increasing leukocyte 11β-HSD1 or local delivery of substrate affords a novel approach for anti-inflammatory therapy.

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