During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept is strengthened by the fact that activated monocytic cells adhere to immobilized recombinant CD13. Furthermore, treatment with anti-CD13 antibodies in a murine model of peritonitis results in a decrease in leukocyte infiltration into the peritoneum, suggesting a potential role for CD13 in leukocyte trafficking in vivo. Therefore, this work supports a new direction for CD13 biology, where these cell surface molecules act as true molecular interfaces that induce and participate in critical inflammatory cell interactions.

CD13/aminopeptidase N is a transmembrane peptidase that is induced in the vasculature of solid tumors and is a potent angiogenic regulator. Here, we demonstrate that CD13 controls endothelial cell invasion in response to the serum peptide bradykinin by facilitating signal transduction at the level of the plasma membrane. Inhibition of CD13 abrogates bradykinin B 2 receptor internalization, leading to the attenuation of downstream events such as bradykinin-induced activation of Cdc42 and filopodia formation, and thus affects endothelial cell motility.Investigation into mechanisms underlying this block led us to focus on B 2 R internalization via membrane-dependent mechanisms. Membrane disruption by depletion of cholesterol or trypsinization halts B 2 R internalization, invasion, and filopodia formation, which can be recovered with addition of cholesterol. However, this functional recovery is severely impaired in the presence of CD13 antagonists, and the distribution of membrane proteins is disordered in treated cells, suggesting a role for CD13 in plasma membrane protein organization. Finally, exogenous expression of wild-type but not mutant CD13 further alters protein distribution, suggesting peptidase activity is required for CD13's regulatory activity. Therefore, CD13 functions as a novel modulator of signal transduction and cell motility via its influence on specific plasma membrane organization, thus regulating angiogenesis. IntroductionBradykinin has long been recognized as a component of an array of potent serum factors that maintain and regulate tissue perfusion by controlling the integrity of endothelial cells. This nonapeptide is the principal effector of the kallikrein-kinin system which functions in many normal and disease-related processes including pain perception, vascular homeostasis, smooth muscle contraction, coagulation, and fibrinolysis (reviewed in Blaukat 1 and Prado et al 2 ). Recently, bradykinin and its kininogen precursor have been implicated in angiogenesis, where the ischemic environment stimulates bradykinin production. In this setting, bradykinin acts immediately as a vasodilator to increase tissue perfusion and later as a long-term angiogenic stimulator. [3][4][5][6][7][8] CD13 is a cell-surface peptidase that was originally defined as a myeloid-specific hematopoietic marker. 9 More recently, however, we have shown that while normal endothelial cells are CD13 Ϫ , neovessels in developing tumors express high levels of CD13. 10 This induction is mediated at the transcriptional level in response to angiogenic stimuli in the tumor microenvironment 11 through signals transduced via the Ras/MAPK pathway. 12 Additional data support the notion that CD13 peptidase activity is required for endothelial invasion and morphogenic phases of tumor neovessel formation. 11 CD13's role as a regulator of angiogenesis is clear: CD13 rescues angiogenesis in the presence of inhibitors of the Ras/MAPK pathway, 12 and CD13 inhibition prevents tumor growth. 10 However, the precise mechanisms mediating CD13'...

Recent reports suggest that commensal bacteria may play a down-regulatory role in autoimmune disease. In the present studies, we demonstrate that phosphorylated dihydroceramides, uniquely structured lipids derived from the common human oral bacterium Porphyromonas gingivalis and from bacteria commonly found in the gastrointestinal tract and other organs, are capable of enhancing autoimmunity. We have previously reported that these lipids have proinflammatory effects on human fibroblasts in vitro and, in preliminary studies, have recovered these lipids from surgically removed human carotid atheroma, suggesting that they may play a role in human inflammatory disease. To investigate whether these lipids have functional effects on autoimmunity, we administered phosphorylated dihydroceramides to mice with the murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). We find that these lipids, and particularly the phosphoethanolamine dihydroceramide (PE DHC) fraction, significantly enhanced EAE. Mechanistically, PE DHC enhances EAE in mice lacking natural killer T cells, fails to enhance EAE in Toll-like receptor 2 (TLR2)-deficient mice and, in vitro, induces dendritic cell interleukin-6 secretion in a TLR2-dependent manner.

The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To address the function of myeloid CD13 directly, we created a CD13 null mouse and assessed the responses of purified primary macrophages or DCs from WT and CD13 null animals in cell assays and inflammatory disease models, where CD13 has been implicated previously. We find that mice lacking CD13 develop normally with normal hematopoietic profiles except for an increase in thymic but not peripheral T cell numbers. Moreover, in in vitro assays, CD13 appears to be largely dispensable for the aspects of phagocytosis, proliferation, and antigen presentation that we tested, although we observed a slight decrease in actin-independent erythrocyte uptake. However, in agreement with our published studies, we show that lack of monocytic CD13 completely ablates anti-CD13-dependent monocyte adhesion to WT endothelial cells. In vivo assessment of four inflammatory disease models showed that lack of CD13 has little effect on disease onset or progression. Nominal alterations in gene expression levels between CD13 WT and null macrophages argue against compensatory mechanisms. Therefore, although CD13 is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis, or myeloid cell function.

CD4+ CD25+ Foxp3+ Tregs are critical regulators of immune responses and autoimmune diseases. nTregs are thymically derived; iTregs are converted in the periphery from CD4+ CD25- Foxp3- Teffs. Recent studies reported that GALT CD103+ DCs mediated enhanced iTreg conversion via the secretion of RA. However, the factors regulating RA secretion and hence, the induction of iTregs by DCs are not yet clear. Activation of the nuclear hormone receptor PPARgamma has been shown to induce RA expression in human DCs, and thus, we postulated that PPARgamma activation in DCs may be an important regulator of RA secretion and iTreg generation. Using in vitro and in vivo approaches, we now demonstrate that PPARgamma activation enhances iTreg generation through increased RA synthesis from murine splenic DCs. In addition, we demonstrate that inhibition of DC PPARgamma decreases iTreg generation, suggesting a role for endogenous PPARgamma ligands in this process. Overall, our findings suggest that PPARgamma may be important as a factor that stimulates DCs to produce RA and as a potential mechanism by which PPARgamma ligands ameliorate autoimmunity.