Monocytes/macrophages are innate immune cells that play a crucial role in the resolution of inflammation. In presence of Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), they display an anti-inflammatory profile and this activation pathway is known as alternative activation. In this study we compare and differentiate pathways mediated by IL-4 and IL-13 activation of human monocytes/macrophage. Here we report differential regulation of IL-4 and IL-13 signaling in monocytes/macrophages starting from IL-4/IL-13 cytokine receptors to Jak-Stat-mediated signaling pathways that ultimately control expression of several infl1ammatory genes. Our data demonstrate that while the receptor-associated tyrosine kinases Jak2 and Tyk2 are activated after the recruitment of IL-13 to its receptor (containing IL-4Rα and IL-13Rα1), IL-4 stimulates Jak1 activation. We further show that Jak2 is upstream of Stat3 activation and Tyk2 controls Stat1 and Stat6 activation in response to IL-13 stimulation. In contrast, Jak1 regulates Stat3 and Stat6 activation in IL-4-induced monocytes. Our results further reveal that while IL-13 utilizes both IL-4Rα-Jak2-Stat3 and IL-13Rα1-Tyk2-Stat1/Stat6 signaling pathways, IL-4 can only use the IL-4Rα-Jak1-Stat3/Stat6 cascade to regulate the expression of some critical inflammatory genes including 15-lipoxygenase (15-LO), monoamine oxidase A (MAO-A) and scavenger receptor CD36. Moreover, we demonstrate here that IL-13 and IL-4 can uniquely affect the expression of particular genes like dual specificity phosphatase 1 (DUSP1) and tissue inhibitor of metalloprotease-3 (TIMP3) and do so through different Jak kinaes. As evidence of differential regulation of gene function by IL-4 and IL-13, we further report that MAO-A-mediated reactive oxygen species (ROS) generation is influenced by different Jak kinases. Collectively, these results have major implications for understanding the mechanism and function of alternatively activated monocytes/macrophages by IL-4 and IL-13 and add novel insights into the pathogenesis and potential treatment of different inflammatory diseases.

The leukocyte integrin ␣ M ␤ 2 is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of ␣ M ␤ 2 , the inter-integrin chimera was created. In the chimeric integrin, the ␤D-␣5 loop-␣5 helix segment comprised of residues

Fibrinogen is a ligand for leukocyte integrin alpha(M)beta2 (CD11b/CD18, Mac-1) and mediates adhesion and migration of leukocytes during the immune-inflammatory responses. The binding site for alpha(M)beta2 resides in gammaC, a constituent subdomain in the D-domain of fibrinogen. The sequence gamma383-395 (P2-C) in gammaC was implicated as the major binding site for alpha(M)beta2. It is unknown why alpha(M)beta2 on leukocytes can bind to immobilized fibrinogen in the presence of high concentrations of soluble fibrinogen in plasma. In this study, we have investigated the accessibility of the binding site in fibrinogen for alpha(M)beta2. We found that the alpha(M)beta2-binding site in gammaC is cryptic and identified the mechanism that regulates its unmasking. Proteolytic removal of the small COOH-terminal segment(s) of gammaC, gamma397/405-411, converted the D100 fragment of fibrinogen, which contains intact gammaC and is not able to inhibit adhesion of the alpha(M)beta2-expressing cells, into the fragment D98, which effectively inhibited cell adhesion. D98, but not D100, bound to the recombinant alpha(M)I-domain, and the alpha(M)I-domain recognition peptide, alpha(M)(Glu253-Arg261). Exposure of the P2-C sequence in fibrinogen, D100, and D98 was probed with a site-specific mAb. P2-C is not accessible in soluble fibrinogen and D100 but becomes exposed in D98. P2-C is also unmasked by immobilization of fibrinogen onto a plastic and by deposition of fibrinogen in the extracellular matrix. Thus, exposure of P2-C by immobilization and by proteolysis correlates with unmasking of the alpha(M)beta2-binding site in the D-domain. These results demonstrate that conformational alterations regulate the alpha(M)beta2-binding site in gammaC and suggest that processes relevant to tissue injury and inflammation are likely to be involved in the activation of the alpha(M)beta2-binding site in fibrinogen.

The leukocyte integrin ␣ M ␤ 2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providing a key link between thrombosis and inflammation. However, the mechanism by which ␣ M ␤ 2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen ␥C domain sequences, P1 (␥190 -202) and P2 (␥377-395), serve as the ␣ M ␤ 2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding ␣ M I-domain of ␣ M ␤ 2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several ␣ M I-domain molecules. To localize the alternative ␣ M I-domain binding sites, we screened peptide libraries covering the complete sequences of the ␥C and ␤C domains, comprising the majority of the D fragment structure, for ␣ M I-domain binding. In addition to the P2 and P1 peptides, the ␣ M I-domain bound to many other sequences in the ␥C and ␤C scans. Similar to P1 and P2, synthetic peptides derived from ␥C and ␤C were efficient inhibitors of ␣ M ␤ 2 -mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the ␣ M I-domain binding depends on basic and hydrophobic residues. These findings establish a new model of ␣ M ␤ 2 binding in which the ␣ M I-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin ␣ M ␤ 2 .Integrins are noncovalently associated cell surface ␣␤ heterodimer receptors that mediate adhesive interactions with the extracellular matrix and with other cells. By providing a link between the cell cytoplasm and the surrounding matrix integrins regulate a diverse range of processes, including cellular differentiation, cell migration, the immune response, and the maintenance of tissue architecture. Integrins also play key roles in a variety of pathological conditions. Many integrins exhibit a very broad binding specificity and are able to recognize diverse ligands representing several protein families.Integrin ␣ M ␤ 2 (Mac-1, CD11b/CD18, and CR3) is the most promiscuous member of the entire family with more than 30 proteins being reported as its ligands. ␣ M ␤ 2 is abundantly expressed on activated leukocytes, primarily neutrophils and monocytes, and mediates critical adhesive reactions during the inflammatory responses. Specifically, it contributes to firm adhesion of neutrophils to endothelial cells, promotes their subsequent diapedesis, and participates in neutrophil migration through the interstitial matrix (reviewed in Ref. 1). Many other neutrophil responses, including phagocytosis, homotypic aggregation, degranulatio...