Role of protease-activated receptors in human skin fibrosis and scleroderma.

Proteases and their receptors have poorly understood roles in skin fibrosis and systemic scleroderma (SSc). We examined the role of protease-activated receptors (PAR(1) and PAR(2) ) in the pathophysiology of human SSc and skin fibrosis. Immunohistochemistry showed that PAR(1) immunoreactivity was positive in fibroblasts of SSc skin and healthy skin. PAR(2) immunoreactivity was positive in SSc skin, but negative in endothelial cells and fibroblasts of healthy skin. Double immunofluorescence using an antibody against smooth muscle actin (α-SMA) as a marker for myofibroblasts verified a certain percentage of myofibroblasts positive for PAR(1) and PAR(2) in SSc skin. In human dermal cultured fibroblasts (HDF), PAR(1) stimulation with or without bleomycin pretreatment mobilized intracellular calcium, indicating that the expressed PARs are functional and have effects on downstream signalling by calcium release. PAR(2) -induced intracellular calcium mobilization was only measurable in HDF after bleomycin pretreatment. Thus, PAR(1) - and PAR(2) -positive fibroblasts are increased in SSc, indicating a regulatory role. Intriguingly, bleomycin activated PAR(2) in HDF indicating that fibrosis-promoting factors have a direct effect on PAR(2) expression and functionality.

Interferon-γ induces upregulation and activation of the interleukin-31 receptor in human dermal microvascular endothelial cells.

Interleukin-31 (IL-31), a recently discovered cytokine derived from T helper cells, is involved in chronic dermatitis and pruritus. This study demonstrates for the first time that the IL-31 receptor complex for IL-31 is substantially upregulated in human dermal microvascular endothelial cells after stimulation with interferon-γ (IFN-γ). Activation of the IL-31 receptor complex results in the induction of the intracellular ERK1/2 signaling pathway and downregulation of IFN-γ-induced monokine induced by IFN-γ expression. Inhibitor studies revealed that the IFN-γ-induced IL-31RA upregulation is processed via JNK and PI3 kinase activation. In sum, our study points toward an interaction between the T(H) 1-derived cytokine IFN-γ and the T(H) 2-derived cytokine IL-31 on endothelial cells.

Terminating the stress: peripheral peptidolysis of proopiomelanocortin-derived regulatory hormones by the dermal microvascular endothelial cell extracellular peptidases neprilysin and angiotensin-converting enzyme.

The skin including the microvascular endothelium is an established peripheral source and target of the immunomodulatory proopiomelanocortin (POMC) peptides ACTH and alpha-MSH. Whereas intracellular POMC peptide generation is well characterized, less is known on their extracellular processing in peripheral tissues by the neuropeptide-specific zinc metalloproteases neprilysin (NEP) and angiotensin-converting enzyme (ACE). This may locally control POMC peptide bioavailability and activation of ACTH/alpha-MSH-specific melanocortin receptors (MCs). In a cell-free system, endothelial cell (EC) membranes prepared from ACE(high)/NEP(low)-expressing primary human dermal microvascular ECs and the ACE(low)/NEP(high) expressing EC line HMEC-1 degraded ACTH(1-39) over time, resulting in temporary increased alpha-MSH immunoreactivity. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy peptide mapping and electrospray ionization-mass spectroscopy sequencing identified several stable fragments generated from ACTH(1-39), ACTH(1-24), and alpha-MSH by EC membranes or recombinant NEP and ACE. Whereas some fragments could be assigned to a cell-specific NEP or ACE activity, other degradation products require additional enzyme activity. Pharmacological NEP inhibition enhanced the ACTH and alpha-MSH-mediated activation of EC ectopically expressing MC(1). Likewise, selected peptides such as alpha-MSH(2-12) generated from ACTH(1-39) and alpha-MSH by recombinant NEP displayed equipotent MC(1)-activating properties in vitro and antiinflammatory activity in murine allergic contact dermatitis in vivo as compared with the parental peptides. Thus, NEP and ACE significantly contribute to the EC processing of stress hormones (ACTH) and antiinflammatory peptides (alpha-MSH), which modulates MC(1) activation but does not completely inactivate the peptide ligand. Because NEP and ACE are regulated by inflammatory mediators and UV light, this may be important for ACTH/MSH-modulated skin inflammation.

Proteinase-activated receptor-2 agonist activates anti-influenza mechanisms and modulates IFNγ-induced antiviral pathways in human neutrophils.

Proteinase-activated receptor-2 (PAR2) is expressed by human leukocytes and participates in the development of inflammatory diseases. Recent studies demonstrated an ability of PAR2 agonist to enhance IFNγ-induced antiviral responses of human leukocytes. However, the precise cellular antiviral defense mechanisms triggered in leukocytes after stimulation with IFNγ and/or PAR2 agonist remain elusive. Therefore, we aimed to identify neutrophil defense mechanisms involved in antiviral resistance. Here we demonstrated that PAR2 agonist enhanced IFNγ-related reduction of influenza A virus (IAV) replication in human neutrophils. PAR2-mediated decrease in IAV replication was associated with reduced NS-1 transcription. Moreover, PAR2-dependent neutrophil activation resulted in enhanced myeloperoxidase degranulation and extracellular myeloperoxidase disrupted IAV. The production of ROS was elevated in response to PAR2 activation. Interestingly, IFNγ did not influence both effects: PAR2 agonist-triggered myeloperoxidase (MPO) release and reactive oxygen species (ROS) production, which are known to limit IAV infections. In contrast, orthomyxovirus resistance gene A (MxA) protein expression was synergistically elevated through PAR2 agonist and IFNγ in neutrophils. Altogether, these findings emphasize two PAR2-controlled antiviral mechanisms that are independent of or modulated by IFNγ.

α-1-Antitrypsin and IFN-γ reduce the severity of IC-mediated vasculitis by regulation of leukocyte recruitment in vivo.

IC-mediated vasculitis (ICV) can be life threatening. The cellular and immune mechanisms controlling ICV are poorly understood. Therefore, we investigated the role of α-1-antitrypsin (α1AT) and IFN-γ in reducing the severity of ICV in a mouse model in vivo. To induce ICV, mice were challenged with the reverse passive Arthus reaction (RPA), the prototypic in vivo model for leukocytoclastic vasculitis (LcV), and the modulation of vascular permeability, edema formation, and leukocyte recruitment was studied. To further analyze the dynamics of RPA, we applied intravital microscopy in the dorsal skinfold chamber. α1AT continuously led to reduced leukocyte recruitment. α1AT interfered with neutrophil recruitment through a KC-dependent mechanism and reduced KC-elicited neutrophil activation. In contrast to α1AT, IFN-γ-reduced leukocyte recruitment during RPA was clearly independent of KC. We also revealed that the recruitment of neutrophils during RPA was a prerequisite for full KC expression. Thus, therapeutic administration of α1AT and IFN-γ might be beneficial for limiting the duration and severity of ICV.