Michaela Fastrich

Expression of Functional Melanocortin Receptors and Proopiomelanocortin Peptides by Human Dermal Microvascular Endothelial Cells

ABSTRACT: Human dermal microvascular endothelial cells (HDMEC) are capable of mediating leukocyte‐endothelial interactions by the expression of cellular adhesion molecules and the release of proinflammatory cytokines and chemokines during cutaneous inflammation. Recent studies support the important role for proopiomelanocortin (POMC) peptides, such as α‐melanocyte stimulating hormone (α‐MSH), as immunomodulators in the cutaneous immune system. The purpose of the studies described here was to determine whether HDMEC serves as both target and source for POMC peptides. RT‐PCR and Northern blot studies demonstrated the constitutive expression of mRNA for the adrenocorticotropin (ACTH) and α‐MSH‐specific melanocortin receptor 1 (MC‐1R) in HDMEC, and the microvascular endothelial cell line HMEC‐1 that could be upregulated by stimulation with IL‐1β and α‐MSH. HDMEC responded to stimulation by α‐MSH with a dose‐ and time‐dependent synthesis and release of the CXC chemokines, IL‐8 and GROα. Likewise, α‐MSH augmented HDMEC chemokine release induced by TNF or IL‐1. HDMEC were found to constitutively express POMC and prohormone convertase 1 (PC‐1); the latter being required to generate ACTH from the POMC prohormone. POMC and PC‐1 mRNA expression are increased as a result of stimulation with UVB and UVA1 radiation, IL‐1, and α‐MSH. In addition, UV‐radiation is capable of inducing the release of HDMEC, ACTH, and α‐MSH in a time‐ and dose‐dependent fashion. Thus, these data provide evidence that HDMEC are capable of expressing functional MC‐1R, POMC, and PC‐1 mRNA; and of releasing POMC peptides with UV light, IL‐1, and α‐MSH as regulatory factors. The expression and regulation of these peptides may be of importance, not only for the autocrine or paracrine regulation of physiologic functions of dermal endothelial cells, but also for the regulation of certain microvascular‐mediated cutaneous or systemic inflammatory responses.

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.

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

Abstract:  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 TH1‐derived cytokine IFN‐γ and the TH2‐derived cytokine IL‐31 on endothelial cells.

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.

Production and processing of POMC peptides by dermal microvascular endothelial cells (EC): modulation of EC biologic functions and implication for skin inflammation.

The availability of neuropeptides or neuroendocrine hormones as important modulators of innate and adaptive immune responses is effectively controlled by neuropeptide‐specific peptidases. In previous studies, drug inhibition or genomic deletion of neutral endopeptidase (NEP, CD10) or of angiotensin‐converting enzyme (ACE, CD143) resulted in a profound augmentation of murine allergic contact dermatitis responses. Likewise, we have identified dermal microvascular endothelial cells (EC) as both source and target of the proopiomelanocortin (POMC) peptides ACTH and α‐melanocyte‐stimulating hormone (α‐MSH), in particular. EC express melanocortin receptor (MC‐) 1 and α‐MSH is capable of profoundly downregulating LPS‐ or cytokine‐induced expression of adhesion molecules in vitro and of endotoxin‐induced cutaneous vasculitis in vivo. In this study, we have tested the hypothesis that NEP or ACE expressed by EC may influence the local bioavailability of POMC peptides. Cell membranes prepared from the high NEP/low ACE expressing human microvascular endothelial cell line 1 (HMEC‐1) or from low NEP/high ACE expressing primary human dermal EC (HDMEC) were incubated for 30–480 min with ACTH1–39 in the presence or absence of NEP or ACE inhibitors, respectively. Analysis of membrane supernatants for ACTH and α‐MSH by radioimmunoassay revealed a decrease in ACTH immunoreactivity (IR) over time that could be partially blocked with NEP inhibitors. In parallel, α‐MSH IR increased peaking after 60 min. Fragments generated by incubation of HMEC‐1 or HDMEC membranes with ACTH1–39, ACHT1–24 or α‐MSH for 1–120 min were further analyzed by Matrix‐assisted‐LASER desorption time‐of‐flight (MALDI‐TOF) spectroscopy. HMEC‐1 membranes generated main peptide products with molecular masses of 2007, 1057 and 945, respectively, from ACTH1–39, and 1057 from ACTH1–24. Inhibition with NEP, but not ACE inhibitors altered the fragmentation profile indicating that NEP is involved in degradation of both ACTH1–39 and ACTH1–24. Likewise, HDMEC membranes fragmented ACTH similar to HMEC‐1 membranes in the presence of NEP inhibitors. Both HMEC‐1 and HDMEC membranes were also capable of slowly degrading α‐MSH suggesting that EC proteolytic peptidases are important for the local control of ACTH/α‐MSH bioavailability, which may play a significant role in controlling local cutaneous inflammatory responses.