Notes: Dermal microvascular endothelial cells (ECs) are both source and target of the pro-opiomelanocortin (POMC) peptides ACTH and α-melanocyte-stimulating hormone (α-MSH). The availability of neuropeptides as important modulators of innate and adaptive immune responses is controlled by neuropeptide-specific peptidases such as neutral endopeptidase (NEP) or angiotensin-converting enzyme (ACE). In this study, we have tested the possibility that NEP or ACE expressed by ECs may influence the local bioavailability of POMC peptides. Incubation of ACTH1−39 with cell membranes prepared from the high NEP-/low ACE-expressing microvascular EC line 1 (HMEC-1) or from low NEP-/high ACE-expressing primary human dermal ECs (HDMECs) for 30–480 min resulted in a decrease in ACTH immunoreactivity (IR) over time in membrane supernatants that could be partially blocked with NEP inhibitors as detected by radioimmunoassay. 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 analysed by mass spectroscopy. HMEC-1 membranes generated peptide products which could be altered by inhibition of NEP, but not ACE. Likewise, HDMEC membranes fragmented ACTH similar to HMEC-1 membranes in the presence of NEP inhibitors. Some of the proteins can be assigned to regular proteolytic cleavage, while others seem to be modified. Importantly, HMEC-1 and HDMEC membranes also slowly degraded α-MSH, suggesting that EC proteolytic peptidases locally control ACTH/α-MSH bioavailability, which may be important in controlling cutaneous inflammation.

Notes: 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.

Notes: AM and the sensory neuropeptide CGRP are potent vasoactive mediators that activate high-affinity G-protein-coupled receptors consisting of receptor-activity modifying proteins (RAMPs) and a seven-transmembrane domain calcitonin receptor-like receptor (CRLR) with RAMP-1/CRLR as CGRP and RAMP2 or -3/CRLR as AM receptors. In this study, we have examined the possibility that AM or CGRP modulate dermal microvascular EC adhesion molecule (ICAM-1 and VCAM-1) expression. Primary HDMEC or cells of the EC line HMEC-1 were transfected with cDNA expression vectors for an EGFP control, RAMP-1, RAMP-2 and CRLR by electroporation, or left untransfected. Stimulation of EC-overexpressing R1/CRLR or R2/CRLR with CGRP or AM (0.01–1000 nm) resulted in a dose-dependent upregulation of intracellular cAMP. Importantly, when HDMEC transfected with R1/CRLR or R2/CRLR were treated with TNFα in combination with CGRP or AM, these peptides interfered with the TNF-induced expression of ICAM-1 and VCAM-1 as well as the adhesion of lymphoblastoid cell lines to HDMEC monolayer in a biphasic manner. Likewise, AM and CGRP modulated the activation of nuclear factor κB (NF-κB) partly by inhibiting the TNFα-induced degradation of cytosolic IκBα. Neither transfection with the orphan CRLR nor RAMPs alone was capable of mediating a full reduction of TNFα-induced ICAM-1 or VCAM-1 expression. In conclusion, CGRP and more pronounced AM are capable of modulating TNFα-induced EC CAM expression, which may be of importance for the regulation of leucocyte–endothelial cell interaction during cutaneous neurogenic inflammation.This study was supported by the “Medizinische Forschungsgesellschaft Salzburg” and a grant of the Austrian Science Foundation (P14906).

Notes: The bioavailability of neuropeptides such as substance P (SP) released from sensory nerves or immune cells during skin inflammation is effectively controlled by proteolytic peptidases. Acute inhibition or genomic deletion of neutral endopeptidase (NEP) results in a SP-dependent augmentation of murine allergic contact dermatitis (ACD) by affecting sensitization and elicitation phase. In this study, we address the hypothesis that absence of NEP may modulate ACD responses by affecting bone marrow-derived dendritic cell (BmDC) maturation and function. BmDCs were generated from NEP-deficient mice (C57BL/6J-NEP–/–) or wild-type controls (C57BL/6J). FACS analysis revealed that d3, d6 and d7 NEP–/– BmDCs expressed significantly more DC cell-surface markers and costimulatory molecules compared to NEP+/+ mice BmDCs, in particular after BmDC maturation with LPS. In MLR utilizing d8 BmDCs pulsed 3 h in vitro with DNBS and T cells from in vivo DNFB-haptenized NEP–/– and NEP+/+ mice, BmDCs from NEP–/– animals promoted proliferation of T cells with higher efficacy compared to wild-type mice BmDCs. Likewise, T cells from NEP–/– mice demonstrated a higher proliferative response to Concavalin A stimulation or CD3/CD28 ligation compared to NEP+/+ mice. In addition, acute systemic NEP inhibition in NEP+/+ mice prior to sensitization with fluorescein isothiocyanate (FITC) after 24 h significantly augmented uptake of FITC in the CD11c-positive DC fraction from regional lymph nodes but not from spleen compared to cells obtained from mice not treated with the NEP inhibitor. These data indicate that functional absence of NEP may significantly control cutaneous ACD inflammatory responses by promoting hapten uptake, DC maturation and T-cell stimulation.