Calcitonin gene-related peptide inhibits chemokine production by human dermal microvascular endothelial cells

Abstract

This study examined whether the sensory neuropeptide calcitonin gene-related peptide (CGRP) inhibits release of chemokines by dermal microvascular endothelial cells. Dermal blood vessels are associated with nerves containing CGRP, suggesting that CGRP-containing nerves may regulate cutaneous inflammation through effects on vessels. We examined CGRP effects on stimulated chemokine production by a human dermal microvascular endothelial cell line (HMEC-1) and primary human dermal microvascular endothelial cells (pHDMECs). HMEC-1 cells and pHDMECs expressed mRNA for components of the CGRP and adrenomedullin receptors and CGRP inhibited LPS-induced production of the chemokines CXCL8, CCL2, and CXCL1 by both HMEC-1 cells and pHDMECs. The receptor activity-modifying protein (RAMP)1/calcitonin receptor-like receptor (CL)-specific antagonists CGRP₈-₃₇ and BIBN4096BS, blocked this effect of CGRP in a dose-dependent manner. CGRP prevented LPS-induced IκBα degradation and NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 in HMEC-1 cells and Bay 11-7085, an inhibitor of NF-κB activation, suppressed LPS-induced production of CXCL1, CXCL8 and CCL2. Thus, the NF-κB pathway appears to be involved in CGRP-mediated suppression of chemokine production. Accordingly, CGRP treatment of LPS-stimulated HMEC-1 cells inhibited their ability to chemoattract human neutrophils and mononuclear cells. Elucidation of this pathway may suggest new avenues for therapeutic manipulation of cutaneous inflammation.

Fig. 1

HMEC-1 cells (A) and pHMDECs (B) express mRNA for components of the AM and CGRP receptors (RAMP1/CL, RAMP2/CL and RAMP3/CL). Total RNA was extracted from unstimulated HMEC-1 cells and subjected to RT-PCR with primers specific for RAMP1, RAMP2, RAMP3, CL and CRCP. The expected sizes for the amplified fragments are 450 bp for RAMP1; 227 bp for RAMP2; 212 bp for RAMP3; 560 bp for CL and 344 bp for CRCP.

Fig. 2

CGRP inhibits LPS-induced CXCL8, CCL2, and CXCL1 production by HMEC-1 cells. (A) Time-dependent chemokine inhibition by CGRP. HMEC-1 cells (0.25 ×10⁶ cells/ml) were exposed to LPS in the presence or absence of CGRP, and supernatants were collected at different time points and assayed for CXCL8, CCL2, and CXCL1 content. (B) CGRP inhibited chemokine production over a range of LPS concentrations (0.001–1 μg/ml). HMEC-1 cells were stimulated with increasing concentrations of LPS in the presence or absence of CGRP (10 nM). Supernatants were collected 72 h after LPS stimulation and assayed for chemokine content. (C) Dose-dependent chemokine inhibition by CGRP. HMEC-1 cells were cultured with various concentrations of CGRP (0.1–100 nM) and stimulated with LPS for 72 h. Chemokine contents in culture supernatants were determined. Each result is the mean ± SD of 3 separate replicates (each with duplicate wells) performed at the same time. Each experiment was performed at least 3 times with the same result except the time-dependent determination of CXCL1 in which the result illustrated was seen in 4 of 6 experiments. (*p<0.05, **p<0.01, ***p<0.001)

Fig. 3

CGRP inhibits LPS-induced CXCL8, CCL2, and CXCL1 production by pHDMECs. pHDMECs (0.25 ×10⁶ cells/ml) were cultured with various concentrations of CGRP (0.1–100 nM) and stimulated with 1 μg/ml of LPS. Supernatants were collected 24 h later and chemokine concentrations were determined. Each result is the mean ± SD of three separate replicates (each with duplicate wells) performed at the same time. This experiment was performed 3 times and the result illustrated by this figure was seen 3 times. (*p<0.05, ***p<0.001)

Fig. 4

CGRP inhibits the expression of LPS-induced chemokines at the mRNA level. HMEC-1 cells (1.5 ×10⁶ cells) were stimulated with LPS (1 μg/ml) in the presence or absence of CGRP (10 nM) for 12 h. Total RNA was extracted and transferred onto nylon membranes. The expression of CXCL8, CCL2, and CXCL1 mRNA in HMEC-1 cells was analyzed by Northern blotting. This experiment was performed 4 times and the result illustrated by this figure was seen in all 4 experiments.

Fig. 5

CGRP_8-37 and BIBN4096BS prevent CGRP-induced suppression of CXCL8, CCL2, and CXCL1 expression. (A) CGRP_8-37. HMEC-1 cells were stimulated with 10 nM of CGRP in the presence or absence of various concentrations (0–1000 nM) of the CGRP antagonist CGRP_8-37 followed by the addition of 1 μg/ml of LPS. Supernatants were collected 48 h later and assayed for chemokine content. (B) BIBN4096BS. This experiment was performed in the same manner except BIBN4096BS (0–1000 nM) was substituted for CGRP_8-37. Each result is the mean ± SD of three separate replicates (each with duplicate wells) performed at the same time. This experiment was performed 3 times and the result illustrated by this figure was seen 3 times. (*p<0.05, **p<0.01, ***p<0.001)

Fig. 6

CGRP prevents NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 and LPS-induced IκBα degradation in HMEC-1 cells. (A) CGRP inhibits NF-κB DNA binding to promoters. Nuclear extracts were prepared from HMEC-1 cells stimulated for 4 h with LPS (1 μg/ml) in the presence or absence of CGRP with CGRP added to cultures 1 hr before LPS. NF-κB binding was assayed by EMSA. Nuclear extracts were incubated with antisera against p50, p65 or IgG control for 15 min before adding the radiolabeled probe. Similar results were observed in 3 independent experiments. (B) CGRP prevents LPS-induced IκBα degradation. HMEC-1 cells were stimulated with LPS in the presence or absence of CGRP as in (A). Cytosolic amounts of IκBα at different time points were determined by Western blot. One representative experiment of 3 is shown. (C) Bay11-7085 blocked LPS-induced CXCL8, CCL2 and CXCL1 production by HMEC-1 cells. HMEC-1 cells were stimulated with LPS (1 μg/ml) in the presence or absence of graded concentrations of Bay11-7085. Supernatants were collected 24 h after LPS stimulation and chemokine content assayed. Each result is the mean ± SD of three separate replicates (each with duplicate wells) performed at the same time. This result is representative of 2 such experiments. (***p<0.001 vs LPS, no Bay11-7085; ^*p<0.05, ^**p<0.01, ^***p<0.001 vs no LPS, no Bay11-7085).

Fig. 7

Exposure of HMEC-1 cells to CGRP during stimulation inhibits chemotaxis of neutrophils and monocunclear cells towards LPS-stimulated HMEC-1 cells or supernatants conditioned by LPS-stimulated HMEC-1 cells. (A) Inhibition of chemotaxis towards stimulated cells. HMEC-1 cells were treated with 1 μg/ml LPS in the presence or absence of CGRP, CGRP alone or medium alone in the lower chambers of the Transwell apparatus for 24 h. Neutrophils or mononuclear cells were then placed in the upper chambers and after 90 min cells that migrated into the lower chambers were enumerated by light microscopy. (B) Inhibition of chemotaxis towards conditioned supernatants. HMEC-1 cells were stimulated with LPS in the presence or absence of CGRP, CGRP alone or medium alone. Supernatants were harvested after 24 h and placed in the lower chambers of the Transwell apparatus. Neutrophils or mononuclear cells were then placed in the upper chambers and after 90 min cells that migrated into the lower chambers were enumerated by light microscopy. The experiment shown is representative of three experiments with similar results (***p<0.001 vs LPS, no CGRP).