Lipopolysaccharide induces calcitonin gene-related peptide in the RAW264.7 macrophage cell line

Abstract

Calcitonin gene-related peptide (CGRP) is widely distributed and plays important roles in a wide array of biological functions. It is enriched in primary sensory neurons and hence involved in nociception and neurogenic inflammation. Recent studies have shown that CGRP can be produced by immune cells such as monocytes/macrophages following inflammatory stimulation, suggesting a role in innate immunity. However, it is unclear how CGRP is up-regulated in macrophages and if it plays a role in macrophage functions such as the production of cytokines and chemokines. Using enzyme-linked immunosorbent assay (ELISA) and multiplex ELISA, lipopolysaccharide (LPS) was found to induce CGRP in the RAW 264.7 macrophage cell line. LPS-induced inflammatory mediators such as nerve growth factor (NGF), interleukin-1beta (IL-1beta), IL-6, prostaglandin E(2) (PGE(2)) and nuclear factor-kappaB (NF-kappaB) signalling are involved in inducing CGRP, whereas the NGF receptor trkA and CGRP receptor signalling pathways are unexpectedly involved in suppressing LPS-induced CGRP, which leads to the fine-tune regulation of CGRP release. Exogenous CGRP and CGRP receptor antagonists, in a concentration-dependent manner, stimulated, inhibited or had no effect on basal or LPS-induced release of monocyte chemoattractant protein-1, IL-1beta, IL-6, tumour necrosis factor-alpha and IL-10 in RAW macrophages. The ligand-concentration-dependent regulation of the production of inflammatory mediators by CGRP receptor signalling is a novel mechanism underlying the stimulating and suppressing role of CGRP in immune and inflammatory responses. Together, our data suggest that monocytes/macrophages are an important source of CGRP. Inflammation-induced CGRP has a positive or negative reciprocal effect on the production of other pro- and anti-inflammatory mediators. Thereby CGRP plays both facilitating and suppressing roles in immune and inflammatory responses.

Figure 1

Lipopolysaccharide (LPS) concentration- and time- dependently increased calcitonin gene-related peptide (CGRP) release from RAW macrophages. (a) LPS concentration- dependently increased CGRP release from RAW 264.7 macrophages. * indicates P < 0·05 or < 0·01, LPS versus vehicle. Co-treatment of LPS (1 μg/ml) with 1 μm cycloheximide (CHX) or 1 μm actinomycin-D (AMD) blocked LPS-induced CGRP release. (b) 1 μg/ml LPS time- dependently increased CGRP release. * indicates P < 0·05 or < 0·01, LPS versus vehicle. The time-point for LPS to induce the maximum CGRP release was 24 hr. Mean ± SEM, n = 3.

Figure 2

Possible inflammatory mediators involved in lipopolysaccharide (LPS) -induced calcitonin gene-related peptide (CGRP) release from RAW macrophages. LPS (1 μg/ml) significantly increased CGRP release from RAW macrophages (a, b, *P < 0·001). (a) Co-treatment of LPS with 1·5 and 5 μg/ml nerve growth factor (NGF) receptor Fc chimera (sequestering NGF), 1 and 10 ng/ml interleukin-6 (IL-6) antiserum or 1 and 10 ng/ml IL-1β antiserum significantly suppressed LPS-induced CGRP release. Higher concentrations of NGF receptor Fc chimera, IL-6 and IL-1β further suppressed CGRP release to lower levels than vehicle. * indicates P < 0·05 or P < 0·001, antisera + LPS versus vehicle. + indicates P < 0·001, antisera + LPS versus LPS. (b) Co-treatment of LPS (L) with receptor-activity-modifying protein 1 (RAMP1) antiserum (1 and 10 ng/ml) or tyrosine receptor kinase A (trkA) receptor antiserum (1 μg/ml) significantly enhanced LPS-induced CGRP release compared with LPS treatment (+P < 0·001). RAMP1 and trkA antisera by themselves had no effect. Mean ± SEM, n = 3.

Figure 3

Cyclooxygenase 2 (COX2) -derived prostaglandin E₂ (PGE2) and transcription factor nuclear factor-κB (NF-κB) are involved in lipopolysaccharide (LPS) -induced calcitonin gene-related peptide (CGRP) release from RAW macrophages. LPS (1 μg/ml) significantly increased CGRP release from RAW macrophages (a–c, *P < 0·001). (a) Co-treatment of LPS with the selective COX2 inhibitor, NS-398, significantly suppressed LPS-induced CGRP release. * indicates P < 0·05 or < 0·001, NS-398 + LPS or LPS versus vehicle. + indicates P < 0·05 or < 0·001, NS-398 + LPS versus LPS. (b) PGE₂ concentration- dependently induced CGRP release or enhanced LPS (L) -induced CGRP release. * indicates P < 0·05 or < 0·001, LPS, PGE₂ or PGE₂ + LPS versus vehicle. + indicates P < 0·05, PGE₂ + LPS versus LPS. Co-treatment of 30 μm PGE₂ (PG) with 1 μm cycloheximide (c) or 1 μm actinomycin-D (a) blocked PGE₂-induced CGRP release. (c) Bay11-7082 concentration dependently suppressed LPS-induced CGRP release. * indicates P < 0·05, Bay11-7082 + LPS versus vehicle. + indicates P < 0·01, Bay11-7082 + LPS versus LPS. Mean ± SEM, n = 3.

Figure 4

Effects of exogenous calcitonin gene-related peptide (CGRP), CGRP8-37 and BIBN4096BS on basal and lipopolysaccharide (LPS) -induced release of monocyte chemotactic protein 1 (MCP-1), interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα) from RAW 264.7 macrophages. Compared with vehicle treatment, LPS (1 μg/ml) significantly increased MCP-1 (b), IL-1β (d) and TNFα (c) release from RAW macrophages (P < 0·001). (a) and (b) show the effect of CGRP, CGRP8-37 and BIBN4096BS on basal and LPS-induced MCP-1 release. * indicates P < 0·05, P < 0·01 or < 0·001, treatment versus vehicle (a) or treatment versus LPS (b). (c) and (d) show the effect of CGRP, CGRP8-37 and BIBN4096BS on basal and LPS-induced IL-1β release. * indicates P < 0·05 or < 0·001, treatment versus vehicle (c) or treatment versus LPS (d). (e) and (f) show the effect of CGRP, CGRO8-37 and BIBN4096BS on basal and LPS-induced TNFα release. * indicates P < 0·05, P < 0·01 or < 0·001, treatment versus vehicle (e) or treatment versus LPS (f). Mean ± SEM, n = 3.

Figure 5

Effects of exogenous calcitonin gene-related peptide (CGRP), CGRP8-37 and BIBN4096BS on basal and lipopolysaccharide (LPS) -induced release of interleukin-6 (IL-6) and IL-10 from RAW 264.7 macrophages. Compared with vehicle treatment, LPS (1 μg/ml) significantly increased IL-6 (b) and IL-10 (d) release from RAW macrophages (P < 0·001). (a) and (b) show the effect of CGRP, CGRP8-37 and BIBN4096BS on basal and LPS-induced IL-6 release. * indicates P < 0·05 P < 0·01 or < 0·001, treatment versus vehicle (a) or treatment versus LPS (b). (c) and (d) show the effect of CGRP, CGRP8-37 and BIBN4096BS on basal and LPS-induced IL-10 release. * indicates P < 0·05, P < 0·01 or P < 0·001, treatment versus vehicle (c) or treatment versus LPS (d). Mean ± SEM, n = 3.