The neuropeptide receptor subunit RAMP1 constrains the innate immune response during acute pancreatitis in mice.

OBJECTIVES: The importance of the Calcitonin-gene-related-peptide-pathway (CGRP) as neuronal modulator of innate immune responses in mice has been previously demonstrated. The CGRP-receptor is composed of two subunits: the receptor-activity-modifying-protein-1 (RAMP1) and the calcitonin-receptor-like-receptor (CLR). CGRP can influence immune cells and their capacity of producing inflammatory cytokines. Using a RAMP1 knockout-mouse (RAMP1-/-) we examined the role of the CGRP-receptor in the acute-phase of cerulein-induced pancreatitis. METHODS: Hourly cerulein-injections for a period of 8 h in RAMP1-/- and wild-type mice were performed. To compare severity and extent of inflammation in RAMP1-/- and wild-type mice, histological analyses were done and cytokine levels were assessed using qRT-PCR 8 h, 24 h, 2 days, and 7 days post-cerulein-treatment. Furthermore, serum activities of LDH and lipase were determined. RESULTS: After 8 h RAMP1-/- mice showed a higher pancreas-to-body-weight-ratio, increased tissue edema and immune cell infiltration with higher amount of F4/80-positive cells as compared to wild-type mice. Overall infiltration of immune cells at 24 h was increased in RAMP1-/- mice and composed predominantly of MPO-positive neutrophils. In addition, after 24 h RAMP1-/- mice presented a higher pancreas-to-body-weight-ratio, higher expression of Ccl3, Il6, and Il1b and increased number of cleaved caspase 3 positive cells. Serum lipase correlated with the extent of tissue damage in RAMP1-/- compared to wild-type mice 24 h post-cerulein treatment. CONCLUSION: Mice lacking RAMP1 showed increased inflammation, tissue edema, and pancreas injury particularly in the early phase of acute pancreatitis. This study highlights the essential role of CGRP for dampening the innate immune response in acute pancreatitis.

CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis.

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1-/-) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1-/- mice; however, the BM repopulation capacity of Ramp1-/- mice was significantly decreased, and the transplanted Ramp1-/- BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

The significance of the relationship between the nervous and immune systems with respect to disease course is increasingly apparent. Immune cells in the liver and spleen are responsible for the development of acute liver injury, yet the regulatory mechanisms of the interactions remain elusive. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, regulates innate immune activation via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. Here, we show that RAMP1 in Kupffer cells (KCs) plays a critical role in the etiology of immune-mediated hepatitis. RAMP1-deficient mice with concanavalin A (ConA)-mediated hepatitis, characterized by severe liver injury accompanied by infiltration of immune cells and increased secretion of pro-inflammatory cytokines by KCs and splenic T cells, showed poor survival. Removing KCs ameliorated liver damage, while depleting T cells or splenectomy led to partial amelioration. Adoptive transfer of splenic T cells from RAMP1-deficient mice led to a modest increase in liver injury. Co-culture of KCs with splenic T cells led to increased cytokine expression by both cells in a RAMP1-dependent manner. Thus, immune-mediated hepatitis develops via crosstalk between immune cells. RAMP1 in KCs is a key regulator of immune responses.

Immunohistochemical localization of the calcitonin gene-related peptide binding site in the primate trigeminovascular system using functional antagonist antibodies.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a neuromodulator implicated in the pathophysiology of migraine. It binds to the extracellular domains of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 1 that together form the CGRP receptor. Antagonist antibodies against CGRP and its binding site at the receptor are clinically effective in preventing migraine attacks. The blood-brain barrier penetration of these antagonist antibodies is limited, suggesting that a potential peripheral site of action is sufficient to prevent migraine attacks. To further understand the sites of CGRP-mediated signaling in migraine, we used immunohistochemical staining with recently developed antagonist antibodies specifically recognizing a fusion protein of the extracellular domains of RAMP1 and CLR that comprise the CGRP binding pocket at the CGRP receptor in monkey and man. We confirmed binding of the antagonist antibodies to human vascular smooth muscle cells (VSMCs) of dural meningeal arteries and neurons in the trigeminal ganglion, both of which are likely sites of action for therapeutic antibodies in migraine patients. We further used one of these antibodies for detailed mapping on cynomolgus monkey tissue and found antagonist antibody binding sites at multiple levels in the trigeminovascular system: in the dura mater VSMCs, in neurons and satellite glial cells in the trigeminal ganglion, and in neurons in the spinal trigeminal nucleus caudalis. These data reinforce and clarify our understanding of CGRP receptor localization in a pattern consistent with a role for CGRP receptors in trigeminal sensitization and migraine pathology.

Calcitonin gene-related peptide regulates type IV hypersensitivity through dendritic cell functions.

Dendritic cells (DCs) play essential roles in both innate and adaptive immune responses. In addition, mutual regulation of the nervous system and immune system is well studied. One of neuropeptides, calcitonin gene-related peptide (CGRP), is a potent regulator in immune responses; in particular, it has anti-inflammatory effects in innate immunity. For instance, a deficiency of the CGRP receptor component RAMP 1 (receptor activity-modifying protein 1) results in higher cytokine production in response to LPS (lipopolysaccharide). On the other hand, how CGRP affects DCs in adaptive immunity is largely unknown. In this study, we show that CGRP suppressed Th1 cell differentiation via inhibition of IL-12 production in DCs using an in vitro co-culture system and an in vivo ovalbumin-induced delayed-type hypersensitivity (DTH) model. CGRP also down-regulated the expressions of chemokine receptor CCR2 and its ligands CCL2 and CCL12 in DCs. Intriguingly, the frequency of migrating CCR2(+) DCs in draining lymph nodes of RAMP1-deficient mice was higher after DTH immunization. Moreover, these CCR2(+) DCs highly expressed IL-12 and CD80, resulting in more effective induction of Th1 differentiation compared with CCR2(-) DCs. These results indicate that CGRP regulates Th1 type reactions by regulating expression of cytokines, chemokines, and chemokine receptors in DCs.

Calcitonin gene-related peptide enhances experimental autoimmune encephalomyelitis by promoting Th17-cell functions.

T(h)17 cells, an inflammatory T helper cell subset, are involved in the pathogenesis of various inflammatory, autoimmune and allergic diseases. Recent evidence supports the idea that immune cell functions and the inflammatory response are finely regulated by various physiological substances. Calcitonin gene-related peptide (CGRP), a neuropeptide released from the sensory nerve endings, is one of these mediators. By binding to its receptor composed of receptor activity-modifying protein 1 (RAMP1) and calcitonin receptor-like receptor, CGRP modulates various immune cell functions, but the function of CGRP in T(h)17 cells is largely unknown. Here, we investigated the effect of CGRP signaling on T(h)17 cells and T(h)17 cell-mediated inflammation and observed that CGRP activates nuclear factor of activated T cells c2 through cAMP/PKA to increase IL-17 production in vitro. In vivo, IL-17 production is suppressed in RAMP1-deficient mice in the experimental autoimmune encephalomyelitis (EAE) model and RAMP1-deficient mice are completely resistant to EAE. Furthermore, T(h)17 cell function and EAE induction are also suppressed in T cell-specific RAMP1-deficient mice. Taken together, our findings indicate that CGRP promotes T(h)17 cell-mediated autoimmune inflammation through the regulation of IL-17 expression.

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract.

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.

Suppression of ovalbumin-induced allergic diarrhea by diminished intestinal peristalsis in RAMP1-deficient mice.

Recent studies have revealed that various neurotransmitters regulate the immune system via their receptors expressed on the immune cells. Calcitonin gene-related peptide (CGRP), a sensory nerve C-fiber neuropeptide, is also known to have the ability to modulate the functions of immune cells in vitro. However, the contribution of CGRP to the immune regulation in vivo remains to be fully elucidated. Here we report that mice deficient in receptor activity-modifying protein 1 (RAMP1), which is a subunit of the CGRP receptor, showed a significantly lower incidence of diarrhea compared with wild-type (WT) mice in the ovalbumin (OVA)-induced food allergic model. Serum OVA-specific IgE levels and the differentiation of T helper cells was comparable in WT mice and RAMP1-deficient mice. Moreover, there were no significant differences between recruitment and degranulation of mast cells in the small intestine of these mice. In contrast, significantly diminished intestinal peristalsis was observed by the allergy induction in RAMP1-deficient mice compared with WT mice. These results suggest that this suppression of allergic diarrhea is due to the diminished intestinal peristalsis in RAMP1-deficient mice.

Expression of functional receptor activity modifying protein 1 by airway epithelial cells with dysregulation in asthma.

BACKGROUND: Epithelial cell expression of calcitonin gene-related peptide (CGRP) is a feature of provoked asthma. Receptor activity modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor combine to form the CGRP1 receptor. OBJECTIVE: To determine whether functional RAMP1 is expressed by airway epithelial cells and whether there are alterations in asthma. METHODS: BEAS-2B and A549 cells lines were studied by RT-PCR, confocal microscopy, a quantitative immunofluorescence assay, and ELISA. Bronchial biopsies from normal subjects and subjects with asthma were examined by immunohistochemistry and in situ hybridization. RESULTS: Inflammatory cytokines induced CGRP release and CGRP mRNA in BEAS-2B and A549 epithelial cell lines. RAMP1 was highly expressed by resting, unstimulated BEAS-2B and A549 cells. CGRP induced internalization of RAMP1 and IL-6 production, both of which were inhibited by the CGRP antagonist, CGRP(8-37). Activation of BEAS-2B and A549 cells by inflammatory cytokines induced CGRP secretion, binding of CGRP to RAMP1, and RAMP1 internalization, which was blocked by CGRP (8-37). RAMP1 immunoreactivity and RAMP1 mRNA expression in bronchial biopsies from subjects with asthma were significantly lower than in normal subjects (P = .002 and P = .007, respectively). Inhalational challenge of atopic subjects with asthma with allergen-derived peptides produced a significant decrease in the numbers of RAMP1-positive epithelial cells in responders (P = .027) but not nonresponders. CONCLUSION: Receptor activity modifying protein 1 was expressed both by airway epithelial cells in culture and in bronchial biopsies from normal subjects and internalized after epithelial cell activation through autocrine feedback of CGRP. There is an apparent dysregulation of RAMP1 in asthmatic epithelium, suggesting continuous stimulation of pathways involving CGRP.

Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion.

Calcitonin gene related peptide (CGRP) has a key role in migraine and recently CGRP receptor antagonists have demonstrated clinical efficacy in the treatment of migraine. However, it remains unclear where the CGRP receptors are located within the CGRP signaling pathway in the human trigeminal system and hence the potential antagonist sites of action remain unknown. Therefore we designed a study to evaluate the localization of CGRP and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP) 1 in the human trigeminal ganglion using immunohistochemistry and compare with that of rat. Antibodies against purified CLR and RAMP1 proteins were produced and characterized for this study. Trigeminal ganglia were obtained at autopsy from adult subjects and sections from rat trigeminal ganglia were used to compare the immunostaining pattern. The number of cells expressing CGRP, CLR and RAMP1, respectively, were counted. In addition, the glial cells of trigeminal ganglion, particularly the satellite glial cell, were studied to understand a possible relation. We observed immunoreactivity for CGRP, CLR and RAMP1, in the human trigeminal ganglion: 49% of the neurons expressed CGRP, 37% CLR and 36% RAMP1. Co-localization of CGRP and the receptor components was rarely found. There were no CGRP immunoreactions in the glial cells; however some of the glial cells displayed CLR and RAMP1 immunoreactivity. Similar results were observed in rat trigeminal ganglia. We report that human and rat trigeminal neurons store CGRP, CLR and RAMP1; however, CGRP and CLR/RAMP1 do not co-localize regularly but are found in separate neurons. Glial cells also contain the CGRP receptor components but not CGRP. Our results indicate, for the first time, the possibility of CGRP signaling in the human trigeminal ganglion involving both neurons and satellite glial cells. This suggests a possible site of action for the novel CGRP receptor antagonists in migraine therapy.