Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma.

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.

Syk-coupled C-type lectins in immunity.

The Syk-coupled C-type lectin receptor Dectin-1 was the first non-Toll like receptor described that could mediate its own intracellular signalling. It was initially identified as important for the innate recognition of and response to fungal pathogens but later studies revealed that it is also involved in triggering adaptive immune responses. It subsequently emerged that Dectin-1 is one of a number of spleen tyrosine kinase-coupled C-type lectin receptors that have been implicated not just in fungal immunity, but also in viral, mycobacterial and helminth infections. Here, we consider the ability of these receptors to trigger different aspects of immunity and highlight their emerging roles in a number of infection scenarios.

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

SUMMARY: 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.

Functional expression of the calcitonin receptor by human T and B cells.

The calcitonin receptor (CTR) is a seven-transmembrane-domain G-protein-coupled receptor that regulates calcium metabolism and bone resorption by osteoclasts. Here we demonstrate that high levels are expressed by normal human T and B lymphocytes from tonsils and peripheral blood in relation to their activation status, as CTR(+) T cells are prone to produce IFN-gamma after TCR stimulation. The receptor is also highly expressed on B cells from chronic lymphocytic leukemia patients, thus suggesting a correlation between its expression, their proliferative extent as well as their memory, antigen-experienced phenotype. Moreover, we found that binding of the receptor with salmon calcitonin induces an increase of intracellular calcium(2+) in peripheral lymphocytes. This effect is involved in several lymphocyte immune functions, as cytosolic calcium(2+) levels regulate both cell proliferation and cytokine production. In our hands, the increase of calcium(2+) levels by CTR binding with sCT induced a dose-dependent cell proliferation. We therefore suppose that expression of this functional receptor may contribute to the modulation of cytoplasmic calcium(2+) levels needed to regulate T and B cell activation and perhaps other immune functions.

Pyrin-only protein 2 modulates NF-kappaB and disrupts ASC:CLR interactions.

NF-kappaB is pivotal for transactivation of cell-cycle regulatory, cytokine, and adhesion molecule genes and is dysregulated in many cancers, neurodegenerative disorders, and inflammatory diseases. Proteins with pyrin and/or caspase recruitment domains have roles in apoptosis, innate immunity, and inflammation. Many pyrin domain (PYD) proteins modulate NF-kappaB activity as well as participate in assembling both the perinuclear "apoptotic speck" and the pro-IL1beta/IL-18-converting inflammasome complex. "Pyrin-only" proteins (POP) are attractive as negative regulators of PYD-mediated functions and one such protein, POP1, has been reported. We report the identification and initial characterization of a second POP. POP2 is a 294 nt single exon gene located on human chromosome 3 encoding a 97-aa protein with sequence and predicted structural similarity to other PYDs. Highly similar to PYDs in CATERPILLER (CLR, NLR, NALP) family proteins, POP2 is less like the prototypic pyrin and ASC PYDs. POP2 is expressed principally in peripheral blood leukocytes and displays both cytoplasmic and nuclear expression patterns in transfected cells. TNF-alpha-stimulated and p65 (RelA)-induced NF-kappaB-dependent gene transcription is inhibited by POP2 in vitro by a mechanism involving changes in NF-kappaB nuclear import or distribution. While colocalizing with ASC in perinuclear specks, POP2 also inhibits the formation of specks by the CLR protein CIAS1/NALP3. Together, these observations demonstrate that POP2 is a negative regulator of NF-kappaB activity that may influence the assembly of PYD-dependent complexes.

Studies on the effects of the N-terminal domain antibodies of calcitonin receptor-like receptor and receptor activity-modifying protein 1 on calcitonin gene-related peptide-induced vasorelaxation in rat uterine artery.

The vascular relaxation sensitivity to calcitonin gene-related peptide (CGRP) is enhanced during pregnancy, compared with nonpregnant human and rat uterine arteries. In the rat uterine artery, two types of CGRP receptors have been shown to coexist, CGRP-A receptor, which is a complex of calcitonin receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP(1)) and CGRP-B receptor, which is different from CRLR. In the present study, we hypothesized that: 1) CGRP-induced vasorelaxation in rat uterine artery is mediated through CGRP-A receptor and 2) N-terminal (Nt) domain of CRLR (Nt-CRLR) has a major contribution in ligand binding and mediating CGRP- induced relaxation effects in rat uterine artery. Polyclonal antibodies against Nt-domain of CRLR and RAMP(1) (Nt-RAMP(1)) were raised in rabbits and characterized for their specificity and were used to inhibit CGRP-induced vasorelaxation in rat uterine artery. For vascular relaxation studies, uterine arteries from Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in Day 18 pregnant rat uterine arteries. These effects were significantly (P < 0.05) inhibited when uterine arteries were incubated with the antibody raised against Nt-CRLR (PD(2) = 6.75 +/- 0.20) and were totally abolished in presence of antibodies for both Nt-CRLR and Nt-RAMP(1) (PD(2) = 6.14 +/- 0.35). In contrast, a monoclonal antibody for CGRP-B receptor had no effect on CGRP-induced rat uterine artery relaxation. These studies suggest that CGRP effects in rat uterine artery are mediated through CGRP-A receptor and that Nt-domain of CRLR may play a predominant role in CGRP binding and thus in causing CGRP-induced uterine artery relaxation.

Amylin: localization, effects on cerebral arteries and on local cerebral blood flow in the cat.

Amylin and adrenomedullin are two peptides structurally related to calcitonin gene-related peptide (CGRP). We studied the occurrence of amylin in trigeminal ganglia and cerebral blood vessels of the cat with immunocytochemistry and evaluated the role of amylin and adrenomedullin in the cerebral circulation by in vitro and in vivo pharmacology. Immunocytochemistry revealed that numerous nerve cell bodies in the trigeminal ganglion contained CGRP immunoreactivity (-ir); some of these also expressed amylin-ir but none adrenomedullin-ir. There were numerous nerve fibres surrounding cerebral blood vessels that contained CGRP-ir. Occasional fibres contained amylin-ir while we observed no adrenomedullin-ir in the vessel walls. With RT-PCR and Real-Time-PCR we revealed the presence of mRNA for calcitonin receptor-like receptor (CLRL) and receptor-activity-modifying proteins (RAMPs) in cat cerebral arteries. In vitro studies revealed that amylin, adrenomedullin, and CGRP relaxed ring segments of the cat middle cerebral artery. CGRP and amylin caused concentration-dependent relaxations at low concentrations of PGF 2alpha-precontracted segment (with or without endothelium) whereas only at high concentration did adrenomedullin cause relaxation. CGRP8-37 blocked the CGRP and amylin induced relaxations in a parallel fashion. In vivo studies of amylin, adrenomedullin, and CGRP showed a brisk reproducible increase in local cerebral blood flow as examined using laser Doppler flowmetry applied to the cerebral cortex of the alpha-chloralose-anesthetized cat. The responses to amylin and CGRP were blocked by CGRP8-37. The studies suggest that there is a functional sub-set of amylin-containing trigeminal neurons which probably act via CGRP receptors.

The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction.

G protein-coupled receptors are usually thought to act as monomer receptors that bind ligand and then interact with G proteins to initiate signal transduction. In this study we report an intracellular peripheral membrane protein named the calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) required for signal transduction at the G protein-coupled receptor for adrenomedullin. Cell lines were made that expressed an antisense construct of the RCP cDNA, and in these cells diminished RCP expression correlated with loss of adrenomedullin signal transduction. In contrast, loss of RCP did not diminish receptor density or affinity, therefore RCP does not appear to act as a chaperone protein. Instead, RCP represents a novel class of protein required to couple the adrenomedullin receptor to the cellular signal transduction pathway. A candidate adrenomedullin receptor named the calcitonin receptor-like receptor (CRLR) has been described, which forms high affinity adrenomedullin receptors when co-expressed with the accessory protein receptor-activity modifying protein 2 (RAMP2). RCP co-immunoprecipitated with CRLR and RAMP2, indicating that a functional adrenomedullin receptor is composed of at least three proteins: the ligand binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP).

Calcitonin receptor antibodies in the identification of osteoclasts.

Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.

Intracellular retention and rapid degradation of human calcitonin receptors overexpressed in COS cells.

Overexpression of native and epitope-tagged human calcitonin (CT) receptors (hCTR-2) in COS-1 cells was performed to permit identification of the receptor protein and begin studies of receptor turnover. Data obtained with immunological techniques and cross-linking of radiolabeled salmon CT ([125I]sCT) revealed two forms of hCTR-2 in transfected cells: a larger, mature cell surface receptor (apparent size, 81 kDa) and a smaller, intracellular form (apparent size, 66 kDa). These conclusions are based on the following observations. 1) Only the larger hCTR-2 was visualized by cell surface [125I]sCT binding, whereas both species were identified by [125I]sCT binding to cell lysates. 2) Immunofluorescence studies with antibodies directed against the epitope confirmed the presence of cell surface and intracellular hCTR-2s; there were apparently many more receptors intracellularly than on the cell surface. 3) Both hCTR-2 forms were changed to a similar size of approximately 57-60 kDa by deglycosylation with endoglycosidase F; this size is consistent with that predicted by the amino acid sequence. Metabolic studies with radioactive amino acids labeled only the intracellular form. This immature form exhibited a rapid half-life of 30 min. We conclude that overexpression of native and epitope-tagged hCTR-2s in COS-1 cells leads to their intracellular retention and rapid degradation.