Svetlana Mojsov

Tissue-specific expression of the human receptor for glucagon-like peptide-I: brain, heart and pancreatic forms have the same deduced amino acid sequences

Glucagon‐like peptide‐I(GLP‐I), encoded by the glucagon gene and released from the gut in response to nutrients, is a potent stimulator of glucose‐induced insulin secretion. In human subjects GLP‐I exerts its physiological effect as an incretin. The incretin effect of GLP‐I is preserved in patients with Type II diabetes mellitus (NIDDM), suggesting that GLP‐I receptor agonist can be used therapeutically in this group of patients. In these studies we addressed the question of whether GLP‐I has broader actions in human physiology. To investigate this issue we examined the tissue distribution of GLP‐I receptor using RNAse protection assay in order to avoid the cross‐reactivities with structurally related receptors and to increase the sensitivity of detection. The riboprobe was synthetized from the human pancreatic GLP‐I receptor cDNA and used in hybridization experiments with total RNA isolated from different human tissues. In addition to the pancreas, we found expression of GLP‐I receptor mRNA in lung, brain, kidney, stomach and heart. Peripheral tissues which are the major sites of glucose turnover, such as liver, skeletal muscle and adipose did not express the pancreatic form of the GLP‐I receptor. We also cloned and sequenced GLP‐I receptor cDNA from human brain and heart. The deduced amino acid sequences are the same as the sequence found in the pancreas. These results indicate that GLP‐I might have effects beyond the pancreas, including the cardiovascular and central nervous systems where a receptor with the same ligand binding specificity is found.

Dendritic cell–expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice

Most treatments that prevent autoimmune diabetes in nonobese diabetic (NOD) mice require intervention at early pathogenic stages, when insulitis is first developing. We tested whether dendritic cell (DC)–expanded, islet antigen–specific CD4+ CD25+ suppressor T cells could treat diabetes at later stages of disease, when most of the insulin-producing islet β cells had been destroyed by infiltrating lymphocytes. CD4+ CD25+ CD62L+ regulatory T cells (T reg cells) from BDC2.5 T cell receptor transgenic mice were expanded with antigen-pulsed DCs and IL-2, and were then injected into NOD mice. A single dose of as few as 5 × 104 of these islet-specific T reg cells blocked diabetes development in prediabetic 13-wk-old NOD mice. The T reg cells also induced long-lasting reversal of hyperglycemia in 50% of mice in which overt diabetes had developed. Successfully treated diabetic mice had similar responses to glucose challenge compared with nondiabetic NOD mice. The successfully treated mice retained diabetogenic T cells, but also had substantially increased Foxp3+ cells in draining pancreatic lymph nodes. However, these Foxp3+ cells were derived from the recipient mice and not the injected T reg cells, suggesting a role for endogenous T reg cells in maintaining tolerance after treatment. Therefore, inoculation of DC-expanded, antigen-specific suppressor T cells has considerable efficacy in ameliorating ongoing diabetes in NOD mice.

Tissue Specific Expression of Different Human ReceptorTypes for Pituitary Adenylate Cyclase Activating Polypeptideand Vasoactive Intestinal Polypeptide: Implications fortheir Role in Human Physiology

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two structurally related peptides with pleiotropic physiological effects. Biochemical and cloning experiments have demonstrated that there are two structurally distinct receptors which recognize PACAP and VIP peptides with similar affinities (PACAP/VIP R‐1, PACAP/VIP R‐2), as well as a receptor that is specific for the PACAP peptide (PACAP‐Type 1 receptor). Using a homology‐based cloning strategy we have identified PACAP/VIP R‐2 receptor in human adipocytes, a tissue which was not previously identified as a target for PACAP and VIP action. This receptor type recognizes PACAP‐38 and VIP with similar affinity with inhibition concentrations of IC50=6.2±4.8u2003nM for PACAP‐38 and IC50=9.4±4.6u2003nM for VIP. Like the other two PACAP receptor types, PACAP/VIP R‐2 is coupled to cAMP‐mediated signal transduction pathway with effective doses ED50=3.2±1.6u2003nM and ED50=2.2±0.9u2003nM for PACAP‐38 and VIP respectively. Transcripts of the common PACAP/VIP R‐2 are also found in human brain and a number of peripheral tissues, such as pancreas, muscle, heart, lung, kidney, stomach and low levels in the liver. Comparison of the tissue distribution of the human PACAP/VIP R‐2 to that of the other two types of human PACAP receptors (PACAP‐Type 1 and the other common PACAP/VIP R‐1) by RNase protection showed that each of the three PACAP receptors is expressed in a unique set of human peripheral tissues. RNA transcripts for all three PACAP receptor types are found in human heart, brain and adipose tissue, while PACAP/VIP R‐2 is the only one of these three receptor types that is expressed in the pancreas and skeletal muscle. These results suggest a novel and not yet characterized role for PACAP and VIP peptides in the neuroendocrine regulation of insulin‐glucose homeostasis.

Calcitonin Gene-Related Peptide Decreases Expression of HLA-DR and CD86 by Human Dendritic Cells and Dampens Dendritic Cell-Driven T Cell-Proliferative Responses Via the Type I Calcitonin Gene-Related Peptide Receptor

These studies were performed to establish whether functional receptors for calcitonin gene-related peptide (CGRP) are present on human dendritic cells (DCs) and to investigate potential immunomodulatory effects of CGRP on DCs other than Langerhans cells. Reverse transcriptase-PCR revealed expression of mRNA for a type 1 CGRP receptor by mature and immature blood-derived DCs. Sequence analysis confirmed the identity of the type 1 CGRP receptor (CGRP-R1). Addition of CGRP (10−7 M) to mature and immature DCs resulted in mobilization of intracellular calcium. Treatment of immature DCs with CGRP (10−7 M), before and after maturation in monocyte-conditioned medium, resulted in decreased cell surface expression of HLA-DR MHC class II and the costimulatory molecule, CD86. Treatment of immature DCs with CGRP (10−7 M) also resulted in decreased expression of CD86, but expression of HLA-DR was unchanged. When CGRP-treated mature DCs were used to stimulate allogeneic T cells, proliferative responses were dampened (∼50%), especially at low DC:T cell ratios (1:360). This effect was not observed with CGRP-treated, immature DCs. In contrast, CGRP-treated mature or immature DCs were no less efficient than untreated DCs in driving syngeneic T cell-proliferative responses to staphylococcal enterotoxin B. We conclude that mature and immature DCs express type 1 CGRP receptors and that signaling through these receptors may dampen mature DC-driven T cell proliferation most likely via down-regulation of CD86 and HLA-DR.

Expression of a Functional Eotaxin (CC Chemokine Ligand 11) Receptor CCR3 by Human Dendritic Cells

Critical to the function of Ag-presenting dendritic cells (DCs) is their capacity to migrate to lymphoid organs and to sites of inflammation. A final stage of development, termed maturation, yields DCs that are strong stimulators of T cell-mediated immunity and is associated with a remodeling of the cell surface that includes a change in the levels of expression of many molecules, including chemokine receptors. We show in this study that CCR3, a chemokine receptor initially discovered on eosinophils, is also expressed by human DCs that differentiate from blood monocytes, DCs that emigrate from skin (epidermal and dermal DCs), and DCs derived from CD34+ hemopoietic precursors in bone marrow, umbilical cord blood, and cytokine-elicited peripheral blood leukapheresis. Unlike other chemokine receptors, such as CCR5 and CCR7, the expression of CCR3 is not dependent on the state of maturation. All DC subsets contain a large intracellular pool of CCR3. The surface expression of CCR3 is not modulated following uptake of particulate substances such as zymosan or latex beads. CCR3 mediates in vitro chemotactic responses to the known ligands, eotaxin and eotaxin-2, because the DC response to these chemokines is inhibited by CCR3-specific mAbs. We postulate that expression of CCR3 may underlie situations where both DCs and eosinophils accumulate in vivo, such as the lesions of patients with Langerhans cell granulomatosis.

Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells

Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.

Multiple Human Receptors for Pituitary Adenylyl Cyclase-Activating Polypeptide and Vasoactive Intestinal Peptide Are Expressed in a Tissue-Specific Mannera

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) share a high degree of structural similarity and exert multiple physiological effects. These effects are mediated by receptors that have different pharmacological properties.*.I There are receptors that are specific for PACAP and VIP, as well as receptors that recognize PACAP and VIP with similar affinities. Recent cloning experiments have identified three distinct types of these receptors that share a high degree of structural homologies with each other.4 The PACAPType 1 receptor is specific for PACAP peptides and recognizes the biologically active PACAP-38 and PACAP-27 with similar affinities, whereas it has a 1000-fold lower affinity for VIP. The other two receptor types, which we designate in this paper as PACAPNIP R-1 and PACAPNIP R-2, recognize PACAP-38, PACAP-27, and VIP with similar affinities. Diverse physiological effects of PACAP have been described, such as, for example, an ability to promote neurite outgrowth,s induce the release of several pituitary hormones,b and stimulate insulin secretion from rat pancreatic P cells. To investigate whether PACAP has a broader role in the regulation of insulin-glucose homeostasis, we concentrated our efforts on identifying PACAP receptors in human tissues that are the site of glucose turnover. Using a homology-based cloning strategy, we have identified in human adipose tissue the human homologue of the rat VIP-2 receptor* isolated from the olfactory bulb cDNA library, and the mouse PACAP-Type 3 receptor isolated from a mouse insulin-secreting P-cell line cDNA library. In competitive binding experiments, the recombinant receptor expressed in the Chinese hamster lung (CHL) cells showed similar binding affinity for PACAP-38 and VIP, with inhibition constants ICso = 6.2

GLUCAGON-LIKE PEPTIDE-1 ACTIVATES THE ADENYLYL CYCLASE SYSTEM IN ROCKFISH ENTEROCYTES AND BRAIN MEMBRANES

Glucagon-like peptide (GLP) exerts important physiological functions in fish liver, but extrahepatic sites of action and physiological roles have been largely ignored. We show here that GLP activates adenylyl cyclase in isolated brain and enterocyte membranes and increases cellular cyclic adenosine monophosphate (cAMP) levels in isolated enterocytes of rockfish (Sebastes caurinus). Following exposure to synthetic zebrafish GLP (zf-GLP) (1 nM-1 microM), a concentration-dependent increase in enterocyte cAMP is noted. The maximum increase in cAMP levels is observed at 1 microM zf-GLP, and represents a 30% increase above control values. Exendin-4, a GLP receptor agonist in mammals, elicits a similar concentration-dependent increase in enterocyte cAMP. In contrast, norepinephrine or prostaglandin E2 (at 1 microM) increased cAMP levels by 2 and 4-fold, respectively. Brain membrane adenylyl cyclase is activated 20-40% by zf-GLP, and to a smaller extent by zf-glucagon, while exendin-4 is as effective as zf-GLP at a dose of 100 nM. These results suggest potential physiological roles of GLP in brain and intestine in piscine systems analogous to GLP-1 functions in these tissues described for mammals.

Short Communication: The Immunodeficiency Virus Coreceptor, Bonzo/STRL33/TYMSTR, Is Expressed by Macaque and Human Skin- and Blood-Derived Dendritic Cells

Dendritic cells (DCs) have been shown to be important in the replication of human and simian immunodeficiency viruses (HIV and SIV, respectively) in vivo and in vitro. DCs express CD4 and several chemokine receptors, such as CCR5 and CXCR4, which are important for viral entry. In vivo, DCs are abundant at body surfaces, where they might be one of the first cells that encounter naturally transmitted virus. Furthermore, DCs pulsed with HIV or SIV in vitro can efficiently transmit virus to T cells, thereby propagating vigorous viral replication. Reports have implicated Bonzo/STRL33/TYM STR to be an additional alternative coreceptor for HIV and especially SIV infection. However, at present there are no reports regarding the expression of Bonzo/STRL33/TYMSTR by human or macaque DCs. Here we demonstrate the presence of Bonzo/STRL33/TYMSTR transcripts in rhesus macaque and human skin-derived DCs, in immature and mature blood monocyte-derived DCs, and in T cells from both skin and blood. Therefore, Bonzo/STRL33/T...

Functional studies of a glucagon receptor isolated from frog Rana tigrina rugulosa: implications on the molecular evolution of glucagon receptors in vertebrates

In this report, the first amphibian glucagon receptor (GluR) cDNA was characterized from the liver of the frog Rana tigrina rugulosa. Functional expression of the frog GluR in CHO and COS‐7 cells showed a high specificity of the receptor towards human glucagon with an EC50 value of 0.8±0.5 nM. The binding of radioiodinated human glucagon to GluR was displaced in a dose‐dependent manner only with human glucagon and its antagonist (des‐His1‐[Nle9‐Ala11‐Ala16]) with IC50 values of 12.0±3.0 and 7.8±1.0 nM, respectively. The frog GluR did not display any affinity towards fish and human GLP‐1s, and towards glucagon peptides derived from two species of teleost fishes (goldfish, zebrafish). These fish glucagons contain substitutions in several key residues that were previously shown to be critical for the binding of human glucagon to its receptor. By RT‐PCR, mRNA transcripts of frog GluR were located in the liver, brain, small intestine and colon. These results demonstrate a conservation of the functional characteristics of the GluRs in frog and mammalian species and provide a framework for a better understanding of the molecular evolution of the GluR and its physiological function in vertebrates.