Biomedical subjects

E J Goetzl

Publications and source records attributed to E J Goetzl.

At least 37 records · Page 2Linked to original sources

PubMed1999

Prostaglandin E2 enhancement of interferon-gamma production by antigen-stimulated type 1 helper T cells.

Prostaglandin E2 (PGE2) is a potent mediator generated in immune tissues by cyclooxygenation of arachidonic acid. PGE2 affects T cell functions through four homologous G protein-coupled receptors termed EP1R, EP2R, EP3R, and EP4R that differ in tissue distribution and signaling. Antigen-evoked secretion of interferon-gamma (IFN-gamma) by sperm whale myoglobin-specific Th1 cells of DBA/2 mouse I-Ed-restricted clones, that express EP3Rs and EP4Rs, was enhanced a maximum of 3-fold by 10(-10) to 10(-8) M PGE2 and 2.5-fold each for the EP1R/EP3R-directed agonist sulprostone (10(-8) and 10(-7) M) and for the EP4R/EP3R/EP2R agonist misoprostol (10(-9) M). Neither PGE2 nor the synthetic analogs affected secretion of IFN-gamma by PMA plus ionomycin-stimulated clones of Th1 cells. Antigen-evoked secretion of IFN-gamma by influenza hemagglutinin-specific mouse lymph node Th1 cells, that also express EP3Rs and EP4Rs, was increased a maximum of 12-fold by 10(-9) to 10(-8) M PGE2, 14-fold by 10(-9) M sulprostone, and 10-fold by 10(-9) M misoprostol. Production of IFN-gamma by either type of Th1 cell was not affected significantly by 10(-6) M PGE2 alone. The generation of IFN-gamma by antigen-stimulated Th1 cells thus is significantly enhanced by physiologically relevant concentrations of PGE2.

Animals↗

PubMed1999

Lysophosphatidic acid and sphingosine 1-phosphate protection of T cells from apoptosis in association with suppression of Bax.

Members of a subfamily of G protein-coupled receptors (GPCRs), encoded by five different endothelial differentiation genes (edgs), specifically mediate effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on cellular proliferation and differentiation. Mechanisms of suppression of apoptosis by LPA and S1P were studied in the Tsup-1 cultured line of human T lymphoblastoma cells, which express Edg-2 and Edg-4 GPCRs for LPA and Edg-3 and Edg-5 GPCRs for S1P. At 10-10 M to 10-7 M, both LPA and S1P protected Tsup-1 cells from apoptosis induced by Abs to Fas, CD2, and CD3 plus CD28 in combination. Apoptosis elicited by C6 ceramide was inhibited by S1P, but not by LPA, in part because ceramide suppressed expression of Edg-2 and Edg-4 surface receptors for LPA without affecting Edg-3 surface receptors for S1P. At 10-9 M to 10-7 M, LPA and S1P significantly suppressed cellular levels of the apoptosis-promoting protein Bax, without altering the levels of Bcl-xL or Bcl-2 assessed by Western blots and immunoassays. Transfections of pairs of antisense plasmids for Edg-2 plus Edg-4 and Edg-3 plus Edg-5, and hygromycin selection of transfectants with reduced expression of the respective Edg R proteins in Western blots, inhibited both protection from apoptosis and reduction in cellular levels of Bax by LPA and S1P. Thus, LPA and S1P protection from apoptosis is mediated by distinct Edg GPCRs and may involve novel effects on Bax regulatory protein.

Animals↗

PubMed1999

A subfamily of G protein-coupled cellular receptors for lysophospholipids and lysosphingolipids.

The results of molecular cloning and homology searches have identified a minimum of five different proteins of the endothelial differentiation gene (edg) encoded subfamily of GPCRs. Edg protein GPCRs show amino acid sequence identity of 31% to 34% as a subfamily, but contain two homology clusters with greater similarity of structures and functions. One cluster of high amino acid sequence homology includes Edg-2 and Edg-4 proteins, that encode GPCRs for LPA, but not lysosphingolipids. A second homology cluster encompasses Edg-1, Edg-3 and Edg-5. Edg-3 and Edg-5 encode GPCRs specific for S1P, but not LPA. Preliminary data suggest that Edg-1 encodes a GPCR for S1P and one or more other lysosphingolipids, but the signals evoked by S1P alone are far weaker than those transduced by Edg-3 and Edg-5. Similarities of the structures of genes for the respective homology clusters supports this tentative classification of the Edg protein GPCRs. Future research will be directed to completion of the elucidation of genomic organization and signaling pathways, and a greater understanding of the breadth of functional roles of Edg proteins in development and activities of the nervous, cardiovascular, endocrine and immune systems.

Animals↗

PubMed1998

Selectivity of effects of vasoactive intestinal peptide on macrophages and lymphocytes in compartmental immune responses.

The major immunoregulatory effects of vasoactive intestinal peptide (VIP) are mediated by structurally distinct type I (VIPR1) and II (VIPR2) G protein-associated receptors on many different types of immune cells. VIP is released in functionally relevant concentrations during many immunologic and inflammatory responses. Mast cells (VIPR1), macrophages (VIPR1 and VIPR2), B cells, and T cells (VIPR1, VIPR2, or VIPR1 and VIPR2) recognize and respond to VIP in patterns that are controlled by the relative levels of expression of VIPR1 and VIPR2. VIPR2 transduces human T-cell chemotaxis, expression of matrix metalloproteinases (MMPs) 2 and 9 and consequently basement membrane and connective tissue transmigration, while signaling suppression of proliferation and cytokine production. In contrast, VIPR1 fails to transduce T-cell chemotaxis but mediates suppression of chemotaxis and MMP expression elicited by some cytokines and chemokines. The relative representation of each type of VIPR, which is presumed to be under cytokine control, thus may determine T-cell responses to VIP and other immune mediators in tissue compartments innervated by VIPergic nerves.

Animals↗

PubMed1998

Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid.

The recent identification of the Vzg-1/Edg2 protein as a functional G protein-coupled receptor for lysophosphatidic acid (LPA) has allowed a sequence-based search for new genes that may encode novel subtypes of LPA receptors. A human cDNA encoding a G protein-coupled receptor, designated Edg4, was identified by searching the GenBankTM for homologs of the human Edg2 LPA receptor. The Edg4 protein is 46% identical and 72% similar in amino acid sequence to human Edg2. When overexpressed in Jurkat T cells, the Edg4 protein mediated LPA-induced activation of a serum response element reporter gene with LPA concentration dependence (EC50 of 10 nM) and specificity. This LPA-induced reporter gene activation could be partially inhibited by pretreatment with pertussis toxin or C3 exoenzyme, suggesting requirements for both a Gi protein and Rho GTPase. Overexpression of Edg4 in Jurkat cells also led to increases in specific binding sites for [3H]LPA. Northern blots revealed that two edg4 mRNA transcripts of 1.8 and 8 kilobases are distributed very differently from edg2 mRNAs in adult human tissues and several cancer cell lines. The existence and distinctive tissue expression of structurally different subtypes of LPA receptors may provide one basis for tissue-specific functions and permit independent regulation of each subtype of LPA receptor.

Amino Acid Sequence↗

PubMed1998

Vasoactive intestinal peptide enhancement of antigen-induced differentiation of a cultured line of mouse thymocytes.

The prominence of vasoactive intestinal peptide (VIP) in rodent thymic neurons suggested that this potent mediator of T cell functions may alter developmental responses of thymocytes to T cell receptor (TCR) -dependent stimulation. CD4+8+ DPK cells derived from a thymic lymphoma of a TCR transgenic mouse respond to pigeon cytochrome C (PCC) antigen in association with distinct I-E MHC II haplotypes on antigen-presenting cells (APCs) by differentiating into CD4+8- T cells. The specific recognition of VIP by two types of homologous G-protein-coupled receptors (VIPR1 and VIPR2) on DPK cells was attributable predominantly to VIPR1 before and to VIPR2 after exposure to APCs and PCC, as assessed by quantification of the respective mRNAs. PCC-evoked differentiation of DPK cells was enhanced significantly by 1 to 100 nM VIP after 3 to 4 days. The effects of VIP analogs with VIPR type selectivity implied that VIP enhancement of differentiation of DPK cells was mediated principally by VIPR2. Differential reduction in the expression of each type of VIPR by transfection of DPK cells with plasmids encoding the respective antisense mRNAs confirmed the central role of VIPR2 in VIP-enhanced conversion to CD4+8- T cells. The suppression of DPK cell differentiation by inhibitors of adenylyl cyclase and protein kinase A suggested a transductional role for VIP-elicited increases in [cAMP]i. That the changes in frequency of CD4+8+ and CD4+8- DPK cells reflected principally differentiation was supported by the lack of consistent differences between the two subsets in the effects of VIP and VIPR2 agonist on cell number, viability, apoptosis, and proliferation. VIP may be one endogenous mediator that explains the unique thymic microenvironment for topographically specific development of T cells.

Animals↗

PubMed1998

Recombinant human G protein-coupled lysophosphatidic acid receptors mediate intracellular calcium mobilization.

Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca2+ levels. In TAg-Jurkat T cells transiently co-transfected with apoaequorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased light emission triggered by increased Ca2+ bound to aequorin. N-Palmitoyl-L-serine-phosphoric acid and N-palmitoyl-L-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis LPA receptors, did not antagonize the Ca2+-mobilizing effects of Edg2 and Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca2+ mobilization by Edg2 and Edg4 was further characterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca2+ flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the initial increase in the Ca2+ concentration was followed by a sustained influx of extracellular Ca2+. The coincident production of inositol phosphates and the inhibition of Ca2+ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca2+ through inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca2+ mobilization by Edg2 but only partially blocked that by Edg4, which suggests that Edg2 transduces Ca2+ mobilization largely through pertussis toxin-sensitive Gi proteins, whereas Edg4 requires both Gi and Gq.

Animals↗

PubMed1998

EP4/EP2 receptor-specific prostaglandin E2 regulation of interleukin-6 generation by human HSB.2 early T cells.

Human leukemic early T cells of the HSB.2 line coexpress the EP2, EP3 and EP4 subtypes of prostaglandin E2 (PGE2) receptors (Rs). EP3 Rs have previously been demonstrated to transduce PGE2 stimulation of secretion of matrix metalloproteinase (MMP)-9 by HSB.2 T cells through Ca++-dependent enhancement of MMP-9 mRNA transcription. We now show that PGE2 and the EP4/EP2/EP3 R-selective agonist misoprostol, but not the EP3 R-directed agonists sulprostone and M&B28767, induced increases in HSB.2 T cell interleukin-6 (IL-6) mRNA and secretion. Pharmacological agents that increase intracellular concentration of cyclic AMP ([cAMP]i) mimicked and synergistically enhanced induction of IL-6 secretion by PGE2, whereas inhibitors of protein kinase A (PKA) but not protein kinase C suppressed PGE2-evoked increases in IL-6 secretion, suggesting that cAMP and PKA are the intracellular messengers of the PGE2 effect. Exposure of HSB.2 T cells to the mitogenic lectin concanavalin A (Con A) increased basal IL-6 secretion, without a change in IL-6 mRNA level. Con A-stimulated HSB.2 T cells responded to PGE2 with greater increases in IL-6 mRNA and secretion of IL-6. Con A also down-regulated mRNA encoding both EP3 Rs and EP2 Rs, and concurrently up-regulated mRNA encoding EP4 Rs of HSB.2 T cells. Therefore, EP4 and EP2 Rs mediate PGE2-induced increases in IL-6 secretion by HSB.2 T cells through a transcriptional and cAMP dependent-mechanism. The increased ratio of EP4 Rs/EP3 Rs may contribute to Con A enhancement of PGE2-elicited increases in IL-6 secretion by HSB.2 T cells.

Animals↗

PubMed1998

Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate.

The lysophospholipid (LPL) mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are generated by enzymatic cleavage of stores of glycerophospholipids and sphingomyelin, respectively, in membranes of stimulated cells. LPLs are albumin bound, distributed widely in mammalian tissues, and increased in concentration by physiological activation of platelets and some other cells, tissue injury, inflammation, and neoplasia. The principal effects of LPA and S1P are growth related, including induction of cellular proliferation, alterations in differentiation and survival, and suppression of apoptosis. LPA and S1P also evoke cellular effector functions, which are dependent on cytoskeletal responses such as contraction, secretion, adhesion, and chemotaxis. The extracellular mediator activities of LPLs are transduced by subfamilies of G-protein-coupled receptors (GPCRs), of which the most completely characterized are those encoded by the endothelial differentiation genes (edgs). One homology cluster composed of Edg-1, -3, and -5 recognizes and responds to S1P, and the other cluster of Edg-2 and -4 is dedicated to LPA. Edg proteins are developmentally regulated and differ in tissue distribution, but couple similarly to multiple types of G-proteins to signal through ras and mitogen-activated protein kinase, rho, phospholipase C, and several protein tyrosine kinases. Numerous interactions between glycerophospholipids and sphingolipids are observed in their biosynthetic and signaling pathways. Many of the cellular effects of LPA and S1P are attributable to modifications in the content and/or activity of a major functional protein. Examples are increases in nuclear levels of transcription factors that regulate the serum response element, suppression of death caspase activities in apoptosis, and elevation of membrane content of heparin binding-epidermal growth factor-like growth factor, which serves as an autocrine and juxtacrine stimulus of proliferation. These ubiquitous LPL mediators of cellular growth, differentiation, and activities thus act directly through complex subfamilies of GPCRs and by regulating expression of biologically critical proteins.

Animals↗

PubMed1998

Signaling mechanisms and molecular characteristics of G protein-coupled receptors for lysophosphatidic acid and sphingosine 1-phosphate.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent phospholipid mediators with diverse biological activities. Their appearance and functional properties suggest possible roles in development, wound healing, and tissue regeneration. The growth-stimulating and other complex biological activities of LPA and S1P are attributable in part to the activation of multiple G protein-mediated intracellular signaling pathways. Several heterotrimeric G proteins, as well as Ras- and Rho-dependent pathways play central roles in the cellular responses to LPA and S1P. Recently, several G protein-coupled receptors encoded by a family of endothelial differentiation genes (edg) have been shown to bind LPA or S1P and transduce responses of cAMP, Ca2+, MAP kinases, Rho, and gene transcription. This review summarizes our current understanding of signaling pathways critical for cellular responses to LPA and S1P and of recent progress in the molecular biological analyses of the Edg receptors.

Amino Acid Sequence↗

PubMed1997

Identification of cDNAs encoding two G protein-coupled receptors for lysosphingolipids.

The structural similarity of lysosphingolipids to lysophosphatidic acid (LPA) prompted a sequence-based search for lysosphingolipid receptors using cDNA sequence of the Edg2 human LPA receptor. Two closely related G protein-coupled receptors, rat H218 and human Edg3, are highly similar to Edg2. When overexpressed in Jurkat cells, H218 and Edg3 activated serum response element-driven transcriptional reporter gene in response to sphingosine 1-phosphate (S1P), dihydro-S1P and sphingosylphosphorylcholine, but not to LPA. H218 and Edg3 expressed in Xenopus oocytes conferred responsiveness to S1P and dihydro-S1P in agonist-triggered 45Ca2+ efflux. Therefore, H218 and Edg3 are functional receptors for S1P and perhaps other closely related lysosphingolipids.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of the human Edg2 protein and its identification as a functional cellular receptor for lysophosphatidic acid.

A cDNA homologous to that encoding sheep Edg2 protein was cloned from a human lung cDNA library. The full-length sequence encodes a 364-amino acid protein which belongs to the superfamily of guanine nucleotide-binding (G) protein-coupled receptors. Human Edg2 mRNA is widely distributed in human tissues with the highest abundance in brain. HEK293 cells expressing the human Edg2 protein showed an elevated response to lysophosphatidic acid (LPA) in a serum response element reporter gene assay, which was LPA concentration-dependent and specific to LPA compared to other lysophospholipids. Over-expression of human Edg2 in CHO cells correlated with increases in specific binding of [3H]-LPA. Recently, the mouse counterpart of Edg2 protein also was identified as a receptor for LPA (Hecht et al., 1996, J. Cell Biol. 135, 1071). Therefore, it is concluded that the human Edg2 protein functions as a cellular receptor for LPA.

Amino Acid Sequence↗

PubMed1997

Upregulation of neuropeptides and neuropeptide receptors in a murine model of immune inflammation in lung parenchyma.

The lung is richly supplied with peptidergic nerves that store and secrete substance P (SP), vasoactive intestinal peptide (VIP), and other neuropeptides known to potently modulate leukocyte function in vitro and airway inflammation in vivo. To investigate and characterize neuromodulation of immune responses compartmentalized in lung parenchyma, neuropeptide release and expression of neuropeptide receptors were studied in lungs of antigen-primed C57BL/6 mice after intratracheal challenge with sheep erythrocytes. The concentrations of cytokines in bronchoalveolar lavage (BAL) fluid rose early and peaked on day 1 for interleukin (IL)-2, interferon gamma, and IL-10; days 1 to 2 for IL-6; and day 3 for IL-4, whereas the total number and different types of leukocytes in BAL fluid peaked subsequently on days 4 to 6 after i.t. antigen challenge. Immunoreactive SP and VIP in BAL fluid increased maximally to nanomolar concentrations on days 1 to 3 and 2 to 7, respectively in lungs undergoing immune responses. The high-affinity SP receptor (NK-1 R), and VIP types I (VIPR1) and II (VIPR2) receptors were localized by immunohistochemistry to surface membranes of mononuclear leukocytes and granulocytes in perivascular, peribronchiolar, and alveolar inflammatory infiltrates during immune responses. As quantified by reverse transcription-polymerase chain reaction, significant increases were observed in levels of BAL lymphocyte mRNA encoding NK-1 R (days 2 to 4), VIPR1 (days 2 to 4), and VIPR2 (days 4 to 6), and in alveolar macrophage mRNA encoding NK-1 R (days 2 to 6) and VIPR1 (days 2 to 4), but not VIPR2. Systemic treatment of mice with a selective, nonpeptide NK-1 R antagonist reduced significantly the total numbers of leukocytes, lymphocytes, and granulocytes retrieved by BAL on day 5 of the pulmonary immune response. The results indicate that SP and VIP are secreted locally during pulmonary immune responses, and are recognized by leukocytes infiltrating lung tissue, and thus their interaction may regulate the recruitment and functions of immune cells in lung parenchyma.

Animals↗

PubMed1997

Novel cyclic peptide agonist of high potency and selectivity for the type II vasoactive intestinal peptide receptor.

Ro 25-1392 [Ac-Glu8,OCH3-Tyr10,Lys12,Nle17,Ala19,A sp25,Leu26,-Lys27,28-vasoactive intestinal peptide(cyclo 21-25)] is a cyclic peptide analog of vasoactive intestinal peptide (VIP) that potently exerts cellular effects typical of VIP. The selectivity of Ro 25-1392 for type I (VIPR1) and type II (VIPR2) VIP receptors was investigated first in competitive binding studies using Chinese hamster ovary cell transfectants stably expressing recombinant human VIPR1 and VIPR2. Nonradioactive Ro 25-1392 was as potent a competitive inhibitor as VIP for the binding of 125I-VIP to VIPR2 transfectants (Ki = 9.6 +/- 1.0 and 16 +/- 1.7 nM, respectively; mean +/- S.E.M., n = 4). In contrast, Ro 25-1392 had a very low affinity for VIPR1, compared with VIP, and attained a maximum of only 40% mean inhibition of binding of 125I-VIP at 1 microM. The affinity of VIP (Ki = 3.4 +/- 1.5 nM, mean +/- S.E.M., n = 4) for binding to VIPR1 was 1000-fold greater than that of Ro 25-1392. Ro 25-1392 evoked concurrent and concentration-dependent increases in intracellular levels of calcium and cyclic AMP (EC50 = 3.0 +/- 0.4 nM, mean +/- S.E.M., n = 4) in VIPR2 transfectants, but not in VIPR1 transfectants. The VIP receptor specificity of Ro 25-1392 was confirmed by preincubation of Chinese hamster ovary transfectants with 0.1 microM Ro 25-1392 for 18 hr at 37 degrees C, to down-regulate each type of VIP receptor. Pretreatment of VIPR2 transfectants with Ro 25-1392 decreased Bmax by a mean of 58% and VIP-induced increases in the intracellular concentration of cyclic AMP by a mean of 65%. In contrast, there was no significant change in VIPR1 transfectants after pretreatment with Ro 25-1392. Ro 25-1392 thus is selectively recognized by VIPR2, with consequent initiation of cyclic AMP and Ca+2 signals and down-regulation of VIPR2. This potent analog of VIP may prove useful for investigations of VIPR2-mediated physiological effects of VIP and exploration of the roles of VIPR2 in diseases.

Animals↗

PubMed1996

Regulation of expression of matrix metalloproteinase-9 in early human T cells of the HSB.2 cultured line by the EP3 subtype of prostaglandin E2 receptor.

The expression by T lymphocytes (T cells) of more than one of the functionally distinct subtypes of prostaglandin E2 (PGE2) receptors (Rs), designated EP1, EP2, EP3, and EP4 Rs, is a principal determinant of specificity and diversity of the immune effects of PGE2. The cultured line of human leukemic T cells, termed HSB.2, co-expresses a total of 7282 +/- 1805 EP3, EP4, and EP2 Rs per cell with a Kd of 3.7 +/- 1.4 nM (mean +/- S.E., n = 9). The EP3/EP1 R-selective agonist sulprostone, EP3/EP2/EP4 R-selective agonists M&B 28767 and misoprostol, and EP2 R-selective agonist butaprost but not the EP1 R-selective antagonist SC-19220 competitively inhibited the binding of [3H]PGE2 to HSB.2 cells. Stimulation of increases in the intracellular concentration of cyclic AMP ([cAMP]i) by PGE2, misoprostol, and butaprost and of increases in the intracellular concentration of calcium ([Ca2+]i) by PGE2 and sulprostone demonstrated the respective involvement of EP2/EP4 Rs and EP3 Rs in transduction of biochemical signals. Matrix metalloproteinase (MMP)-9 was identified by zymography and Western blots as the principal MMP secreted by HSB.2 cells. The cytosolic level and secretion of MMP-9 were increased maximally after 24 h of incubation of HSB.2 cells with 10(-8)-10(-6) M PGE2, sulprostone, M&B 28767, and misoprostol but not with 10(-6) M PGF2alpha, PGD2, PGI2, or butaprost, suggesting a principal dependence on EP3 Rs. That stimulation of MMP-9 secretion by PGE2 was not diminished in Ca2+-free medium but was suppressed significantly and dose-dependently by thapsigargin, an inhibitor of endomembrane Ca2+-ATPase, suggested that MMP-9 expression by HSB.2 cells is mediated by increases in [Ca2+]i attributable to release of Ca2+ from intracellular stores. The lack of effect of dibutyryl cAMP, forskolin, and SQ 22536, an adenylyl cyclase inhibitor, on MMP-9 secretion by HSB.2 cells argued against any role for cAMP-dependent mechanisms linked to EP2/EP4 Rs. Cycloheximide and actinomycin D, which respectively inhibited protein and RNA synthesis, suppressed basal and PGE2 induction of MMP-9 production by HSB.2 cells. Northern analysis indicated that PGE2 and sulprostone time-dependently increased expression of MMP-9 mRNA. Thus, stimulation of MMP-9 in HSB.2 T cells by PGE2 is attributable to [Ca2+]i-dependent EP3 R-mediation of increases in message transcription.

Adenine↗

PubMed1996

Transduction of specific inhibition of HuT 78 human T cell chemotaxis by type I vasoactive intestinal peptide receptors.

The major immunoregulatory effects of vasoactive intestinal peptide (VIP) are mediated by structurally distinct types I (VIPR1) and II (VIPR2) G protein-associated receptors on some T cells, B cells, and macrophages. Identification of the separate immunologic activities of each type of VIPR has been complicated by the usual expression of only VIPR2 or of VIPR1 and VIPR2 together by most human T cells obtainable in sufficient number for functional analyses. The results of reverse-transcription PCR, Western blot, and [125I]VIP-binding studies have established that HuT 78 cultured human lymphoma T cells bear a mean of 75,000 VIPR1s per cell with a mean Kd of 3.3 nM, which transduce mean maximal increases in intracellular concentration of cAMP of 2.1-fold (ED50 = 72 nM), but no VIPR2s. HuT 78 T cells, in contrast to T cells that express VIPR2, did not respond to VIP by chemotaxis through micropore filters without or with a top layer of basement membrane-like Matrigel. Matrix metalloproteinase (MMP)-dependent in situ cleavage of [3H]type IV human collagen in the layer of Matrigel by HuT 78 T cells also was not stimulated by VIP. In contrast, IL-4 and TNF-alpha both stimulated HuT 78 T cell chemotaxis and in situ MMP activity at respective optimal concentrations ranging from 3 x 10(-10) M to 3 x 10(-9) M and 10(-10) M to 3 x 10(-10) M. VIP inhibited significantly HuT 78 T cell chemotaxis through Matrigel in response to both IL-4 and TNF-alpha, as a result of suppression of both chemotactic mobility, assessed by migration through micropore filters without Matrigel, and in situ MMP activity. The transduction of opposite effects of VIP on T cell migration through a model basement membrane by VIPR1 and VIPR2 suggests that the net chemotactic response of most T cells to VIP is determined by the VIPR2/VIPR1 ratio and that the predominant expression of VIPR1 would stabilize T cell populations in lymphoid follicles and tissue infiltrates.

Base Sequence↗

PubMed1996

Selective regulation of RNK-16 cell matrix metalloproteinases by the EP4 subtype of prostaglandin E2 receptor.

Cell surface expression of multiple structurally and functionally distinct prostaglandin E2 (PGE2) receptors (Rs), designated the EP1, EP2, EP3, and EP4 Rs, is a principal determinant of the diverse cellular effects of PGE2. The RNK-16 line of rat large granular lymphocytes, which has served as a model for natural killer cells, coexpresses a mean of 1092 EP3 Rs and EP4 Rs per cell with a mean Kd of 2.7 nM. The presence of the EP3 and EP4 Rs and the absence of the EP1 and EP2 Rs were revealed by inhibition of [3H]PGE2 binding by the EP3/EP1R agonist sulprostone, the EP3/EP2/EP4R agonist M&B 28767, and the EP2/EP4/EP3R agonist misoprostol but not by the EP1R antagonist SC-19220 or the EP2R agonist butaprost. Functional EP4 R expression was confirmed by finding that PGE2 and misoprostol, but not butaprost or sulprostone, evoked increases in the intracellular concentration of cyclic AMP ([cAMP]) in RNK-16 cells. Matrix metalloproteinase (MMP)-1 and -3 were identified by zymography and Western blots as the principal MMPs secreted by RNK-16 cells. Secretion of both MMPs by RNK-16 cells attained a maximal level after 24 h of incubation and was enhanced significantly by 10(-9) to 10(-7) M PGE2, 10(-6) M misoprostol, and 10(-4) M dibutyryl cyclic AMP, but not by the EP3R agonist sulprostone. Thus, the effect of PGE2 on RNK-16 cell MMP secretion is mediated by an EP4 R-dependent mechanism involving increases in [cAMP]i. The migration of RNK-16 cells across micropore filters, without or with a layer of Matrigel, was stimulated chemokinetically by PGE2 and misoprostol. PGE2-elicited chemokinesis of RNK-16 cells across a Matrigel model basement membrane, but not across a microfilter alone, was suppressed by the GM 6001 inhibitor of MMP activities. Stimulation of MMP activities in RNK-16 cells by the EP4R thus facilitates migration of the NK cells across vascular basement membranes.

Alprostadil↗

PubMed1996

Integrin-dependent role of human T cell matrix metalloproteinase activity in chemotaxis through a model basement membrane.

Human T lymphoblastoma cells of the CD4+ 8+ Tsup-1 line, that express alpha 4 and alpha 5 but not alpha 6 integrins of the beta 1 family, and CD4+ human blood T cells bind vasoactive intestinal peptide (VIP) with high affinity, leading to increased adherence, secretion of matrix metalloproteinases (MMPs), and chemotaxis. VIP-enhanced adherence of T cells to fibronectin was inhibited significantly by neutralizing monoclonal antibodies to beta 1 > alpha 4 > > alpha 5, but not to alpha 6. Antibodies to beta 1 and alpha 4 suppressed to a similarly significant extent VIP stimulation of both MMP-dependent T cell chemotaxis through fibronectin-enriched Matrigel and T cell degradation of 3H-type IV collagen in the Matrigel, without affecting VIP-evoked secretion of MMP by suspensions of T cells. The lesser inhibition of VIP-enhanced adherence of T cells to fibronectin by anti-alpha 5 antibody, than antibodies to beta 1 or alpha 4 chains, was associated with lesser or no suppression of MMP-dependent T cell chemotaxis through Matrigel and T cell degradation of type IV collagen in the Matrigel in response to VIP. Specific beta 1 integrins thus mediate interactions of stimulated T cells with basement membranes, including adherence, localized digestion by MMPs, and chemotactic passage, that promote entry of T cells into extravascular tissues.

Antibodies, Blocking↗