Microarray Profiling of Lymphocytes in Internal Diseases With an Altered Immune Response: Potential and Methodology

Recently it has become possible to investigate expression of all human genes with microarray technique. The authors provide arguments to consider peripheral white blood cells and in particular lymphocytes as a model for the investigation of pathophysiology of asthma, RA, and SLE diseases in which inflammation is a major component. Lymphocytes are an alternative to tissue biopsies that are most often difficult to collect systematically. Lymphocytes express more than 75% of the human genome, and, being an important part of the immune system, they play a central role in the pathogenesis of asthma, RA, and SLE. Here we review alterations of gene expression in lymphocytes and methodological aspects of the microarray technique in these diseases. Lymphocytic genes may become activated because of a general nonspecific versus disease-specific mechanism. The authors suppose that in these diseases microarray profiles of gene expression in lymphocytes can be disease specific, rather than inflammation specific. Some potentials and pitfalls of the array technologies are discussed. Optimal clinical designs aimed to identify disease-specific genes are proposed. Lymphocytes can be explored for research, diagnostic, and possible treatment purposes in these diseases, but their precise value should be clarified in future investigation

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

Background: In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.Methods: Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase ( MAPK) and cyclooxygenase ( COX) to these reponses was established, using the inhibitors Y-27632 ( 1 mu M), U-0126 ( 3 mu M) and indomethacin ( 3 mu M), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F-2 alpha (PGF(2 alpha)) and prostaglandin E-2 (PGE(2)) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 mu M) and the selective EP1-antagonist AH-6809 (10 mu M) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF(2 alpha)- and PGE(2)-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.Results: Epidermal growth factor (EGF)- and platelet-derived growth factor ( PDGF)- induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF(2 alpha)- and PGE(2)-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2a- and PGE(2)-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 ( 10 mu M) significantly reduced ( approximately 50 %) and the EP1-antagonist AH-6809 ( 10 mu M) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.Conclusion: The results indicate that growth factors induce ASM contraction through contractile prostaglandins - not derived from the epithelium - which in turn rely on Rho-kinase for their contractile effects.</p

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

Changes in the ECM and increased airway smooth muscle ( ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle ( BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction ( Emax) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I- induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin- induced depression of contractility was blocked by the integrin antagonist Arg- Gly- Asp- Ser ( RGDS) but not by its negative control Gly- Arg- Ala- Asp- Ser- Pro ( GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM- induced changes in Emax in BTSM strips and their proliferative responses in BSTM cells and for Emax and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM

Muscarinic M3-receptors mediate cholinergic synergism of mitogenesis in airway smooth muscle

Muscarinic receptor agonists have been considered to act syner- and cytoskeletal reorganization has been proposed (4–6). gistically in combination with growth facors on airway smooth Furthermore, M2-receptors may stimulate nonselective catmuscle growth. Characterization of the proliferative responses ion channels through Gi/G(o) proteins, resulting in a rise in and of the receptor subtype(s) involved has not yet been stud- [Ca2�]i (7). ied. Therefore, we investigated mitogenesis induced by stimula- Muscarinic receptor agonists have been reported to be tion of muscarinic receptors, alone and in combination with mitogenic for human ASM cells, though at most modestly, stimulation by platelet-derived growth factor (PDGF). For this and to respond synergistically in combination with growth purpose, [ 3H]thymidine-incorporation was measured at differfactors (8, 9). Although carbachol-induced mitogenesis has ent culture stages in bovine tracheal smooth muscle cells. Funcbeen reported to be pertussis toxin (PTX)-sensitive (8, 10), tional muscarinic M3-receptors, as measured by formation of suggesting a role for the Gi-protein–coupled muscarinic M2inositol phosphates, were present in unpassaged cells, but were receptor, measurements were performed using human AS

Bradykinin augments EGF-induced airway smooth muscle proliferation by activation of conventional protein kinase C isoenzymes

This study aims to investigate the effects of bradykinin, alone and in combination with growth factors on proliferation of cultured bovine tracheal smooth muscle cells. Bradykinin did not induce mitogenic responses by itself, but concentration-dependently augmented growth factor-induced [H-3]thymidine incorporation and cell proliferation. The bradykinin effect was mediated by bradykinin B? receptors, and not dependent on cyclo-oxygenase. Bradykinin-induced synergism with epidermal growth factor (EGF) could be suppressed by the protein kinase C (PKC) inhibitors GF 109203X (Bisindolylmaleimide 1; specific for conventional and novel PKCs) and Go 6976 (12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; specific for conventional PKCs). In addition, sole activation of PKC using Phorbol 12-myristate 13-acetate (PMA) was sufficient for a synergistic interaction with EGF. In contrast to bradykinin however, PMA was mitogenic by itself which was not at all affected by Go 6976, but abolished by GF 109203X. Bradykinin transiently activated the p42/p44 MAP kinase pathway, whereas PMA-induced activation of p42/p44 mitogen activated protein (MAP) kinase was sustained. Neither the combination of bradykinin and EGF nor that of PMA and EGF induced synergistic activation of p42/p44 MAP kinase, however. These results show that bradykinin B, receptor-stimulation augments growth factor-induced mitogenic responses of airway smooth muscle cells through activation of conventional PKC isozymes. In addition, the results show that PKC isozyme-specificity underlies stimulus-specific differences in mitogenic capacity for bradykinin and PMA. Published by Elsevier B.V

Cloning, pharmacological characterization, and polymorphism screening of the guinea pig β2-adrenoceptor

In asthma, beta(2)-adrenoceptor agonist responsiveness has been associated with Arg16Gly and Gln27Glu polymorphisms of the beta(2)-adrenoceptor. Since the guinea pig is extensively used as an animal model for asthma, we investigated the occurrence of possible polymorphism of the guinea pig beta(2)-adrenoceptor. The guinea pig beta(2)-adrenoceptor coding region was amplified by sequence homology-based cloning. Homology of the translated protein with the human beta(2)-adrenoceptor was 88% with Ala at position 16 and Glu at position 27. Radioligand binding and cAMP-accumulation experiments of Chinese hamster ovary (CHO) cells transfected with the guinea pig beta(2)-adrenoceptor revealed a homogeneous population of functional receptors. Five degenerate single nucleotide polymorphisms were found within the beta(2)-adrenoceptor coding region of outbred Dunkin Hartley guinea pigs, at residues 354, 453, 483, 534 and 642. In conclusion, we have cloned the guinea pig beta(2)-adrenoceptor, which shows to be functional upon expression in a recombinant system and contains five single nucleotide polymorphisms dissimilar to human polymorphisms. (C) 2002 Elsevier Science B.V All rights reserved

(Endo)cannabinoids mediate different Ca(2+) entry mechanisms in human bronchial epithelial cells

In human bronchial epithelial (16HBE14o) cells, CB(1) and CB(2) cannabinoid receptors are present, and their activation by the endocannabinoid virodhamine and the synthetic non-selective receptor agonist CP55,940 inhibits adenylyl cyclase and cellular interleukin-8 release. Here, we analyzed changes in intracellular calcium ([Ca(2+)](i)) evoked by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), CP55,940, and virodhamine in 16HBE14o(-) cells. Delta(9)-THC induced [Ca(2+)](i) increase and a large transient [Ca(2+)](i) mobilization, the latter probably reflecting store-depletion-driven capacitative Ca(2+) entry (CCE). In contrast, CP55,940 induced a rather moderate Ca(2+) influx and a sustained [Ca(2+)](i) mobilization. CP55,940-induced Ca(2+) influx was inhibited by Ni(2+), indicating CCE, possibly mediated by transient receptor potential channel TRPC1, the mRNA of which is expressed in 16HBE14o(-) cells. CP55,940-induced calcium alterations were mimicked by virodhamine concentrations below 30 mu M. Interestingly, higher virodhamine induced an additional Ca2+ entry, insensitive to Ni(2+), but sensitive to the TRPV1 antagonist capsazepine, the TRPV1-TRPV4 inhibitor ruthenium red, and the non-CCE (NCCE) inhibitors La(3+) and Gd(3+). Such pharmacological profile is supported by the presence of TRPV1, TRPV4, and TRPC6 mRNAs as well as TRPV1 and TRPC6 proteins in 16HBE14o(-) cells. Cannabinoid receptor antagonists increased virodhamine-induced Ca(2+) entry. Virodhamine also enhanced arachidonic acid release, which was insensitive to cannabinoid receptor antagonism, but sensitive to the phospholipase A(2) inhibitor quinacrine, and to capsazepine. Arachidonic acid induced [Ca(2+)](i) increase similar to virodhamine. Collectively, these observations suggest that [Ca(2+)](i) alterations induced by Delta(9)-THC, CP55,940 and by low concentrations of virodhamine involve mobilization and subsequent CCE mechanisms, whereas such responses by high virodhamine concentrations involve NCCE pathways