Role and regulation of interleukin-1 molecules in pro-asthmatic sensitised airway smooth muscle.

Interleukin (IL)-1beta is a pleiotropic, pro-inflammatory cytokine that has been importantly implicated in driving the inflammatory response and resultant changes in airway smooth muscle (ASM) responsiveness in asthma. IL-1beta belongs to a family of molecules, known as the IL-1 axis, which exert both pro- and anti-inflammatory effects. Since dysregulation of IL-1 axis molecules may be critical in the pathobiology of asthma, the present study examined the expression and activation of both the inhibitory and stimulatory IL-1 axis molecules in human ASM cells and their roles in modulating cytokine and immunoglobulin (Ig)E immune complex (IgE cx)-mediated changes in rabbit ASM constrictor and relaxant responsiveness. The results demonstrate the following. 1) Pre-treatment of isolated rabbit tracheal rings with the inhibitory IL-1 axis members, IL-1 receptor antagonist and IL-1 type-II receptor abrogated both IL-5- and IgE cx-induced changes in ASM responsiveness. 2) Administration of IL-5, IL-1beta and IgE cxs to human ASM cells increased mRNA and protein expressions of both stimulatory and inhibitory IL-1 axis molecules. 3) The time course of IL-5-induced IL-1 axis molecule expression preceded that of both IL-1beta and IgE immune cxs. Collectively, these findings suggest that modulation at the level of the interleukin-1 axis of molecules may have significant therapeutic potential in the treatment of asthma.

IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle.

In testing the hypothesis that interleukin-4 receptor alpha-subunit (IL-4R alpha)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4R alpha blocking antibody (anti-IL-4R alpha Ab). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4R alpha Ab. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to naïve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4R alpha-coupled release and action of IL-5 in the sensitized ASM itself.

Rhinovirus elicits proasthmatic changes in airway responsiveness independently of viral infection.

BACKGROUND: Rhinovirus (RV), the principal pathogen responsible for the common cold, is importantly implicated in triggering attacks of asthma secondary to changes in airway responsiveness. OBJECTIVE: Because the airway histopathologic features of RV infection are relatively modest, we tested the hypothesis that RV can directly elicit proasthmatic-like changes in airway smooth muscle (ASM) responsiveness independently of actual viral infection and its associated cytopathic effects. METHODS: Isolated ASM tissues and cultured ASM cells were inoculated with either infectious or noninfectious (UV-irradiated) RV16 and RV2, the latter serotypes belonging to the "major" and "minor" groups of RV subtypes, respectively. ASM constrictor and relaxant responsiveness, G(i) protein expression, and proinflammatory cytokine release were subsequently compared under the different treatment conditions. RESULTS: In contrast to RV2, which had no effect, RV16 inoculation elicited enhanced ASM contractility and impaired relaxation to cholinergic and beta-adrenergic agonists, respectively, in association with increased ASM membrane G(i) protein expression and induced release of the proinflammatory cytokines IL-5 and IL-1beta. These proasthmatic-like effects were also observed in ASM exposed to UV-irradiated RV16, wherein viral replication was completely inhibited. In contrast, pretreatment of ASM with a neutralizing antibody directed against ICAM-1, the host receptor for the "major" group of RVs, completely abrogated the proasthmatic effects of RV16. CONCLUSIONS: The results demonstrate that (1) RV16 elicits proasthmatic changes in ASM responsiveness that can occur independently of actual viral infection of the ASM and (2) the effects of RV16 are attributed solely to binding of the virus to its host receptor (ICAM-1) on the ASM cell surface. Collectively, these findings support the notion that RV-induced exacerbation of wheezing in asthmatic individuals can occur even in the absence of any cytopathology associated with viral infection.

Association between IL-1beta/TNF-alpha-induced glucocorticoid-sensitive changes in multiple gene expression and altered responsiveness in airway smooth muscle.

The pleiotropic cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha have been implicated in the pathophysiology of asthma. To elucidate the role of these cytokines in the pro-asthmatic state, the effects of IL-1beta and TNF-alpha on airway smooth muscle (ASM) responsiveness and ASM expression of multiple genes, assessed by high-density oligonucleotide array analysis, were examined in the absence and presence of the glucocorticoid dexamethasone (DEX). Administration of IL-1beta/TNF-alpha increased ASM contractility to acetylcholine and impaired ASM relaxation to isoproterenol. These pro-asthmatic- like changes in ASM responsiveness were associated with IL-1beta/ TNF-alpha-induced mRNA expression of a host of proinflammatory genes that regulate transcription, cytokines and chemokines, cellular adhesion molecules, and various signal transduction molecules that regulate ASM responsiveness. In the presence of DEX, the changes induced in ASM responsiveness were abrogated, and most of the IL-1beta/TNF-alpha-mediated changes in proinflammatory gene expression were repressed, although mRNA expression of a small number of genes was enhanced by DEX. Collectively, the observations support the concept that, together with its role as a regulator of airway tone, in response to IL-1beta/TNF-alpha, the ASM expresses a host of glucocorticoid-sensitive genes that contribute to the altered structure and function of the airways in the pro-asthmatic state. We speculate that glucocorticoid-sensitive, cytokine-induced pathways involved in ASM cell signaling represent important targets for new therapeutic interventions.

Autocrine signaling by IL-10 mediates altered responsiveness of atopic sensitized airway smooth muscle.

To elucidate the role and mechanism of action of interleukin (IL)-10 in regulating airway smooth muscle (ASM) responsiveness in the atopic asthmatic state, isolated rabbit tracheal ASM segments were passively sensitized with serum from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects in both the absence and presence of an anti-IL-10 receptor blocking antibody (Ab). Relative to control ASM, atopic asthmatic serum-sensitized tissues exhibited enhanced isometric constrictor responses to administered acetylcholine and attenuated the relaxation responses to isoproterenol. These proasthmatic effects were prevented in atopic asthmatic serum-sensitized ASM that was pretreated with anti-IL-10 receptor Ab. In complementary experiments, exposure of cultured human ASM cells to atopic asthmatic serum induced upregulated expression of IL-10 mRNA. Moreover, extended studies demonstrated that 1) exogenous IL-10 administration to naive ASM elicited augmented contractility to acetylcholine and impaired relaxation to isoproterenol, 2) these effects of IL-10 were prevented by pretreating the tissues with an IL-5 receptor Ab, and 3) IL-10 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an intrinsic T helper type 2-type autocrine mechanism involving IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.

Methacholine-induced temporal changes in airway geometry and lung density by CT.

PURPOSE: Electron-beam CT (EBCT) was utilized to assess the time course of changes in airways cross-sectional area (CSA) and lung density during methacholine-induced bronchoconstriction. MATERIALS AND METHODS: EBCT scans (200 ms, 3-mm thickness, 2 mm increments) were obtained before (baseline) and 30 s, 2 min, and 4 min after bolus IV injection of methacholine to pigs receiving mechanical ventilation. A total of seven experiments were analyzed using custom-made image analysis software. With each challenge, five different airways and 50 lung regions of interest were studied. RESULTS: The time course of lung density changes paralleled the time course for CSA changes. The maximal response to methacholine, measured in terms of both CSA and lung density changes, occurred 30 s after injection. Lung density changes were unaffected by reconstruction algorithm, normal (standard) or sharp (high resolution). Overall, there was increased air content in the lung during bronchoconstriction. This effect was significantly greater at the dependent lung regions. CONCLUSIONS: EBCT is an effective tool to assess temporal and regional changes in the lung during bronchoconstriction. Measurements of lung density during bronchoconstriction allow for assessment of peripheral changes that are beyond the CT spatial resolution of airways anatomy.

Mechanism of cooperative effects of rhinovirus and atopic sensitization on airway responsiveness.

To elucidate the mechanistic interplay between rhinovirus (RV) exposure and atopic sensitization in regulating airway smooth muscle (ASM) responsiveness, isolated rabbit ASM tissue and cultured human ASM cells were passively sensitized with sera from atopic asthmatic or nonatopic nonasthmatic (control) subjects in the absence and presence of inoculation with RV serotype 16. Relative to control subjects, atopic asthmatic serum-sensitized and RV-inoculated ASM exhibited significantly increased contractility to acetylcholine, impaired relaxation to isoproterenol, and enhanced release of the proinflammatory cytokine interleukin-1beta. These effects were potentiated in atopic asthmatic serum-sensitized ASM concomitantly inoculated with RV and inhibited by pretreating the tissues with monoclonal blocking antibodies against intercellular adhesion molecule (ICAM)-1 (CD54), the host receptor for RV serotype 16, or lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18), the endogenous counterreceptor for ICAM-1. Moreover, RV inoculation was found to potentiate the induction of mRNA and surface protein expression of FcepsilonRII (CD23), the low-affinity receptor for IgE, in atopic asthmatic serum-sensitized ASM. Collectively, these observations provide new evidence demonstrating that 1) RV exposure and atopic sensitization act cooperatively to potentiate induction of proasthmatic changes in ASM responsiveness in association with upregulated proinflammatory cytokine release and FcepsilonRII expression and 2) the effects of RV exposure and atopic sensitization are mediated by cooperative ICAM-1-coupled LFA-1 signaling in the ASM itself.

Bi-directional activation between human airway smooth muscle cells and T lymphocytes: role in induction of altered airway responsiveness.

Because both T lymphocyte and airway smooth muscle (ASM) cell activation are events fundamentally implicated in the pathobiology of asthma, this study tested the hypothesis that cooperative intercellular signaling between activated T cells and ASM cells mediates proasthmatic changes in ASM responsiveness. Contrasting the lack of effect of resting human T cells, anti-CD3-activated T cells were found to adhere to the surface of naive human ASM cells, increase ASM CD25 cell surface expression, and induce increased constrictor responsiveness to acetylcholine and impaired relaxation responsiveness to isoproterenol in isolated rabbit ASM tissues. Comparably, exposure of resting T cells to ASM cells prestimulated with IgE immune complexes reciprocally elicited T cell adhesion to ASM cells and up-regulated T cell expression of CD25. Extended studies demonstrated that: 1) ASM cells express mRNAs and proteins for the cell adhesion molecules (CAMs)/costimulatory molecules, CD40, CD40L, CD80, CD86, ICAM-1 (CD54), and LFA-1 (CD11a/CD18); 2) apart from LFA-1, ASM cell surface expression of the latter molecules is up-regulated in the presence of activated T cells; and 3) pretreatment of ASM cells and tissues with mAbs directed either against CD11a or the combination of CD40 and CD86 completely abrogated both the activated T cell-induced changes in expression of the above CAMs/costimulatory molecules in ASM cells and altered ASM tissue responsiveness. Collectively, these observations identify the presence of bi-directional cross-talk between activated T cells and ASM cells that involves coligation of specific CAMs/costimulatory molecules, and this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness.

Autocrine cytokine signaling mediates effects of rhinovirus on airway responsiveness.

The airway responses to allergen exposure in allergic asthma are qualitatively similar to those elicited by specific viral respiratory pathogens, most notably rhinovirus (RV), suggesting that the altered airway responsiveness seen in allergic asthma and that elicited by viral respiratory tract infection may share a common underlying mechanism. To the extent that T helper cell type 2 (Th2) cytokines have been implicated in the pathogenesis of allergic asthma, this study examined the potential role(s) of Th2-type cytokines in mediating pro-asthmatic-like changes in airway smooth muscle (ASM) responsiveness after inoculation of naive ASM with human RV. Isolated rabbit ASM tissues and cultured human ASM cells were exposed to RV (serotype 16) for 24 h in the absence and presence of monoclonal blocking antibodies (MAbs) or antagonists directed against either the Th2-type cytokines interleukin (IL)-4 and IL-5, intercellular adhesion molecule (ICAM)-1 (the endogenous host receptor for most RVs), or the pleiotropic proinflammatory cytokine IL-1beta. Relative to control (vehicle-treated) tissues, RV-exposed ASM exhibited significantly enhanced isometric contractility to acetylcholine and impaired relaxation to isoproterenol. These pro-asthmatic-like changes in ASM responsiveness were ablated by pretreating the RV-exposed tissues with either IL-5-receptor-alpha blocking antibody or human recombinant IL-1-receptor antagonist, whereas IL-4 neutralizing antibody had no effect. Extended studies further demonstrated that inoculation of ASM cells with RV elicited 1) an increased mRNA expression and release of IL-5 protein, which was inhibited in the presence of anti-ICAM-1 MAb, and 2) an enhanced release of IL-1beta protein, which was inhibited in the presence of IL-5 receptor-alpha antibody. Collectively, these observations provide new evidence demonstrating that RV-induced changes in ASM responsiveness are largely attributed to ICAM-1-dependent activation of a cooperative autocrine signaling mechanism involving upregulated IL-5-mediated release of IL-1beta by the RV-exposed ASM itself.

Intrinsic ICAM-1/LFA-1 activation mediates altered responsiveness of atopic asthmatic airway smooth muscle.

Cell adhesion molecules (CAMs) have been importantly implicated in the pathobiology of the airway responses in allergic asthma, including inflammatory cell recruitment into the lungs and altered bronchial responsiveness. To elucidate the mechanism of CAM-related mediation of altered airway responsiveness in the atopic asthmatic state, the expressions and actions of intercellular adhesion molecule-1 (ICAM-1) and its counterreceptor ligand lymphocyte function-associated antigen-1 (LFA-1; i.e., CD11a/CD18) were examined in isolated rabbit airway smooth muscle (ASM) tissues and cultured human ASM cells passively sensitized with sera from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with a monoclonal blocking antibody (MAb) to either ICAM-1 or CD11a, whereas a MAb directed against the related beta(2)-integrin Mac-1 had no effect. Moreover, relative to control tissues, atopic asthmatic sensitized ASM cells displayed an autologously upregulated mRNA and cell surface expression of ICAM-1, whereas constitutive expression of CD11a was unaltered. Extended studies further demonstrated that 1) the enhanced expression and release of soluble ICAM-1 by atopic sensitized ASM cells was prevented when cells were pretreated with an interleukin (IL)-5-receptor-alpha blocking antibody and 2) administration of exogenous IL-5 to naive (nonsensitized) ASM cells induced a pronounced soluble ICAM-1 release from the cells. Collectively, these observations provide new evidence demonstrating that activation of the CAM counterreceptor ligands ICAM-1 and LFA-1, both of which are endogenously expressed in ASM cells, elicits autologously upregulated IL-5 release and associated changes in ICAM-1 expression and agonist responsiveness in atopic asthmatic sensitized ASM.

Altered expression and action of the low-affinity IgE receptor FcepsilonRII (CD23) in asthmatic airway smooth muscle.

BACKGROUND: Changes in cell surface expression of certain immunoglobulin Fc receptors have been demonstrated in leukocytes isolated from the lungs of atopic asthmatic individuals. This, together with emerging evidence that Fc receptors can also be expressed and activated in non-bone marrow-derived cell types, including airway smooth muscle (ASM), raises the hypothesis that the atopic asthmatic ASM phenotype is associated with an altered endogenous expression and action of specific Fc receptors present in the ASM itself. OBJECTIVE: The current study addressed the above hypothesis by examining (1) whether the expression of certain key Fc receptor subtypes for IgE and IgG is altered in ASM tissue isolated from human atopic asthmatic individuals and (2) whether this altered Fc receptor expression is comparably induced in naive human ASM tissue and cultured cells after their passive sensitization with human atopic asthmatic serum or IgE immune complexes. METHODS: Messenger RNA and cell surface protein expression of the individual IgG receptor subtypes FcgammaRI, FcgammaRII, and FcgammaRIII, as well as the IgE receptor subtypes FcepsilonRI and FcepsilonRII, were examined in human ASM tissue isolated from atopic asthmatic and control (nonatopic/nonasthmatic) individuals. In addition, we examined the effects of passive sensitization of ASM tissue and cultured ASM cells with control serum, atopic asthmatic serum, or exogenously administered IgE immune complexes on Fc receptor expression and action (ie, induction of proinflammatory cytokine release). RESULTS: The observations demonstrate that (1) human ASM tissue expresses messenger RNA and surface protein for FcepsilonRII, as well as for all the Fcgamma receptor subtypes, (2) in contrast to unaltered Fcgamma subtype expression, however, relative to control human ASM, FcepsilonRII is significantly up-regulated in inherently asthmatic ASM tissue, (3) up-regulated expression of FcepsilonRII represents, at least in part, an inducible phenomenon that is largely attributed to IgE immune complex-coupled activation of the receptor, and (4) the latter action is associated with FcepsilonRII-induced autologous elaboration of the proinflammatory cytokine, IL-1beta, by the atopic sensitized ASM. CONCLUSION: These observations provide new evidence that human ASM tissue expresses FcepsilonRII in addition to all 3 subtypes of Fcgamma receptors and that the expression of FcepsilonRII is selectively increased in atopic asthmatic ASM, a phenomenon associated with IgE immune complex/FcepsilonRII-mediated elaboration of IL-1beta by the ASM itself.

Autocrine interaction between IL-5 and IL-1beta mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle.

T-helper type 2 (Th2) cytokines have been implicated in the pathogenesis of the pulmonary inflammatory response and altered bronchial responsiveness in allergic asthma. To elucidate the mechanism of Th2-dependent mediation of altered airway responsiveness in the atopic asthmatic state, the expression and actions of specific cytokines were examined in isolated rabbit and human airway smooth muscle (ASM) tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal isometric contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with either an IL-5 receptor blocking antibody (IL-5ra) or the human recombinant IL-1 receptor antagonist (IL-1ra), whereas an IL-4 neutralizing antibody had no effect. Moreover, relative to controls, atopic asthmatic sensitized ASM cells demonstrated an initial, early (after 3 hours of incubation) increased mRNA expression and protein release of IL-5. This was followed (after 6 hours of incubation) by an enhanced mRNA expression and release of IL-1beta protein, an effect that was inhibited in sensitized cells pretreated with IL-5ra. Extended studies demonstrated that naive ASM exposed to exogenously administered IL-5 exhibited an induced upregulated mRNA expression and protein release of IL-1beta associated with proasthmatic-like changes in ASM constrictor and relaxant responsiveness, and that these effects were ablated in tissues pretreated with IL-1ra. Taken together, these observations provide new evidence that (a) the Th2 cytokine IL-5 and the pleiotropic proinflammatory cytokine IL-1beta are endogenously released by atopic asthmatic sensitized ASM and mechanistically interact to mediate the proasthmatic perturbations in ASM responsiveness; and (b) the nature of this interaction is given by an initial endogenous release of IL-5, which then acts to induce the autologous release of IL-1beta by the sensitized ASM itself, resulting in its autocrine manifestation of the proasthmatic phenotype.

Rhinovirus-mediated changes in airway smooth muscle responsiveness: induced autocrine role of interleukin-1beta.

An important interplay exists between specific viral respiratory pathogens, most commonly rhinovirus (RV), and altered airway responsiveness in the development and exacerbations of asthma. Given that RV infection reportedly induces the release of various cytokines in different cell types and that the reported effects of RV on airway smooth muscle (ASM) responsiveness are highly comparable to those obtained in ASM exposed to the proinflammatory cytokine interleukin (IL)-1beta, this study examined whether RV (serotype 16)-mediated pertubations in ASM responsiveness are mechanistically coupled to altered induced expression and action of IL-1beta in RV-exposed isolated rabbit and human ASM tissue and cultured cells. Relative to control tissues, ASM inoculated with RV exhibited significantly increased maximal isometric contractility to ACh (P < 0.01) and attenuated relaxation to isoproterenol (P < 0. 005). In extended studies, we found that 1) the RV-induced changes in ASM responsiveness were ablated by pretreating the tissues with the IL-1 recombinant human receptor antagonist; 2) in contrast to their respective controls, RV-inoculated ASM tissue and cultured cells exhibited progressively induced expression of IL-1beta mRNA and elaboration of IL-1beta protein at 6 and 24 h after viral exposure; and 3) the latter effect of RV was inhibited in the presence of a monoclonal antibody to intercellular adhesion molecule-1, the endogenous receptor for most RV. Collectively, these observations provide new evidence demonstrating that "pro-asthmatic-like" pertubations in agonist responsiveness elicited in RV-exposed ASM are largely attributed to the induced autologous expression and autocrine action of IL-1beta in the virus-infected ASM.

Regulation of TH1- and TH2-type cytokine expression and action in atopic asthmatic sensitized airway smooth muscle.

CD4(+) T helper (TH)1- and TH2-type cytokines reportedly play an important role in the pathobiology of asthma. Recent evidence suggests that proasthmatic changes in airway smooth muscle (ASM) responsiveness may be induced by the autocrine release of certain proinflammatory cytokines by the ASM itself. We examined whether TH1- and TH2-type cytokines are expressed by atopic asthmatic sensitized ASM and serve to autologously regulate the proasthmatic phenotype in the sensitized ASM. Expression of these cytokines and their receptors was examined in isolated rabbit and human ASM tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or control subjects. Relative to controls, atopic sensitized ASM cells exhibited an early increased mRNA expression of the TH2-type cytokines, interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and their receptors. This was later followed by enhanced mRNA expression of the TH1-type cytokines, IL-2, IL-12, and interferon-gamma (IFN-gamma), as well as their respective receptors. In experiments on isolated ASM tissue segments (a) exogenous administration of IL-2 and IFN-gamma to atopic asthmatic serum-sensitized ASM ablated both their enhanced constrictor responsiveness to acetylcholine (ACh) and their attenuated relaxation responsiveness to beta-adrenoceptor stimulation with isoproterenol, and (b) administration of IL-5 and GM-CSF to naive ASM induced significant increases in their contractility to ACh and impaired their relaxant responsiveness to isoproterenol. Collectively, these observations provide new evidence demonstrating that human ASM endogenously expresses both TH1- and TH2-type cytokines and their receptors, that these molecules are sequentially upregulated in the atopic asthmatic sensitized state, and that they act to downregulate and upregulate proasthmatic perturbations in ASM responsiveness, respectively.

Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle.

We have previously demonstrated that the asthma-associated proinflammatory eicosanoid leukotriene D4 (LTD4) is co-mitogenic with insulin-like growth factors (IGFs) in airway smooth-muscle (ASM) cells in vitro. This synergistic effect of LTD4 and IGF on ASM cell growth involves proteolysis of ASM-produced IGF binding proteins (IGFBPs), which are cell growth-inhibitory proteins. We also identified this IGFBP protease to be the matrix metalloproteinase-1 (MMP-1), and showed that this enzyme had a significant role in modulating IGF action in ASM cells. In the present study, we tested the hypothesis that ASM hyperplasia in vivo involves induction of MMP-1 leading to IGFBP proteolysis. We detected the presence of MMP-1 and measured its levels in human airway tissue sections prepared from nonasthmatic and asthmatic subjects. Six nonasthmatic and six asthmatic airway tissue samples were analyzed for immunoreactive MMP-1 through an immunohistochemical detection method. Both the bronchial and tracheal smooth-muscle cells from different regions of the same sample were examined and documented. The immunostaining for MMP-1 was significantly elevated in both the bronchial and tracheal smooth-muscle cells of the airway sections from asthmatic samples relative to that of the nonasthmatic samples. The differences in levels of MMP-1, IGFBP-2, IGFBP-3, and IGFBP proteolytic activity were quantified using densitometric analyses of the ASM tissue extracts that were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MMP-1 levels in the asthmatic airway tissue extracts were 12-fold higher than those found in control samples. In addition, IGFBP-2 and IGFBP-3, which we have previously demonstrated to be proteolytic substrates of MMP-1, were found to be cleaved in asthmatic airway tissue extracts. Furthermore, the asthmatic airway extracts contained IGFBP proteolytic activity that was shown by immunodepletion studies to be due to MMP-1. These observations demonstrate that MMP-1 may play a significant role in inducing ASM hyperplasia and airway obstruction in asthma by modulating the IGF axis.

Regulation of second messengers associated with airway smooth muscle contraction and relaxation.

Agonist-receptor interactions regulate airway smooth muscle tone through activation of guanine nucleotide binding proteins (G proteins), which are coupled to second messenger pathways that mediate changes in the tissue's contractile state. With respect to airway smooth muscle (ASM) contraction, receptor activation elicits phosphatidylinositol turnover that results in the formation of the second messengers, 1,2,-diacylglyserol, which activates protein kinase C (PKC), and inositol 1,4,5,-trisphosphate (Ins[1,4,5]P3), which binds to its intracellular receptor to mobilize intracellular calcium (Ca2+). Both the mobilization of Ca2+ and activation PKC play critical roles in initiating and acutely modulating the intensity and duration of the ASM contraction response. In contrast, bronchodilator agonist-mediated receptor activation is typically coupled to an enhanced accumulation of the second messenger, adenosine 3',5'-cyclic monophosphate (cAMP) which, through activation of cAMP-dependent protein kinase, induces the phosphorylation of specific proteins, leading to ASM relaxation. For activation of both of these functionally distinct signal transduction pathways, the agonist-receptor complexes interact with specific G proteins, which in turn modulate the enzymes regulating the production of their respective second messengers. Perturbations in Ins(1,4,5)P3 accumulation, its metabolism and intracellular binding may underlie changes in ASM contractility. Comparably, changes in ASM relaxation responsiveness, secondary to perturbations in cAMP accumulation, may be due to altered receptor/G protein modulation of adenylate cyclase activity, as well as to altered binding of Ins(1,4,5)P3 to its Ca2+-mobilizing intracellular receptor. This review begins with an overview of the structural and functional characteristics of G protein-linked receptors, followed by descriptions of the role of G proteins, their transmembrane signaling processes, and mechanisms regulating second messenger-coupled ASM contraction and relaxation, and concludes with new information underscoring the important roles of altered receptor/G protein-coupled expression and regulatory interactions between signaling pathways in modulating second-messenger accumulation and action in the "pro-asthmatic" sensitized airway smooth muscle.

Altered lung mechanics after double-lung transplantation.

We studied lung mechanics and small airways function in 15 patients after double-lung (DL) transplantation. Patients were classified as stable (DL-S, n = 11), or having obliterative bronchiolitis syndrome (DL-OBS, n = 4). We performed pulmonary function tests (PFT), measured slope of phase 3 of the single-breath nitrogen test (N2SP3), and obtained pressure-volume curves and values: chord compliance (Cst,L), specific chord compliance (SCst,L), and elastic recoil pressure at 90% TLC. PFT showed mild restrictive pattern in DL-S and severe obstructive lung disease in DL-OBS. The N2SP3 measurement indicated small airways dysfunction in 82% of DL-S and in all DL-OBS patients. The Cst,L was 0.24 +/- 0.08 L/cm H2O in DL-S and 0.16 +/- 0.05 L/cm H2O in DL-OBS, both lower than control subjects 0.34 +/- 0. 09 L/cm H2O (p < 0.01; p < 0.001). Moreover, SCst,L was 0.09 +/- 0. 03 cm H2O-1 in DL-S, and 0.05 +/- 0.02 cm H2O-1 in DL-OBS, significantly lower than control subjects 0.12 +/- 0.02 cm H2O-1 (p < 0.05; p < 0.001). Elastic recoil at 90% TLC was normal in 14 of 15 patients. We found a linear correlation between N2SP3 and FEV1, and between FEV1 and Cst,L and SCst,L for combined DL-S and DL-OBS. Reduced compliance near FRC with normal elastic recoil at high lung volumes does not suggest changes in lung parenchyma. We speculate that structural or functional alterations in small airways may have contributed to low compliance measurements. Of special concern are our findings that DL-S had significant small airways dysfunction and reduced compliance in a pattern similar to the DL-OBS, only smaller in magnitude.

Mechanism of rhinovirus-induced changes in airway smooth muscle responsiveness.

An important interplay exists between specific viral respiratory infections and altered airway responsiveness in the development and exacerbations of asthma. However, the mechanistic basis of this interplay remains to be identified. This study addressed the hypothesis that rhinovirus (RV), the most common viral respiratory pathogen associated with acute asthma attacks, directly affects airway smooth muscle (ASM) to produce proasthmatic changes in receptor-coupled ASM responsiveness. Isolated rabbit and human ASM tissue and cultured ASM cells were inoculated with human RV (serotype 16) or adenovirus, each for 6 or 24 h. In contrast to adenovirus, which had no effect, inoculation of ASM tissue with RV induced heightened ASM tissue constrictor responsiveness to acetylcholine and attenuated the dose-dependent relaxation of ASM to beta-adrenoceptor stimulation with isoproterenol. These RV-induced changes in ASM responsiveness were largely prevented by pretreating the tissues with pertussis toxin or with a monoclonal blocking antibody to intercellular adhesion molecule-1 (ICAM-1), the principal endogenous receptor for most RVs. In extended studies, we found that the RV-induced changes in ASM responsiveness were associated with diminished cAMP accumulation in response to dose-dependent administration of isoproterenol, and this effect was accompanied by autologously upregulated expression of the Gi protein subtype, Gialpha3, in the ASM. Finally, in separate experiments, we found that the RV-induced effects on ASM responsiveness were also accompanied by autologously induced upregulated mRNA and cell surface protein expression of ICAM-1. Taken together, these findings provide new evidence that RV directly induces proasthmatic phenotypic changes in ASM responsiveness, that this effect is triggered by binding of RV to its ICAM-1 receptor in ASM, and that this binding is associated with the induced endogenously upregulated expression of ICAM-1 and enhanced expression and activation of Gi protein in the RV-infected ASM.

Autologously up-regulated Fc receptor expression and action in airway smooth muscle mediates its altered responsiveness in the atopic asthmatic sensitized state.

To elucidate the role of IgE-dependent mechanisms in inducing altered airway responsiveness in the atopic asthmatic state, the expression and actions of Fc receptor activation were examined in isolated rabbit tracheal smooth muscle (TSM) tissue and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic-sensitized TSM exhibited significantly increased maximal isometric contractility to acetylcholine (P < 0. 01) and attenuated maximal relaxation responses and sensitivity (i.e.,-log ED50) to isoproterenol (P < 0.005). These changes in agonist responsiveness in atopic sensitized TSM were ablated by pretreating the tissues with a blocking mAb to the low affinity receptor for IgE, FcepsilonRII (i.e., CD23) or by depleting the sensitizing serum of its immune complexes. Moreover, in complimentary experiments, exogenous administration of IgE immune complexes to naive TSM produced changes in agonist responsiveness that were qualitatively similar to those obtained in the atopic asthmatic-sensitized state. Extended studies further demonstrated that, in contrast to their respective controls, atopic asthmatic serum-sensitized human and rabbit TSM tissue and cultured cells exhibited markedly induced mRNA and cell surface expression of FcepsilonRII, whereas constitutive expression of the IgG receptor subtype, FcgammaRIII, was unaltered. Finally, the up-regulated mRNA expression of FcepsilonRII observed following exposure of TSM to atopic asthmatic serum or to exogenously administered IgE immune complexes was significantly inhibited by pretreating the tissues or cells with anti-CD23 mAb. Collectively, these observations provide evidence demonstrating that the altered agonist responsiveness in atopic asthmatic sensitized airway smooth muscle is largely attributed to IgE-mediated induction of the autologous expression and activation of FcepsilonRII receptors in the airway smooth muscle itself.

Autocrine role of interleukin 1beta in altered responsiveness of atopic asthmatic sensitized airway smooth muscle.

The role of IL-1beta in regulating altered airway responsiveness in the atopic/asthmatic sensitized state was examined in isolated rabbit tracheal smooth muscle (TSM) tissue and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. During half-maximal isometric contraction of the tissues with acetylcholine, relative to control TSM, the atopic sensitized TSM exhibited significant attenuation of both their maximal relaxation (P < 0.001) and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol and PGE2 (P < 0.05), whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in both tissue groups. The impaired relaxation responses to isoproterenol and PGE2 were ablated in sensitized TSM that were pretreated with either the IL-1 recombinant human receptor antagonist or an IL-1beta-neutralizing antibody. Moreover, extended studies demonstrated that, in contrast to their respective controls, both passively sensitized rabbit TSM tissue and cultured cells exhibited markedly induced expression of IL-1beta mRNA at 6 h after exposure to the sensitizing serum, a finding similar to that also obtained in passively sensitized human bronchial smooth muscle tissue. Finally, unlike their respective controls, passively sensitized TSM tissue and cultured cells also displayed progressively enhanced release of IL-1beta protein into the culture media for up to 24 h after exposure to atopic/asthmatic serum. Collectively, these observations provide new evidence demonstrating that the altered responsiveness of atopic/asthmatic sensitized airway smooth muscle is largely attributed to its autologously induced expression and autocrine action of IL-1beta.