T lymphocyte-mediated changes in airway smooth muscle responsiveness are attributed to induced autocrine release and actions of IL-5 and IL-1beta

BACKGROUND: Bidirectional stimulatory cross-talk was recently found to exist between activated T cells and airway smooth muscle (ASM) cells, a process that involves coligation of specific cellular adhesion-costimulatory molecules that results in the induction of proasthmatic-like changes in ASM responsiveness. OBJECTIVE: The present study examined whether the cooperative intercellular signaling between activated T cells and ASM cells is coupled to the induced expression and actions of IL-5 and IL-1beta. METHODS: Agonist-induced constrictor and relaxant responses were examined in rabbit ASM segments exposed to resting and anti-CD3-activated T cells in the absence and presence of either an anti-IL-5 receptor mAb or the recombinant human IL-1 receptor antagonist. In addition, mRNA and protein expression of IL-5 and IL-1beta were assayed under control and anti-CD3-stimulated conditions. RESULTS: Relative to inactive T cells, incubation of ASM tissues with anti-CD3-activated T cells induced proasthmatic-like changes in agonist-mediated ASM responsiveness. This T cell-induced perturbation in ASM responsiveness was ablated by pretreating the tissues with either an anti-IL-5 receptor mAb or IL-1 receptor antagonist. Moreover, exposure of ASM cells to anti-CD3-activated T cells elicited an initial increased mRNA expression and release of IL-5, followed by an enhanced expression and release of IL-1beta, and the induced release of these cytokines was prevented in ASM cells that were pretreated with an anti-IL-5 receptor mAb. CONCLUSION: Collectively, these observations provide new evidence demonstrating that exposure of naive ASM cells to activated T cells induces the sequential release of IL-5 and IL-1beta from the ASM cells and that the latter cytokines act in an autocrine manner to elicit the proasthmatic phenotype of altered ASM responsiveness.     

MAPK regulation of gene expression in airway smooth muscle

Mitogen-activated protein kinases (MAPK) are important components of signaling modules activated by neurotransmitters, cytokines, and growth factors, as well as chemical and mechanical stressors. In the airway, these external signals produce acute responses that modify smooth muscle contraction and may also induce chronic responses that modify airway structure. Both acute and chronic events in airway remodeling result from altered expression of multiple genes encoding protein mediators of cell-cell signaling, extracellular matrix remodeling, cell cycle control and intracellular signaling pathways. This review will focus on inflammatory and growth factor mediators of cell-cell signaling regulated by the ERK and p38 MAPK pathways in airway smooth muscle (ASM). These signaling mediators affect ASM tissue mechanics, cell migration, and gene expression patterns in a paracrine and autocrine fashion, although the relative importance of each MAPK pathway varies with the stimulus. These events thereby contribute to normal airway function and participate in pathological changes in ASM that accompany symptoms of asthma.     

MKK3/6-p38 MAPK signaling is required for IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

Coupled bone turnover is directed by the expression of receptor-activated NF-kappaB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1beta treatment and subsequently reduced approximately 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1beta or TNF-alpha treatment. IL-1beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.     

Maturational changes in airway smooth muscle shortening and relaxation. Implications for asthma

Greater airway responsiveness in healthy juveniles is considered a factor in the higher asthma prevalence at a young age compared with adults. Several studies on the contractile response of airway smooth muscle (ASM) from birth to adulthood have addressed the hypothesis that a maturation of ASM plays a role in juvenile airway hyperresponsiveness. Maturation of distinct ASM properties, i.e. force generation, shortening, and relaxation, has been reported, although the majority of the studies have focused on maturation of maximum force and/or sensitivity to contractile agonists. However, in most animal species maturation of the ability to generate force does not correlate with maturation of airway responsiveness. Ontogenesis of ASM shortening has been less extensively studied and the existing reports emphasize an increase during maturation of tissue passive forces opposing shortening. ASM spontaneous relaxation has been very minimally investigated. We have recently demonstrated that the ability of ASM to spontaneously relax during stimulation is sharply reduced in juvenile airway tissue. It remains to be determined the role of these ASM properties in the onset of childhood asthma and whether specific alterations are induced by the occurrence of obstructive airway diseases in young individuals.     

Relationship between intrapulmonary airway diameter and smooth muscle tone in excised lungs.

1. Intrapulmonary bronchi in excised dog lungs were outlined with tantalum dust and stereoscopic radiographs taken during deflation and inflation of the lung after rinsing with solutions of saline, histamine, isoprenaline or EDTA. Dimensions of airways were calculated from measurements of the stereoscopic X-ray images. 2. After treatment with EDTA to minimize bronchial smooth muscle activity, airway diameters increased at all transpulmonary pressures (Ptp) and lung volumes relative to their diameter after treatment with histamine; airway hysteresis in relation to Ptp decreased. 3. At low lung volumes, the per cent increase from histamine to EDTA for airways of different sizes was the same (24-30%) but at high volumes (30 cm H2O distending pressure) the dilatation induced by EDTA was 30% for airways less than 3.0 mm diameter and 13% for those greater than 5.0 mm diameter. 4. Even at high lung volumes, intrapulmonary airways are free to constrict or dilate in spite of the stiffness of the supporting parenchyma.     

ABCB1基因多态性和P-糖蛋白表达的相关性

p-糖蛋白作为一种广泛分布于全身各组织和器官的重要转运体,影响多种药物的体内的吸收、分布、代谢和排泄过程,还能防止外源性有害物质的侵入.p-糖蛋白由ABCB1基因编码,ABCB1基因多态性决定p-糖蛋白的蛋白表达量,进而影响p-糖蛋白的体内的功能作用.单核苷酸多态性(SNP)是P-糖蛋白产生变异的重要影响因素,可决定不同个体对药物治疗的不同应答,人体中存在的非同义和同义SNP均可对P-糖蛋白的转运功能产生重要影响.本文将对各组织器官中,ABCB1基因多态性与p-糖蛋白表达量的相关性进行综述.     

Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice

The amine- and peptide-producing pulmonary neuroendocrine cells (PNEC) are widely distributed within the airway mucosa of mammalian lung as solitary cells and innervated clusters, neuroepithelial bodies (NEB), which function as airway O2 sensors. These cells express Cftr and hence could play a role in the pathophysiology of cystic fibrosis (CF) lung disease. We performed confocal microscopy and morphometric analysis on lung sections from Cftr-/- (null), Cftr+/+, and Cftr+/- (control) mice at developmental stages E20, P5, P9, and P30 to determine the distribution, frequency, and innervation of PNEC/NEB, innervation and cell mass of airway smooth muscle, and neuromuscular junctions using synaptic vesicle protein 2, smooth muscle actin, and synaptophysin markers, respectively. The mean number of PNEC/NEB in Cftr-/- mice was significantly reduced compared with control mice at E20, whereas comparable or increased numbers were observed postnatally. NEB cells in Cftr null mice showed a significant reduction in intracorpuscular nerve endings compared with control mice, which is consistent with an intrinsic abnormality of the PNEC system. The airways of Cftr-/- mice showed reduced density (approximately 20-30%) of smooth muscle innervation, decreased mean airway smooth muscle mass (approximately 35%), and reduced density (approximately 20%) of nerve endings compared with control mice. We conclude that the airways of Cftr-/- mice exhibit heretofore unappreciated structural alterations affecting cellular and neural components of the PNEC system and airway smooth muscle and its innervation resulting in blunted O2 sensing and reduced airway tonus. Cftr could play a role in the development of the PNEC system, lung innervation, and airway smooth muscle.     

Homologous serum increases fibronectin expression and cell adhesion in airway smooth muscle cells

This study describes altered patterns of growth and upregulation of fibronectin expression of cultured canine airway smooth muscle cells grown in homologous serum, which provides a model of the vascular leakage occurring in asthma, compared to fetal bovine serum (FBS). Cells were incubated in increasing concentrations of serum (2.5–40%) for 72 hours. Both homologous serum and FBS caused cellular proliferation which reached a maximum increase at 2.5–5% serum concentration. Differences in the cellular responses to the two types of sera were noted at higher concentrations of sera. At a concentration of 40% FBS, airway smooth muscle cells increased in number by 307±16% (n=5) compared to serum-free control cells, whereas in canine serum the increase in growth was significantly smaller, 239±25% (n=7) (P<0.05). Airway fibrocytes similarly treated increased in number by 256±43% (n=3) in 40% FBS, but exhibited a reduction in cell number to 80 ±10% (n=3) of controls in 40% homologous serum (P<0.05). Smooth muscle cells demonstrated a dose-dependent increase in fibronectin expression when grown in homologous serum, but not in FBS, suggesting phenotypic change occurred in these cells when exposed to homologous serum. These data suggest that the leakage of plasma in the asthmatic airway may trigger phenotypic change in both airway smooth muscle cells and airway fibrocytes leading to cellular proliferation and expression of extracellular matrix molecules. These in vitro changes are consistent with the histological findings in clinical asthma.     

Regulation of G protein-coupled receptor-adenylyl cyclase responsiveness in human airway smooth muscle by exogenous and autocrine adenosine

Adenosine is a mediator of bronchoconstriction in asthmatics and is believed to mediate its effects through adenosine receptor activation in inflammatory cells. In this study, we identify human airway smooth muscle (ASM) as a direct target of adenosine. Acute exposure of human ASM cultures to adenosine receptor (AR) agonists resulted in rapid accumulation of cyclic adenosine monophosphate (cAMP) with a pharmacologic profile consistent with A(2b)AR activation. Little or no evidence of A1AR or A3AR expression was suggested on acute addition of various AR ligands, although a low level of A1ARs was identified in radioligand binding studies. Treatment with adenosine deaminase suggested that human ASM cultures secrete adenosine that feeds back on A(2b)ARs and regulates basal cAMP levels as well as a small degree of A(2b)AR, beta(2)AR, and prostaglandin E(2) receptor desensitization. When subjected to chronic treatment with AR agonists or agents that enhance accumulation of endogenous, extracellular adenosine, a dual effect of A(2b)AR desensitization and adenylyl cyclase (AC) sensitization was observed. This AC sensitization was eliminated by pertussis toxin and partially reversed by the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine, suggesting a contributory role for the A1AR. Overexpression of A1ARs and A(2b)ARs in human ASM cultures resulted in differential effects on basal, agonist-, and AC-mediated cAMP production. These data demonstrate that human ASM is a direct target of exogenous and autocrine adenosine, with effects determined by differential contributions of A(2b) and A1 adenosine receptors that are time-dependent. Accordingly, the relative distribution and activation of AR subtypes in ASM in vivo may influence airway function in diseases such as asthma and warrant consideration in therapeutic strategies that target ARs or alter nucleotide/ nucleoside levels in the airway.     

Differential expression of beta 1 integrins in nonneoplastic smooth and striated muscle cells and in tumors derived from these cells.

Integrins are a superfamily of transmembrane alpha beta heterodimers that play an important role in cell-matrix and cell-cell interactions by acting as receptors for extracellular matrix proteins and for cell adhesion molecules. Using monoclonal antibodies against beta 1, alpha 1 to alpha 6, and alpha v subunits, the in situ distribution pattern of beta 1 integrins was examined immunohistochemically in nonneoplastic smooth and striated muscle cells and in their tumors. Nonneoplastic smooth muscle cells were beta 1+, alpha 1+, alpha 3+, alpha v+ and, in diverse localizations, also alpha 5+ or even alpha 6+. The expression of the beta 1 chain was conserved in all leiomyomas and leiomyosarcomas. The distribution pattern of the alpha subunits by contrast underwent several changes during malignant transformation of smooth muscle cells. These alterations consisted in a neoexpression of alpha 2, alpha 4, and alpha 6 as well as in an abnormal abrogation of alpha 1 and alpha 3 in some leiomyosarcomas. Except for the absence of alpha 5 in the majority of epithelioid leiomyosarcomas, expression of the alpha 5 and alpha v subunits was mainly conserved. In addition, tumors with epithelioid differentiation differed from typical cases by the absence of alpha 1 and the simultaneous presence of alpha 4. Adult striated muscle cells were beta 1+ but alpha 1- to alpha 6- and alpha v-, whereas fetal striated muscle cells were not only beta 1+ but also alpha 3+/-, alpha 4+/-, alpha 5+ and alpha 6+. In all rhabdomyosarcomas the expression of beta 1 was retained. Furthermore, the majority of cases showed the expression of one or more alpha subunits most of which, ie, alpha 4, alpha 5, and alpha 6, were also found in fetal striated muscle cells. In conclusion, beta 1 integrins exhibited a differential expression pattern along the two lines of myogenic differentiation. This integrin profile underwent characteristic changes during malignant transformation. Nevertheless, the compiled distribution patterns of the alpha 1, alpha 3, and alpha v subunits allowed in most instances the discrimination between tumors of smooth (alpha 1+/alpha 3+/alpha v+) and striated muscle (alpha 1-/alpha 3-/alpha v-) differentiation.     

Transforming growth factor beta from multiple myeloma cells inhibits proliferation and IL-2 responsiveness in T lymphocytes

Multiple myeloma (MM) is a cancer of plasma cells, characterized by profound suppression of host immune responses. Here we show that MM cell lines significantly suppress the proliferation, blasting, response to interleukin-2 (IL-2), and expression of CD25 by concanavalin A (Con A)-activated or allostimulated peripheral blood T lymphocytes. T cells arrest in the G1 stage of the cell cycle, and do not enter the IL-2 autocrine growth pathway. T cell inhibition was mediated by a soluble factor. MM cell lines did not produce IL-10 but did produce large amounts of transforming growth factor beta1 (TGF-beta1). T cells were assessed for their ability to respond to IL-2 when co-cultured with MM cells in the presence or absence of the TGF-beta inhibitor, TGF-beta latency-associated peptide (LAP). MM cells suppressed IL-2 responses but this inhibition was completely reversed by TGF-beta LAP. A CD25-, IL-2-dependent blast cell line was not inhibited by MM cells or rhTGF-beta, confirming the specificity of the inhibition mechanism for the IL-2 autocrine growth pathway. We conclude that MM cells suppress T cells in their entry into the autocrine IL-2/CD25 pathway and in response to IL-2, and that TGF-beta has a significant role to play.     

GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha.

The role of mucosal fibroblasts in intestinal inflammatory reactions is not known. In this study, we demonstrate that fibroblasts grown from histologically normal human duodenal biopsy tissues expressed mRNA genes for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) when stimulated with lipopolysaccharide (LPS) or IL-1 alpha. The increased mRNA expression of GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and IL-8 in response to IL-1 alpha and LPS stimulation was time- and dose-dependent. In contrast, IL-10 was weakly expressed when fibroblasts were stimulated with LPS, IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha), but the expression was enhanced in the presence of cycloheximide combined with optimal concentrations of LPS, IL-1 alpha or TNF-alpha, IL-1 alpha was a more potent stimulator than LPS for GM-CSF, IL-6, IL-8 and IL-10 expression, but not for IL-1 alpha and IL-1 beta. Increased GM-CSF, IL-6 and IL-8 gene expression was associated with the production of cytokine proteins in culture supernatant, but IL-1 alpha and IL-1 beta remained undetectable. Dexamethasone suppressed both gene expression and protein production of GM-CSF, IL-6 and IL-8 when fibroblasts were exposed to IL-1 alpha. TNF-alpha stimulated the release of GM-CSF, IL-6 and IL-8 and, combined with IL-1 alpha, cytokine production was enhanced synergistically. Finally, both LPS and IL-1 alpha up-regulated ICAM-1 and VCAM-1 gene expression. These findings implicate duodenal fibroblasts in the initiation and/or regulation of intestinal inflammation.