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Background: Severe neutrophilic asthma is resistant to treatment with glucocorticoids. The immunomodulatory protein macrophage migration inhibitory factor (MIF) promotes neutrophil recruitment to the lung and antagonises responses to glucocorticoids. We hypothesised that MIF promotes glucocorticoid resistance of neutrophilic inflammation in severe asthma.Methods: We examined whether sputum MIF protein correlated with clinical and molecular characteristics of severe neutrophilic asthma in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. We also investigated whether MIF regulates neutrophilic inflammation and glucocorticoid responsiveness in a murine model of severe asthma in vivo.Results: MIF protein levels positively correlated with the number of exacerbations in the previous year, sputum neutrophils and oral corticosteroid use across all U-BIOPRED subjects. Further analysis of MIF protein expression according to U-BIOPRED-defined transcriptomic-associated clusters (TACs) revealed increased MIF protein and a corresponding decrease in annexin-A1 protein in TAC2, which is most closely associated with airway neutrophilia and NLRP3 inflammasome activation. In a murine model of severe asthma, treatment with the MIF antagonist ISO-1 significantly inhibited neutrophilic inflammation and increased glucocorticoid responsiveness. Coimmunoprecipitation studies using lung tissue lysates demonstrated that MIF directly interacts with and cleaves annexin-A1, potentially reducing its biological activity.Conclusion: Our data suggest that MIF promotes glucocorticoid-resistance of neutrophilic inflammation by reducing the biological activity of annexin-A1, a potent glucocorticoid-regulated protein that inhibits neutrophil accumulation at sites of inflammation. This represents a previously unrecognised role for MIF in the regulation of inflammation and points to MIF as a potential therapeutic target for the management of severe neutrophilic asthma. [ABSTRACT FROM AUTHOR]

Introduction: Glaucoma is a progressive neurodegenerative disease associated with age. Accumulation of amyloid-beta (Aß) proteins in the ganglion cell layer (GCL) and subsequent retinal ganglion cell (RGC) loss is an established pathological hallmark of the disease. The mechanism through which Aß provokes RGC loss remains unclear. The receptor for the advanced glycation end product (RAGE), and its ligand Aß, have been shown to mediate neuronal loss via internalizing Aß within the neurons. In this study, we investigated whether the RAGE--Aß axis plays a role in RGC loss in experimental glaucoma. Methods: Retinal ischemia was induced by an acute elevation of intraocular pressure in RAGE [ABSTRACT FROM AUTHOR]

In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, [PGE.sub.2], forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) [[beta].sub.2]-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1[beta] in ASM cells. IL-1[beta]-induced IL-8 release was also repressed by [PGE.sub.2] and forskolin, whereas the [[beta].sub.2]-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKI[alpha], a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-[kappa]B-dependent genes, we used an adenoviral-delivered NF-[kappa]B-dependent luciferase reporter to examine the effects of forskolin and the [[beta].sub.2]-adrenoceptor agonists on NF-[kappa]B activation. There was no effect on luciferase activity measured in the presence of forskolin or [[beta].sub.2]-adrenoceptor agonists. This finding is consistent with the observation that IL-1[beta]-induced expression of IL-6, a known NF-[kappa]B-dependent gene in ASM, was also unaffected by [[beta].sub.2]-adrenoceptor agonists, forskolin, [PGE.sub.2], 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1[beta]-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including [[beta].sub.2]-adrenoceptor agonists, may not occur through a generic effect on NF-[kappa]B. human airway smooth muscle cells; asthma; inflammation; prostaglandin

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-[beta] is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-[beta] mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-[beta]1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-[beta] 1 on ASM cells was inhibited by selective inhibitors of TGF-[beta] receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-[kappa]B (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-[beta]1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-[beta]1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-[beta]1-induced proliferation. Our findings suggest that TGF-[beta]1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-[kappa] B is also involved in the TGF-[beta] 1 mitogenic signaling, but Smad pathway does not appear important. laser capture microdissection; transforming growth factor-[beta]1 expression; airway smooth muscle cells; asthma; corticosteroids doi:10.1152/ajplung.00068.2007

Airway smooth muscle cells (ASMC) are a source of inflammatory chemokines that may propagate airway inflammatory responses. We investigated the production of the CXC chemokine growth-related oncogene protein-[alpha] (GRO-[alpha]) from ASMC induced by cytokines and the role of MAPK and NF-[kappa]B pathways. ASMC were cultured from human airways, grown to confluence, and exposed to cytokines IL-1[beta] and TNF-[alpha] after growth arrest. GRO-[alpha] release, measured by ELISA, was increased by >50-told after IL- 1[beta] (0.1 ng/ml) or 5-fold after TNF-[alpha] (1 ng/ml) in a dose- and time-dependent manner. GRO-[alpha] release was not affected by the T helper type 2 cytokines IL-4, IL-10, and IL-13. IL-1[beta] and TNF-[alpha] also induced GRO-[alpha] mRNA expression. Supernatants from IL-1[beta]-stimulated ASMC were chemotactic for neutrophils; this effect was inhibited by anti-GRO-[alpha] blocking antibody. AS-602868, an inhibitor of IKK-2, and PD-98059, an inhibitor of ERK, inhibited GRO-[alpha] release and mRNA expression, whereas SP-600125, an inhibitor of JNK, reduced GRO-[alpha] release without effect on mRNA expression. SB-203580, an inhibitor of p38 MAPK, had no effect. AS-602868 but not PD-98059 or SP-600125 inhibited p65 DNA-binding induced by IL-1[beta] and TNF-[alpha] By chromatin immunoprecipitation assay, IL-1[beta] and TNF-[alpha] enhanced p65 binding to the GRO-[alpha] promoter, which was inhibited by AS-602868. IL-1[beta]- and TNF-[alpha]-stimulated expression of GRO-[alpha] from ASMC is regulated by independent pathways involving NF-[kappa]B activation and ERK and JNK pathways. GRO-[alpha] released from ASMC participates in neutrophil chemotaxis. growth-related oncogene protein-[alpha]; extracellular signal-regulated kinase; c-Jun [NH.sub.2]-terminal kinase; nuclear factor-[kappa]B

Transforming growth factor (TGF)-[beta] may play an important role in airway remodeling, and the fibrogenic effect of TGF-[beta] may be mediated through connective tissue growth factor (CTGF) release. We investigated the role of MAPKs and phosphatidylinositol 3-kinase (PI3K) and the effects of inllammatory cytokines on TGF-[beta]-induced CTGF expression in human airway smooth muscle cells (ASMC). We examined whether Smad signal was involved in the regulatory mechanisms. TGF-[beta]1 induced a time- and concentration-dependent expression of CTGF gene and protein as analyzed by real-time RT-PCR and Western blot. Inhibition of ERK and c-jun N[H.sub.2]-terminal kinase (JNK), but not of p38 MAPK and PI3K, blocked the effect of TGF-[beta]1 on CTGF mRNA and protein expression and on Smad2/3 phosphorylation. T helper lymphocyte 2-derived cytokines, IL-4 and IL-13, attenuated TGF-[beta]1-stimulated mRNA and protein expression of CTGF and inhibited TGF-[beta]1-stimulated ERK1/2 and Smad2/3 activation in ASMC. The proinflammatory cytokines tumor necrosis factor-[alpha] and 1L-1[beta] reduced TGF-[beta]1-stimulated mRNA expression of CTGF but did not inhibit TGF-[beta]-induced Smad2/3 phosphorylation. TGF-[beta]1-stimulated CTGF expression is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-[beta] through modulation of Smad and ERK signals. mitogen-activated protein kinases; signal transduction; Smad; cytokines; asthma; transforming growth factor

Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX3C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, C[X.sub.3]CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1[beta], TNF-[alpha], and IFN-[gamma], the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-[beta]. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-[gamma] and TNF-[alpha] induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-[beta] had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone ([10..sup.-8]-[10.sup.-6] M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun N[H.sub.2]terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 [micro]M) and SB-203580 (20 [micro]M), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-[gamma]- and TNF-[alpha]-induced JNK phosphorylation remained unaltered in the presence of TGF-[beta] but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-[beta]- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors. chemokine; airway inflammation; asthma; chronic obstructive pulmonary disease

Background: The receptor for advanced glycation end products (RAGE) and Toll‐like receptor 4 (TLR4) is implicated in COPD. Although these receptors share common ligands and signalling pathways, it is not known whether they act in concert to drive pathological processes in COPD. We examined the impact of RAGE and/or TLR4 gene deficiency in a mouse model of COPD and also determined whether expression of these receptors correlates with airway neutrophilia and airway hyperresponsiveness (AHR) in COPD patients. Methods: We measured airway inflammation and AHR in wild‐type, RAGE−/−, TLR4−/− and TLR4−/−RAGE−/− mice following acute exposure to cigarette smoke (CS). We also examined the impact of smoking status on AGER (encodes RAGE) and TLR4 bronchial gene expression in patients with and without COPD. Finally, we determined whether expression of these receptors correlates with airway neutrophilia and AHR in COPD patients. Results: RAGE−/− mice were protected against CS‐induced neutrophilia and AHR. In contrast, TLR4−/− mice were not protected against CS‐induced neutrophilia and had more severe CS‐induced AHR. TLR4−/−RAGE−/− mice were not protected against CS‐induced neutrophilia but were partially protected against CS‐induced mediator release and AHR. Current smoking was associated with significantly lower AGER and TLR4 expression irrespective of COPD status, possibly reflecting negative feedback regulation. However, consistent with preclinical findings, AGER expression correlated with higher sputum neutrophil counts and more severe AHR in COPD patients. TLR4 expression did not correlate with neutrophilic inflammation or AHR. Conclusions: Inhibition of RAGE but not TLR4 signalling may protect against airway neutrophilia and AHR in COPD. [ABSTRACT FROM AUTHOR]

Abstract Background Bacterial infections are a cause of exacerbation of airway disease. Airway smooth muscle cells (ASMC) are a source of inflammatory cytokines/chemokines that may propagate local airway inflammatory responses. We hypothesize that bacteria and bacterial products could induce cytokine/chemokine release from ASMC. Methods Human ASMC were grown in culture and treated with whole bacteria or pathogen associated molecular patterns (PAMPs) for 24 or 48 h. The release of eotaxin-1, CXCL-8 or GMCSF was measured by ELISA. Results Gram-negative E. coli or Gram-positive S. aureus increased the release of CXCL-8, as did IL-1β, LPS, FSL-1 and Pam3CSK4, whereas FK565, MODLys18 or Poly I:C did not. E. coli inhibited eotaxin-1 release under control conditions and after stimulation with IL-1β. S. aureus tended to inhibit eotaxin-1 release stimulated with IL-1β. E. coli or LPS, but not S. aureus, induced the release of GMCSF. Conclusion Gram-positive or Gram-negative bacteria activate human ASMC to release CXCL-8. By contrast Gram-negative bacteria inhibited the release of eotaxin-1 from human ASMCs. E. coli, but not S. aureus induced GMCSF release from cells. Our findings that ASMC can respond directly to Gram-negative and Gram-positive bacteria by releasing the neutrophil selective chemokine, CXCL-8, is consistent with what we know about the role of neutrophil recruitment in bacterial infections in the lung. Our findings that bacteria inhibit the release of the eosinophil selective chemokine, eotaxin-1 may help to explain the mechanisms by which bacterial immunotherapy reduces allergic inflammation in the lung.

Abstract Background The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases. Methods Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance. Results We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction. Conclusion Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.