J. Margaret Hughes

Interleukin-4 inhibits mitogen-induced proliferation of human airway smooth muscle cells in culture

The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which factors regulate growth and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived growth factor, basic fibroblast growth factor, and thrombin. IL-4 (5, 15, 50, and 150 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived growth factor (20 and 40 ng/ml), basic fibroblast growth factor (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by 19, 21, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 μM) and forskolin (10 μM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 μM), and forskolin (10 μM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by 21%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which factors regulate growth and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived growth factor, basic fibroblast growth factor, and thrombin. IL-4 (5, 15, 50, and 150 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived growth factor (20 and 40 ng/ml), basic fibroblast growth factor (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by 19, 21, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 microM) and forskolin (10 microM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 microM), and forskolin (10 microM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by 21%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.

Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation

BACKGROUND The receptor for advanced glycation end products (RAGE) shares common ligands and signaling pathways with TLR4, a key mediator of house dust mite (Dermatophagoides pteronyssinus) (HDM) sensitization. We hypothesized that RAGE and its ligand high-mobility group box-1 (HMGB1) cooperate with TLR4 to mediate HDM sensitization. OBJECTIVES To determine the requirement for HMGB1 and RAGE, and their relationship with TLR4, in airway sensitization. METHODS TLR4(-/-), RAGE(-/-), and RAGE-TLR4(-/-) mice were intranasally exposed to HDM or cockroach (Blatella germanica) extracts, and features of allergic inflammation were measured during the sensitization or challenge phase. Anti-HMGB1 antibody and the IL-1 receptor antagonist Anakinra were used to inhibit HMGB1 and the IL-1 receptor, respectively. RESULTS The magnitude of allergic airway inflammation in response to either HDM or cockroach sensitization and/or challenge was significantly reduced in the absence of RAGE but not further diminished in the absence of both RAGE and TLR4. HDM sensitization induced the release of HMGB1 from the airway epithelium in a biphasic manner, which corresponded to the sequential activation of TLR4 then RAGE. Release of HMGB1 in response to cockroach sensitization also was RAGE dependent. Significantly, HMGB1 release occurred downstream of TLR4-induced IL-1α, and upstream of IL-25 and IL-33 production. Adoptive transfer of HDM-pulsed RAGE(+/+)dendritic cells to RAGE(-/-) mice recapitulated the allergic responses after HDM challenge. Immunoneutralization of HMGB1 attenuated HDM-induced allergic airway inflammation. CONCLUSION The HMGB1-RAGE axis mediates allergic airway sensitization and airway inflammation. Activation of this axis in response to different allergens acts to amplify the allergic inflammatory response, which exposes it as an attractive target for therapeutic intervention.

Effect of fenoterol on immunological release of leukotrienes and histamine from human lung in vitro: Selective antagonism by β-adrenoceptor antagonists

The inhibitory effects of fenoterol, a beta 2-adrenoceptor agonist, on the release of SRS-A leukotrienes and histamine from chopped human lung tissue were measured and selective beta-adrenoceptor antagonists used to investigate the nature of the receptors involved. Fenoterol 0.01-1.0 microM inhibited the antigen-induced release of SRS-A and histamine, but not the release induced by the calcium ionophore A23187. Propranolol 0.1 and 1.0 microM and butoxamine 10 and 100 microM significantly antagonized the effects of fenoterol 0.1 microM on SRS-A and histamine at concentrations which affect (beta 2-adrenoceptors, while atenolol 0.1 to 10 microM showed no antagonism at concentrations which affect beta 1-adrenoceptors. These results suggest that adrenoceptors in human lung which modulate the immunological release of SRS-A leukotrienes are of the beta 2-subtype as for histamine release.

RAGE: a new frontier in chronic airways disease.

Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous inflammatory disorders of the respiratory tract characterized by airflow obstruction. It is now clear that the environmental factors that drive airway pathology in asthma and COPD, including allergens, viruses, ozone and cigarette smoke, activate innate immune receptors known as pattern‐recognition receptors, either directly or indirectly by causing the release of endogenous ligands. Thus, there is now intense research activity focused around understanding the mechanisms by which pattern‐recognition receptors sustain the airway inflammatory response, and how these mechanisms might be targeted therapeutically. One pattern‐recognition receptor that has recently come to attention in chronic airways disease is the receptor for advanced glycation end products (RAGE). RAGE is a member of the immunoglobulin superfamily of cell surface receptors that recognizes pathogen‐ and host‐derived endogenous ligands to initiate the immune response to tissue injury, infection and inflammation. Although the role of RAGE in lung physiology and pathophysiology is not well understood, recent genome‐wide association studies have linked RAGE gene polymorphisms with airflow obstruction. In addition, accumulating data from animal and clinical investigations reveal increased expression of RAGE and its ligands, together with reduced expression of soluble RAGE, an endogenous inhibitor of RAGE signalling, in chronic airways disease. In this review, we discuss recent studies of the ligand–RAGE axis in asthma and COPD, highlight important areas for future research and discuss how this axis might potentially be harnessed for therapeutic benefit in these conditions.

Tumour necrosis factor-α potentiates contraction of human bronchus in vitro

Chronic inflammation of the airways is an important component in the induction of airway hyperresponsiveness (AHR) in asthma. The pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) have been implicated in the induction of AHR. Whether these cytokines directly modulate the contractile properties of human airway smooth muscle (ASM) has not been fully investigated.

Mast Cells Produce Novel Shorter Forms of Perlecan That Contain Functional Endorepellin: A ROLE IN ANGIOGENESIS AND WOUND HEALING*

Background: Mast cells modulate events in wound healing. Results: Shorter forms of perlecan are produced by mast cells via proteolytic processing and alternative splicing, which contain domain V and functional endorepellin. Conclusion: The production of these shorter forms modulates endothelial cell adhesion, proliferation, and migration. Significance: Mast cells produce specific forms of perlecan that affect endothelial cell behavior. Mast cells are derived from hematopoietic progenitors that are known to migrate to and reside within connective and mucosal tissues, where they differentiate and respond to various stimuli by releasing pro-inflammatory mediators, including histamine, growth factors, and proteases. This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL-2H3 and HMC-1, produce the heparan sulfate proteoglycan, perlecan, with a molecular mass of 640 kDa as well as smaller molecular mass species of 300 and 130 kDa. Utilizing domain-specific antibodies coupled with N-terminal sequencing, it was confirmed that both forms contained the C-terminal module of the protein core known as endorepellin, which were generated by mast cell-derived proteases. Domain-specific RT-PCR experiments demonstrated that transcripts corresponding to domains I and V, including endorepellin, were present; however, mRNA transcripts corresponding to regions of domain III were not present, suggesting that these cells were capable of producing spliced forms of the protein core. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the α2β1 integrin and stimulate the migration of cells in “scratch assays,” both activities of which were inhibited by incubation with either anti-endorepellin or anti-perlecan antibodies. This study shows for the first time that mast cells secrete and process the extracellular proteoglycan perlecan into fragments containing the endorepellin C-terminal region that regulate angiogenesis and matrix turnover, which are both key events in wound healing.

Effect of lipoxygenase inhibitors on release of slow-reacting substances from human lung

Abstract The effects of the lipoxygenase inhibitors nordihydroguaiaretic acid (NDG) and 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C) on the release of slow-reacting substances (SRS) from human lung tissue were investigated in vitro. NDG (5 × 10 −6 M and 5 × 10 −5 M) and BW 755C (10 −5 and 10 −4 M) caused a dose-dependent inhibition of SRS release. There was a small reduction of histamine release with the higher concentration of each drug. These results suggest that lipoxygenase iny be useful in determining the role of SRS in inflammatory processes in vivo.

Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations.

The extension of airway smooth muscle cell (ASMC) functions, from just contractile, to synthetic and/or proliferative states, is an important component of airway remodelling and inflammation in asthma. Whereas all these functions have been demonstrated in ASM, currently, it is not known whether ASMC can be differentiated on the basis of their proliferative and synthetic functions. We used flow‐cytometric techniques to determine, first, whether human ASMC are phenotypically heterogenous with regard to their secretory function, and second, the proliferative status of secretory cells. ASMC were induced to synthesize GM‐CSF by stimulation with IL‐1β and TNF‐α followed by 10% human serum. Flow‐cytometric detection of intracellular GM‐CSF revealed that only a proportion of cells in culture (∼ 20–60%) synthesize GM‐CSF. To determine the proliferative status of GM‐CSF producing cells, ASMC were pretreated with 5,6‐carboxyfluorescein diacetate succinimidyl ester (CFSE), a fluorescein based dye used to track cell division, prior to cytokine/serum stimulation. Simultaneous analysis of intracellular GM‐CSF and CFSE revealed that GM‐CSF producing cells were present in both the divided and undivided ASMC populations. Thus, cytokine production and proliferation occurred in overlapping ASMC populations and prior progression through the cell cycle was not essential for ASMC cytokine production.

TLR2 ligand engagement upregulates airway smooth muscle TNFα-induced cytokine production

Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 μg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα-induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CRE-mediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease.