K. Fan Chung

Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsìveness, and the late asthmatic response

BACKGROUND Interleukin-5 (IL-5) is essential for the formation of eosinophils, which are thought to have a major role in the pathogenesis of asthma and other allergic diseases. We aimed to assess the effects of monoclonal antibody to IL-5 on blood and sputum eosinophils, airway hyper-responsiveness, and the late asthmatic reaction to inhaled allergen in patients with mild asthma. METHODS We did a double-blind randomised placebo-controlled trial, in which a single intravenous infusion of humanised (IgG-K) monoclonal antibody to IL-5 (SB-240563) was given at doses of 2.5 mg/kg (n=8) or 10.0 mg/kg (n=8). The effects of treatment on responses to inhaled allergen challenge, sputum eosinophils, and airway hyper-responsiveness to histamine were measured at weeks 1 and 4 with monitoring of blood eosinophil counts for up to 16 weeks. FINDINGS Monoclonal antibody against IL-5 lowered the mean blood eosinophil count at day 29 from 0.25x10(9)/L (95% CI 0.16-0.34) in the placebo group to 0.04x10(9)/L (0.00-0.07) in the 10 mg/kg group (p<0.0001), and prevented the blood eosinophilia that follows allergen challenge. After inhaled allergen challenge, 9 days after treatment, the percentage sputum eosinophils were 12.2% in the placebo group and lowered to 0.9% (-1.2 to 3.0; p=0.0076) in the 10 mg/kg group, and this effect persisted at day 30 after the dose. There was no significant effect of monoclonal antibody to IL-5 on the late asthmatic response or on airway hyper-responsiveness to histamine. INTERPRETATION A single dose of monoclonal antibody to IL-5 decreased blood eosinophils for up to 16 weeks and sputum eosinophils at 4 weeks, which has considerable therapeutic potential for asthma and allergy. However, our findings question the role of eosinophils in mediating the late asthmatic response and causing airway hyper-responsiveness.

A molecular mechanism of action of theophylline: Induction of histone deacetylase activity to decrease inflammatory gene expression

The molecular mechanism for the anti-inflammatory action of theophylline is currently unknown, but low-dose theophylline is an effective add-on therapy to corticosteroids in controlling asthma. Corticosteroids act, at least in part, by recruitment of histone deacetylases (HDACs) to the site of active inflammatory gene transcription. They thereby inhibit the acetylation of core histones that is necessary for inflammatory gene transcription. We show both in vitro and in vivo that low-dose theophylline enhances HDAC activity in epithelial cells and macrophages. This increased HDAC activity is then available for corticosteroid recruitment and predicts a cooperative interaction between corticosteroids and theophylline. This mechanism occurs at therapeutic concentrations of theophylline and is dissociated from phosphodiesterase inhibition (the mechanism of bronchodilation) or the blockade of adenosine receptors, which are partially responsible for its side effects. Thus we have shown that low-dose theophylline exerts an anti-asthma effect through increasing activation of HDAC which is subsequently recruited by corticosteroids to suppress inflammatory genes.

Bradykinin–evoked sensitization of airway sensory nerves: A mechanism for ACE–inhibitor cough

Cough accompanied by an increased sensitivity of the cough reflex is the most common symptom of inflammatory airway disease1,5. This symptom is also frequently reported in patients receiving angiotensin–converting enzyme (ACE) inhibitors as therapy for heart failure or hypertension2–4, although the underlying mechanism is unknown. We have investigated the possibility that the inflammatory peptide bradykinin, normally degraded by ACE, causes sensitization of airway sensory nerves and an enhancement of the cough reflex in conscious guinea pigs. Treatment of guinea pigs for two weeks with captopril led to an increased cough response to inhaled citric acid, which was prevented by concomitant treatment with the bradykinin receptor antagonist icatibant. A similar icatibant–sensitive enhancement of citric acid–evoked cough was seen in untreated animals after prior inhalation of bradykinin, although cough evoked by hypertonic saline was unaffected. In electrophysiological studies performed in vitro, responses of single vagal C fibers to capsaicin, applied to receptive fields of single–fiber units in the trachea, were also markedly increased after perfusion with bradykinin, whereas Aδ fiber responses to hypertonic saline were unaffected. These results indicate that bradykinin–evoked sensitization of airway sensory nerves may underlie the pathogenesis of ACE–inhibitor cough. Bradykinin receptor antagonists may be of benefit in treating chronic cough seen with this and other inflammatory conditions.

Platelet-activating factor as a mediator of allergic disease

Many different mediators have been implicated in allergic responses and allergic diseases.14 It is becoming increasingly apparent that no single mediator can account for allergic diseases, such as asthma, rhinitis, eczema, or anaphylaxis, and that the clinical manifestation depends on a complex interaction of mediators that are released from a variety of cells. Although previous research concentrated on the mast cell as the key cell in allergic reactions, it now appears likely that other inflammatory cells, such as macrophages, eosinophils , neutrophils, platelets, and lymphocytes, may also contribute to the inflammatory response. The recognition that cells, other than mast cells, such as eosinophils,5 platelets,6 and macrophages,7 may be activated by IgE-dependent mechanisms has focused attention on these other cells as sources of mediators. Histamine,8 prostaglandinsg and leukotrienes3 have been previously proposed as playing a role in mediating allergic responses. Another mediator that is receiving increasing attention is PAF, a highly potent inflammatory mediator formed, like prostaglandins and leukotrienes, by the action of phospholipase A, on membrane phospholipids. The purpose of this article is to review the various effecs of PAF that may be relevant to allergic diseases and to speculate on its possible involvement in allergy. With the introduction of specific PAF antagonists during the next few years, the role of this inflammatory mediator will be clarified. had previously been demonstrated in the rabbit that platelets provided the major source of histamine among the blood elements,” but it was not known whether histamine release during allergen challenge represented a direct or indirect consequence of the recognition of allergen by antibody. Histamine release in sensitized rabbits undergoing antigen challenge was demonstrated to be the consequence of IgE-dependent activation of basophils that, in turn, released a soluble product capable of eliciting platelet activation.““S ‘* This product was termed PAF and was subsequently demonstrated to be a phospholipid.‘3 The chemical structure was then identified in 1979 by three independent groups as 1-o-alkyl-2-acetyl-sn-glyceryl-3phosphorylcholine (Fig. 1). 14-16 This phospholipid has been variously referred to as PAF-acether,14 acetyl glyceryl ether phosphorylcholine,‘5 or antihypertensive polar renomedullary lipid.16 The original nomenclature (PAF) will be used throughout this article, although it is now apparent that PAF has many other actions in addition to activation of platelets.

New targets for drug development in asthma

Asthma is a chronic inflammatory disease that affects about 300 million people worldwide, a total that is expected to rise to about 400 million over the next 15-20 years. Most asthmatic individuals respond well to the currently available treatments of inhaled corticosteroids and beta-adrenergic agonists; however, 5-10% have severe disease that responds poorly. Improved knowledge of asthma mechanisms has led to the recognition of different asthma phenotypes that might reflect distinct types of inflammation, explaining the effectiveness of anti-leucotrienes and the anti-IgE monoclonal antibody omalizumab in some patients. However, more knowledge of the inflammatory mechanisms within the airways is required. Improvements in available therapies-such as the development of fast-onset, once-a-day combination drugs with better safety profiles-will occur. Other drugs, such as inhaled p38 MAPK inhibitors and anti-oxidants, that target specific pathways or mediators could prove useful as monotherapies, but could also, in combination with corticosteroids, reduce the corticosteroid insensitivity often seen in severe asthma. Biological agents directed against the interleukin-13 pathway and new immunoregulatory agents that modulate functions of T-regulatory and T-helper-17 cells are likely to be successful. Patient-specific treatments will depend on the development of discriminatory handprints of distinct asthma subtypes and are probably over the horizon. Although a cure is unlikely to be developed in the near future, a greater understanding of disease mechanisms could bring such a situation nearer to reality.

IL-17-induced cytokine release in human bronchial epithelial cells in vitro : role of mitogen-activated protein (MAP) kinases

Recent data indicate that interleukin (IL)‐17 may contribute to neutrophilic airway inflammation by inducing the release of neutrophil‐mobilizing cytokines from airway cells. The aim of this study was to evaluate the role of mitogen activated protein kinases in IL‐17 induced release of IL‐8 and IL‐6 in bronchial epithelial cells. Transformed human bronchial epithelial cells (16HBE) were stimulated with either IL‐17 or vehicle. Both groups were treated either with SB202190 (inhibitor of p38 MAP kinase), PD98059 (inhibitor of extracellular‐signal‐regulated kinase [ERK] pathway), Ro‐31‐7549 (protein kinase C [PKC] inhibitor), LY 294002 (a phosphatidylinositol 3‐kinase [PI 3‐kinase] inhibitor) or vehicle. IL‐6 and IL‐8 levels were measured in conditioned media by ELISA. The IL‐17‐induced release of IL‐6 and IL‐8 was concentration‐dependently inhibited by SB202190 and by PD98059 in bronchial epithelial cells without affecting cell proliferation or survival. Ro‐31‐7549 and LY294002 had no significant effect on IL‐17‐induced IL‐6 or IL‐8 release in bronchial epithelial cells. Taken together, these data indicate a role for p38 and ERK kinase pathways in IL‐17‐induced release of neutrophil‐mobilizing cytokines in human bronchial epithelial cells. These mechanisms constitute potential pharmacotherapeutical targets for inhibition of the IL‐17‐mediated airway neutrophilia.

Characterization of allergen-induced bronchial hyperresponsiveness and airway inflammation in actively sensitized Brown-Norway rats

Bronchial responsiveness to inhaled acetylcholine (ACh) and inflammatory cell recruitment in bronchoalveolar lavage fluid (BALF) were studied in inbred Brown-Norway rats actively sensitized to, and later exposed to, ovalbumin (OA). We examined animals 21 days after initial sensitization at 18 to 24 hours, or 5 days after a single challenge, or after the last of seven repeated exposures administered every 3 days. BALF was examined as an index of inflammatory changes within the lung. Animals repeatedly exposed to OA aerosols had an increased baseline lung resistance and a significant increase in bronchial responsiveness to inhaled ACh compared to control animals at both 18 to 24 hours and 5 days after the last OA exposure. Sensitized animals receiving a single OA aerosol also demonstrated bronchial hyperresponsiveness (BHR) to inhaled ACh (p less than 0.01) at 18 to 24 hours of a similar order as the multiple-exposed group. There was a significant increase in eosinophils, lymphocytes, and neutrophils in BALF at 18 to 24 hours but not at 5 days after single or multiple exposure to OA aerosol in the sensitized groups. Control animals demonstrated no changes in bronchial responsiveness, although a small but significant increase in inflammatory cells was observed compared to saline-only treated animals. There was a significant correlation between bronchial responsiveness and eosinophil counts in the BALF in the single allergen-exposed group (Rs = 0.68; p less than 0.05). We conclude that (1) BHR after allergen exposure in sensitized rats is associated with the presence of pulmonary inflammation but persists despite the regression of inflammatory cells in BALF after multiple OA exposures, and (2) this rat model has many characteristics of human allergen-induced BHR.

Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca2+ in the cytosol ([Ca2+]i). A rise in [Ca2+]i is normalized by rapid reuptake of Ca2+ into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca2+ (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca2+]i, cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca2+]i following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca2+ re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca]i after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.

Induction of eotaxin expression and release from human airway smooth muscle cells by IL‐1β and TNFα: effects of IL‐10 and corticosteroids

Eotaxin is a novel C‐C chemokine with selective chemoattractant activity for eosinophils. We determined whether eotaxin could be produced by human airway smooth muscle (HASM) cells in culture and examined its regulation by interleukin‐10 (IL‐10) and the corticosteroid, dexamethasone. Stimulation of the cells with interleukin‐1β (IL‐1β) or tumour necrosis factor (TNFα) each at 10 ng ml−1 induced the release of eotaxin protein with maximal accumulation by 24 h. Interferon‐γ (IFNγ) alone at 10 ng ml−1 had no effect and there was no synergy between these cytokines on the release of eotaxin. Reverse phase high performance liquid chromatographic (HPLC) analysis of supernatents from cells treated with TNFα (10 ng ml−1 for 96 h showed immunoreactivity to eotaxin which eluted with the expected retention time of 34.5–35 min. Both IL‐1β and TNFα‐induced release of eotaxin was not inhibited by dexamethasone (1 μM), however IL‐10 (10 ng ml−1) had a significant inhibitory effect. Dexamethasone and IL‐10 did not inhibit the induction of eotaxin mRNA induced by IL‐1β or TNFα. Thus, human airway smooth muscle cells can release eotaxin and could be an important source of chemokine production during airway inflammatory events.

Effect of inhaled l-arginine on exhaled nitric oxide in normal and asthmatic subjects

BACKGROUND Nitric oxide (NO) plays an important part in the regulation of many physiological functions and may also be involved in several pulmonary diseases. Endogenous NO is synthesised by different isoforms of NO synthase (NOS) from l-arginine. METHODS The effect of inhaledl-arginine 0.75 g (six normal and six asthmatic subjects), 1.5 g (six normal and six asthmatic subjects), and 3 g (seven normal and six asthmatic subjects) has been studied in a double blind placebo controlled, randomised, parallel group design study. In addition, the effect of a single dose (3 g) of inhaled l-alanine has been assessed in five normal and five asthmatic subjects. RESULTS l-arginine increased exhaled NO in a dose-dependent fashion with a maximum at 60 minutes. The cumulative effect of l-arginine (3 g) on NO in asthmatic subjects, expressed as the area under the curve in arbitrary units (au) and compared with the effect of placebo (0.9% NaCl), was significantly higher (mean 0.11 au; 95% confidence interval (CI) 0.03 to 0.19) than in normal subjects (0.012 au; 95% CI 0.002 to 0.022). There was a negative correlation (r = –0.72) between the increase in exhaled NO and the fall in forced expiratory volume in one second (FEV1) (0.034 au, 95% CI 0.030 to 0.038) after 3 gl-arginine in asthmatic subjects. Inhalation of 3  g ofl-alanine produced a similar reduction in FEV1(0.033 au, 95% CI 0.007 to 0.059) but no significantly different changes in exhaled NO (0.017 au, 95% CI 0.001 to 0.039) compared with placebo (0.020 au, 95% CI 0.001 to 0.042). CONCLUSIONS An increase in the amount of substrate for NOS increases the formation of endogenous NO.l-arginine may have therapeutic potential in diseases in which there is defective production of NO, but in asthma it may amplify the inflammatory response in the airways.