Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response. Recent studies have suggested a prominent role for mast cells in host defense. In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells. Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria. Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1). Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-κB) nuclear binding activity. In some experiments, NF-κB inhibitor, Bay-11 was added to determine functional significance. Both M. catarrhalis and N. cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1. This was accompanied by NF-κB activation. Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells. Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces. The NF-κB inhibitor, Bay-11, inhibited expression of MCP-1. These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, characterized by progressive and largely irreversible airflow limitation due to alveolar destruction (emphysema), small airway narrowing, and chronic bronchitis. It is one of the leading causes of morbidity and mortality worldwide and in the UK, it may affect approximately 1.5 per cent of the population; and up to one in eight emergency admissions may be due to COPD,corresponding to over one million bed days, with some 24160 people in the UK dying as a result of COPD in 2005 (Burden of Lung Disease 2nd Edition,British Thoracic Society 2006). Most cases of COPD are triggered by chronic inhalation of cigarette smoke.However, some people do not suffer from COPD even if they smoke for many years. COPD cannot be cured, and patients usually live with poor life quality. Treatments include giving up smoking, medication and oxygen therapy. Genetic factors contribute to the development of COPD. In Northern Europe,Z-AT homozygotes (342Glu Lys) develop emphysema in their third or forth decade. One explanation is AT deficiency because they form inactive polymers. However, this cannot explain why bronchoalveolar lavage fluid (BALF) from Z-AT homozygotes with emphysema contains more neutrophils than BALF from individuals with emphysema and normal AT (M-AT). Inhaling pollutants which include smoking (cigarettes, pipes, cigars, etc.) and other fumes such as those found in many industrial work environments probably also plays a role in an individual’s development of COPD. Previously, it has been shown that the polymeric conformer of AT is present in BALF from Z-AT homozygotes and that it is a chemoattractant for neutrophils in vitro (Parmar JS, 2002). These findings have been confirmed by others (Mulgrew AT, 2004). However, it is unknown where the polymers form and if 4 they are chemotactic in vivo. My colleague Dr Carl Atkison† showed that polymers of Z 1-AT are present in the alveolar wall of Z-AT homozygotes with emphysema, which accounts for 20% of the total AT from lung homogenates.These Z-AT individuals also have an excess of neutrophils in the alveolar wall compared with M-AT homozygotes. Furthermore, neutrophils and polymeric AT co-localize in the alveolar wall (Mahadeva R, 2005). To investigate whether there was a direct relationship between polymers of Z-AT and the excess neutrophils, polymers of AT were instilled into the lungs of wild-type mice (Mahadeva R, 2005). This produced a significant increase in neutrophil influx into the lungs compared with instillation of the native protein.Examination of the time course demonstrated that the influx of neutrophils was closely linked to the presence of polymeric AT. The mechanism of neutrophil recruitment in this mouse model was subsequently shown to be a direct chemotactic effect rather than stimulation of IL-8 homologues or other CXC chemokines. Oxidized AT (Ox-AT) promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF- B and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the anti-elastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway † My colleagues’ contributions are acknowledged in future text where appropriate by the following superscripts: (a) Dr Sam Alam, (b) Dr Jichun Wang, (c) Dr Carl Atkinson, (d) Dr Sabina Janciauskiene. 5 interacts directly with epithelial cells to release chemokines IL-8 and MCP-1,which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells oxidizes AT, perpetuating the cycle, potentially contributing to the pathogenesis of COPD. Furthermore, this demonstrates that molecules such as oxidants, anti-proteinases, and chemokines, rather than acting independently, collectively interact to cause emphysema (Li Z, 2009). To investigate the molecular basis for the interaction between Z-AT and Ox-AT associated with cigarette smoking, female mice transgenic for normal (MAT)or Z-AT on CBA background were exposed to cigarette smoke (CS). Transgenic mice for Z-AT developed a significant increase in pulmonary polymers following acute CS exposure. Increased levels of neutrophils in CSZ lungs were tightly correlated with polymer concentrations. Oxidation of human plasma Z-AT by CS or -chlorosuccinimide greatly accelerated polymerization, which could be abrogated by antioxidants. The results showed that cigarette smoke accelerated polymerization of Z-AT by oxidative modification, which in so doing further reduced pulmonary defense and increased neutrophil influx into the lungs. These novel findings provided a molecular explanation for the striking observation of premature emphysema in ZZ homozygote smokers, and raised the prospect of anti-oxidant therapy in ZAT related COPD (Alam S, 2011).