Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

Background

Previous work has shown that disruption of the gene for group X secreted phospholipase A₂ (sPLA₂-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA₂s in the mammalian genome, the involvement of other sPLA₂s in the asthma model is possible – in particular, the group V sPLA₂ (sPLA₂-V) that like sPLA₂-X is highly active at hydrolyzing membranes of mammalian cells.

Methodology and Principal Findings

The allergen-driven asthma phenotype was significantly reduced in sPLA₂-V-deficient mice but to a lesser extent than observed previously in sPLA₂-X-deficient mice. The most striking difference observed between the sPLA₂-V and sPLA₂-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA₂-V^−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V^−/− mice diminishes Th2 cytokine responses in the airways.

Conclusions

This paper illustrates the diverse roles of sPLA₂s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA₂-V as a potential new therapy to treat asthma and other allergic disorders.     

Function of secreted phospholipase A2 group-X in asthma and allergic disease

Elevated secreted phospholipase A₂ (sPLA₂) activity in the airways has been implicated in the pathogenesis of asthma and allergic disease for some time. The identity and function of these enzymes in asthma is becoming clear from work in our lab and others. We focused on sPLA₂ group X (sPLA₂-X) after identifying increased levels of this enzyme in asthma, and that it is responsible for a large portion of sPLA₂ activity in the airways and that the levels are strongly associated with features of airway hyperresponsiveness (AHR). In this review, we discuss studies that implicated sPLA₂-X in human asthma, and murine models that demonstrate a critical role of this enzyme as a regulator of type-2 inflammation, AHR and production of eicosanoids. We discuss the mechanism by which sPLA₂-X acts to regulate eicosanoids in leukocytes, as well as effects that are mediated through the generation of lysophospholipids and through receptor-mediated functions. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.     

IgE production in CD40/CD40L cross-talk of B and mast cells and mediator release via TGase 2 in mouse allergic asthma

TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 on IgE production and signaling pathways in mast cell activation related to OVA-induced allergic asthma. Bone marrow-derived mast cells (BMMCs) isolated from WT or TGase 2^?/? mice were activated with Ag/Ab (refer to act-WT-BMMCs and act-KO-BMMCs, respectively). B cells isolated from splenocytes were activated with anti-mouse IgM (act-B cells), and B cells were co-cultured with BMMCs. WT and TGase 2^?/? mice were sensitized and challenged with OVA adsorbed in alum hydroxide. Intracellular Ca^2 + ([Ca^2 +]_i) levels were determined by fluorescence intensity; IgE, mediators and TGase 2 activity by ELISA; the CD138 expression by FACS analyzer; cell surface markers and signal molecules by Western blot; NF-κB by EMSA; co-localization of mast cells and B cells by immunohistochemistry; Fcε RI-mediated mast cell activation by PCA test; expression of cytokines, MMPs, TIMPs, TLR2 and Fc?RI by RT-PCR. In vitro, act-KO-BMMCs reduced the [Ca^2 +]i levels, NF-κB activity, expression of CD40/CD40L, plasma cells, total IgE levels and TGase 2 activity in act-B cells co-cultured with act-BMMCs, expression of inflammatory cytokines and MMPs2/9, release of mediators (TNF-α, LTs and cytokines), and activities of signal molecules (PKCs, MAP kinases, I-κB and PLA2), which were all increased in act-WT-BMMCs. TGase 2 siRNA transfected/activated-BMMCs reduced all responses as same as those in act-KO-BMMCs. In allergic asthma model, TGase 2^?/? mice protected against PCA reaction, OVA-specific IgE production and AHR, and they reduced co-localization of mast cells and B cells or IgE in lung tissues, expression and co-localization of surface molecules in mast cells (c-kit and CD40L) and B cells (CD23 and CD40), inflammatory cells including mast cells, goblet cells, amounts of collagen and mediator release in BAL fluid and/or lung tissues, which were all increased in WT mice. TLR expression in TGase 2^?/? mice did not differ from those in WT mice. Our data suggest that TGase 2 expression and Ca^2 + influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via up-regulating mast cell CD40L expression, and induce the expression of numerous signaling molecules associated with airway inflammation and remodeling in allergic asthma.     

Allergic rhinitis and asthma: inflammation in a one-airway condition

Background

Allergic rhinitis and asthma are conditions of airway inflammation that often coexist.

Discussion

In susceptible individuals, exposure of the nose and lungs to allergen elicits early phase and late phase responses. Contact with antigen by mast cells results in their degranulation, the release of selected mediators, and the subsequent recruitment of other inflammatory cell phenotypes. Additional proinflammatory mediators are released, including histamine, prostaglandins, cysteinyl leukotrienes, proteases, and a variety of cytokines, chemokines, and growth factors. Nasal biopsies in allergic rhinitis demonstrate accumulations of mast cells, eosinophils, and basophils in the epithelium and accumulations of eosinophils in the deeper subepithelium (that is, lamina propria). Examination of bronchial tissue, even in mild asthma, shows lymphocytic inflammation enriched by eosinophils. In severe asthma, the predominant pattern of inflammation changes, with increases in the numbers of neutrophils and, in many, an extension of the changes to involve smaller airways (that is, bronchioli). Structural alterations (that is, remodeling) of bronchi in mild asthma include epithelial fragility and thickening of its reticular basement membrane. With increasing severity of asthma there may be increases in airway smooth muscle mass, vascularity, interstitial collagen, and mucus-secreting glands. Remodeling in the nose is less extensive than that of the lower airways, but the epithelial reticular basement membrane may be slightly but significantly thickened.

Conclusion

Inflammation is a key feature of both allergic rhinitis and asthma. There are therefore potential benefits for application of anti-inflammatory strategies that target both these anatomic sites.

     

Regulation of the inflammatory response in asthma by mast cell products

In airways, mast cells lie adjacent to nerves, blood vessels and lymphatics, which highlights their pivotal importance in regulating allergic inflammatory processes. In asthma, mast cells are predominantly activated by IgE receptor cross linking. In response to activation, preformed mediators that are stored bound to proteoglycans, for example, TNF-alpha, IL-4, IL-13, histamine, tryptase and chymase, are released. New synthesis of arachidonic acid metabolites (leukotriene C4 (LTC4), leukotriene B4 (LTB4) and prostaglandin D2 (PGD2)) and further cytokines is stimulated. Mediators from degranulating mast cells are critical to the pathology of the asthmatic lung. Mast cell proteases stimulate tissue remodelling, neuropeptide inactivation and enhanced mucus secretion. Histamine stimulates smooth muscle cell contraction, vasodilatation and increased venular permeability and further mucus secretion. Histamine induces IL-16 production by CD8+ cells and airway epithelial cells; IL-16 is an important early chemotactic factor for CD4+ lymphocytes. LTC4, LTB4 and PGD2 affect venular permeability and can regulate the activation of immune cells. The best characterized mast cell cytokine in asthmatic inflammation is TNF-alpha, which induces adhesion molecules on endothelial cells and subsequent transmigration of inflammatory leucocytes. IL-13 is critical to development of allergic asthma, although its mode of action is less clear.     

The Impact of Allergic Rhinitis and Asthma on Human Nasal and Bronchial Epithelial Gene Expression

Background

The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium.

Objective

Defining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles.

Methods

This cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used.

Results

The study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions.

Conclusions

Differences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.     

Oleanolic Acid Controls Allergic and Inflammatory Responses in Experimental Allergic Conjunctivitis

Pollen is the most common aeroallergen to cause seasonal conjunctivitis. The result of allergen exposure is a strong Th2-mediated response along with conjunctival mast cell degranulation and eosinophilic infiltration. Oleanolic acid (OA) is natural a triterpene that displays strong anti-inflammatory and immunomodulatory properties being an active anti-allergic molecule on hypersensitivity reaction models. However, its effect on inflammatory ocular disorders including conjunctivits, has not yet been addressed. Hence, using a Ragweed pollen (RWP)-specific allergic conjunctivitis (EAC) mouse model we study here whether OA could modify responses associated to allergic processes. We found that OA treatment restricted mast cell degranulation and infiltration of eosinophils in conjunctival tissue and decreased allergen-specific Igs levels in EAC mice. Th2-type cytokines, secreted phospholipase A₂ type-IIA (sPLA₂-IIA), and chemokines levels were also significantly diminished in the conjunctiva and serum of OA-treated EAC mice. Moreover, OA treatment also suppressed RWP-specific T-cell proliferation. In vitro studies, on relevant cells of the allergic process, revealed that OA reduced the proliferative and migratory response, as well as the synthesis of proinflammatory mediators on EoL-1 eosinophils and RBL-2H3 mast cells exposed to allergic and/or crucial inflammatory stimuli such as RWP, sPLA₂-IIA or eotaxin. Taken together, these findings demonstrate the beneficial activity of OA in ocular allergic processes and may provide a new intervention strategy and potential therapy for allergic diseases.     

A Novel Model of IgE-Mediated Passive Pulmonary Anaphylaxis in Rats

Mast cells are central effector cells in allergic asthma and are augmented in the airways of asthma patients. Attenuating mast cell degranulation and with it the early asthmatic response is an important intervention point to inhibit bronchoconstriction, plasma exudation and tissue oedema formation. To validate the efficacy of novel pharmacological interventions, appropriate and practicable in vivo models reflecting mast cell-dependent mechanisms in the lung, are missing. Thus, we developed a novel model of passive pulmonary anaphylaxis in rats. Rats were passively sensitized by concurrent intratracheal and intradermal (ear) application of an anti-DNP IgE antibody. Intravenous application of the antigen, DNP-BSA in combination with Evans blue dye, led to mast cell degranulation in both tissues. Quantification of mast cell degranulation in the lung was determined by (1) mediator release into bronchoalveolar lavage, (2) extravasation of Evans blue dye into tracheal and bronchial lung tissue and (3) invasive measurement of antigen-induced bronchoconstriction. Quantification of mast cell degranulation in the ear was determined by extravasation of Evans blue dye into ear tissue. We pharmacologically validated our model using the SYK inhibitor Fostamatinib, the H₁-receptor antagonist Desloratadine, the mast cell stabilizer disodium cromoglycate (DSCG) and the β₂-adrenergic receptor agonist Formoterol. Fostamatinib was equally efficacious in lung and ear. Desloratadine effectively inhibited bronchoconstriction and ear vascular leakage, but was less effective against pulmonary vascular leakage, perhaps reflecting the differing roles for histamine receptor sub-types. DSCG attenuated both vascular leakage in the lung and bronchoconstriction, but with a very short duration of action. As an inhaled approach, Formoterol was more effective in the lung than in the ear. This model of passive pulmonary anaphylaxis provides a tissue relevant readout of early mast cell activity and pharmacological benchmarking broadly reflects responses observed in patients with asthma.     

The Role of Secretory Phospholipase A2 in the Central Nervous System and Neurological Diseases

Secretory phospholipase A₂ (sPLA₂s) are small secreted proteins (14–18 kDa) and require submillimolar levels of Ca²⁺ for liberating arachidonic acid from cell membrane lipids. In addition to the enzymatic function, sPLA₂ can exert various biological responses by binding to specific receptors. Physiologically, sPLA₂s play important roles on the neurotransmission in the central nervous system and the neuritogenesis in the peripheral nervous system. Pathologically, sPLA₂s are involved in the neurodegenerative diseases (e.g., Alzheimer’s disease) and cerebrovascular diseases (e.g., stoke). The common pathology (e.g., neuronal apoptosis) of Alzheimer’s disease and stroke coexists in the mixed dementia, suggesting common pathogenic mechanisms of the two neurological diseases. Among mammalian sPLA₂s, sPLA₂-IB and sPLA₂-IIA induce neuronal apoptosis in rat cortical neurons. The excess influx of calcium into neurons via l-type voltage-dependent Ca²⁺ channels mediates the two sPLA₂-induced apoptosis. The elevated concentration of intracellular calcium activates PKC, MAPK and cytosolic PLA₂. Moreover, it is linked with the production of reactive oxygen species and apoptosis through activation of the superoxide producing enzyme NADPH oxidase. NADPH oxidase is involved in the neurotoxicity of amyloid β peptide, which impairs synaptic plasticity long before its deposition in the form of amyloid plaques of Alzheimer’s disease. In turn, reactive oxygen species from NADPH oxidase can stimulate ERK1/2 phosphorylation and activation of cPLA₂ and result in a release of arachidonic acid. sPLA₂ is up-regulated in both Alzheimer’s disease and cerebrovascular disease, suggesting the involvement of sPLA₂ in the common pathogenic mechanisms of the two diseases. Thus, our review presents evidences for pathophysiological roles of sPLA₂ in the central nervous system and neurological diseases.     

Suppression of Th2-driven, allergen-induced airway inflammation by sauchinone

Sauchinone, a lignan compound isolated from the root of Saururus chinensis, has been recently demonstrated to exhibit anti-inflammatory activity via the suppression of NF-kB p65 activity in vitro. In an effort to evaluate the in vivo anti-inflammatory function of sauchinone, we have evaluated the effects of sauchinone on allergen-induced airway inflammation using a murine model of allergic asthma. We observed that marked eosinophilic and lymphocyte infiltration in the BAL fluid were suppressed to a significant degree by sauchinone, and that mucus-secreting goblet cell hyperplasia and collagen deposition in the airways were also ameliorated by administration of sauchinone treatment. Moreover, gene expression of the inflammatory cytokines, IL-13, and IL-5 and eotaxin in the lung, and IL-5 in the draining lymph node were significantly decreased in sauchinone-treated mice. We demonstrated that sauchinone repressed Th2 cell development in vitro and IL-4 production by Th2 cells, and also inhibited GATA-3-mediated IL-5 promoter activity in a dose-dependent manner. Collectively, sauchinone ameliorated allergen-induced airway inflammation, in part, by repressing GATA-3 activity for Th2 cell development, indicating the possible therapeutic potential of sauchinone in airway inflammatory diseases including allergic asthma and rhinitis.     

CD1d expressed in mast cell surface enhances IgE production in B cells by up-regulating CD40L expression and mediator release in allergic asthma in mice

Mast cells play important roles via FcεRI-mediated activation in allergic asthma. A nonpolymorphic MHC I-like molecule CD1d, which is mainly expressed in APCs, presents glycolipid Ag to iTCR on iNKT cells and modulates allergic responses. This study aimed to investigate the role of CD1d on IgE production and mast cell activation related to allergic asthma. Bone marrow-derived mast cells (BMMCs) from C57BL/6 Wild type (WT) or KO (CD1d^−/−) mice were activated with Ag/Ab (refer to WT-act-BMMCs and KO-act-BMMCs, respectively) or α-Galactosylceramide (WT-αGal-BMMCs, KO-αGal-BMMCs) in the presence of iNKT cells. WT, KO or BMMC-transferred KO mice were sensitized and/or challenged by OVA or α-Gal to induce asthma. KO-act-BMMCs reduced intracellular Ca^2 + levels, expression of signaling molecules (Ras, Rac1/2, PLA₂, COX-2, NF-κB/AP-1), mediator release (histamines, leukotrienes and cytokines/chemokines), and total IgE levels versus the corresponding WT-BMMCs. KO mice reduced total and OVA-specific serum IgE levels, number of mast cells, recruiting molecules (CCR2/CCL2, VCAM-1, PECAM-1), expression of tryptase, c-kit, CD40L and cytokine mRNA, co-localization of c-kit and CD1d or iNKT cells in BAL cells or lung tissues, and PCA responses, compared with the corresponding WT mice. BMMC-transferred KO-both mice showed the restoration of all allergic responses versus KO-both mice (Ag/Ab reaction plus α-Gal). KO-αGal-BMMCs or KO-αGal mice did not show any responses. Our data suggest that CD1d-expressed mast cells may function as APC cells for iNKT cells and exacerbate airway inflammation and remodeling through up-regulating IgE production via B cell Ig class switching and mediator release in mast cells of OVA-challenged mice.     

Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice

Among the emerging phospholipase A₂ (PLA₂) superfamily, the secreted PLA₂ (sPLA₂) family consists of low-molecular-mass, Ca²⁺-requiring extracellular enzymes with a His-Asp catalytic dyad. To date, more than 10 sPLA₂ enzymes have been identified in mammals. Individual sPLA₂s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Despite numerous enzymatic and cell biological studies on this enzyme family in the past two decades, their precise in vivo functions still remain largely obscure. Recent studies using transgenic and knockout mice for several sPLA₂ enzymes, in combination with lipidomics approaches, have opened new insights into their distinct contributions to various biological events such as food digestion, host defense, inflammation, asthma and atherosclerosis. In this article, we overview the latest understanding of the pathophysiological functions of individual sPLA₂ isoforms fueled by studies employing transgenic and knockout mice for several sPLA₂s.     

Phospholipase A2 in Experimental Allergic Bronchitis: A Lesson from Mouse and Rat Models

Background

Phospholipases A₂ (PLA₂) hydrolyzes phospholipids, initiating the production of inflammatory lipid mediators. We have previously shown that in rats, sPLA₂ and cPLA₂ play opposing roles in the pathophysiology of ovalbumin (OVA)-induced experimental allergic bronchitis (OVA-EAB), an asthma model: Upon disease induction sPLA₂ expression and production of the broncho-constricting CysLTs are elevated, whereas cPLA₂ expression and the broncho-dilating PGE₂ production are suppressed. These were reversed upon disease amelioration by treatment with an sPLA₂ inhibitor. However, studies in mice reported the involvement of both sPLA₂ and cPLA₂ in EAB induction.

Objectives

To examine the relevance of mouse and rat models to understanding asthma pathophysiology.

Methods

OVA-EAB was induced in mice using the same methodology applied in rats. Disease and biochemical markers in mice were compared with those in rats.

Results

As in rats, EAB in mice was associated with increased mRNA of sPLA₂, specifically sPLA₂gX, in the lungs, and production of the broncho-constricting eicosanoids CysLTs, PGD₂ and TBX₂ in bronchoalveolar lavage (BAL). In contrast, EAB in mice was associated also with elevated cPLA₂ mRNA and PGE₂ production. Yet, treatment with an sPLA₂ inhibitor ameliorated the EAB concomitantly with reverting the expression of both cPLA₂ and sPLA₂, and eicosanoid production.

Conclusions

In both mice and rats sPLA₂ is pivotal in OVA-induced EAB. Yet, amelioration of asthma markers in mouse models, and human tissues, was observed also upon cPLA₂ inhibition. It is plausible that airway conditions, involving multiple cell types and organs, require the combined action of more than one, essential, PLA₂s.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.     

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background

Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E₂ (PGE₂) are probably also released and could explain the refractory period observed in patients with EIB.

Objective

This study aimed to evaluate the protective effect of PGE₂ on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction.

Methods

We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE₂ and prostanoid receptor (EP) antagonists for EP_1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed.

Results

Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE₂ significantly reduced mannitol-induced degranulation through EP₂ and EP₄ receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone.

Conclusions

Our data show a protective role for the PGE₂ receptors EP₂ and EP₄ following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.     

Structures and mechanisms of enzymes in the leukotriene cascade

Leukotrienes are a family of proinflammatory lipid mediators of the innate immune response and are important signaling molecules in inflammatory and allergic conditions. The leukotrienes are formed from arachidonic acid, which is released from membranes by cPLA₂, and further converted by 5-lipoxygenase to form the labile epoxide leukotriene (LT) A₄. This intermediate is converted by either of the two enzymes, LTA₄ hydrolase or LTC₄ synthase, to form LTB₄ or LTC₄, respectively. In order for 5-lipoxygenase to work efficiently in cells, five-lipoxygenase-activating protein needs to be present. LTB₄ is one of the most powerful chemotactic agents whereas LTC₄ induces smooth muscle contractions, for example in the airways causing bronchoconstriction in asthmatic patients. The leukotrienes and the five enzymes/proteins involved in their formation have been subject to intense studies including drug design programs. Compounds blocking the formation or action of leukotrienes are potentially beneficial in treatment of several acute and chronic inflammatory diseases of the cardiovascular and respiratory systems. In order to succeed with drug development studies, knowledge of the molecular characteristics of the targets is indispensable. This chapter reviews the biochemistry, catalytic, and structural properties of the enzymes in the leukotriene cascade.     

Association of Lung Inflammatory Cells with Small Airways Function and Exhaled Breath Markers in Smokers – Is There a Specific Role for Mast Cells?

Background

Smoking is associated with a mixed inflammatory infiltrate in the airways. We evaluated whether airway inflammation in smokers is related to lung function parameters and inflammatory markers in exhaled breath.

Methods

Thirty-seven smokers undergoing lung resection for primary lung cancer were assessed pre-operatively by lung function testing including single-breath-nitrogen washout test (sb-N2-test), measurement of fractional exhaled nitric oxide (FeNO) and pH/8-isoprostane in exhaled breath condensate (EBC). Lung tissue sections containing cancer-free large (LA) and small airways (SA) were stained for inflammatory cells. Mucosal (MC_T) respectively connective tissue mast cells (MC_TC) and interleukin-17A (IL-17A) expression by mast cells was analysed using a double-staining protocol.

Results

The median number of neutrophils, macrophages and mast cells infiltrating the lamina propria and adventitia of SA was higher than in LA. Both MCTC and MC_T were higher in the lamina propria of SA compared to LA (MC_TC: 49 vs. 27.4 cells/mm²; MC_T: 162.5 vs. 35.4 cells/mm²; P<0.005 for both instances). IL-17A expression was predominantly detected in MC_TC of LA. Significant correlations were found for the slope of phase III % pred. of the sb-N2-test (r_s= -0.39), for the FEV₁% pred. (r_s= 0.37) and for FEV₁/FVC ratio (r_s=0.38) with MC_T in SA (P<0.05 for all instances). 8-isoprostane concentration correlated with the mast cells in the SA (r_s=0.44), there was no correlation for pH or FeNO with cellular distribution in SA.

Conclusions

Neutrophils, macrophages and mast cells are more prominent in the SA indicating that these cells are involved in the development of small airway dysfunction in smokers. Among these cell types, the best correlation was found for mast cells with lung function parameters and inflammatory markers in exhaled breath. Furthermore, the observed predominant expression of IL-17A in mast cells warrants further investigation to elucidate their role in smoking-induced lung injury, despite the lack of correlation with lung function and exhaled breath parameters.     

Blocking KV1.3 Channels Inhibits Th2 Lymphocyte Function and Treats a Rat Model of Asthma

Allergic asthma is a chronic inflammatory disease of the airways. Of the different lower airway-infiltrating immune cells that participate in asthma, T lymphocytes that produce Th2 cytokines play important roles in pathogenesis. These T cells are mainly fully differentiated CCR7⁻ effector memory T (T_EM) cells. Targeting T_EM cells without affecting CCR7⁺ naïve and central memory (T_CM) cells has the potential of treating T_EM-mediated diseases, such as asthma, without inducing generalized immunosuppression. The voltage-gated K_V1.3 potassium channel is a target for preferential inhibition of T_EM cells. Here, we investigated the effects of ShK-186, a selective K_V1.3 channel blocker, for the treatment of asthma. A significant proportion of T lymphocytes in the lower airways of subjects with asthma expressed high levels of K_V1.3 channels. ShK-186 inhibited the allergen-induced activation of peripheral blood T cells from those subjects. Immunization of F344 rats against ovalbumin followed by intranasal challenges with ovalbumin induced airway hyper-reactivity, which was reduced by the administration of ShK-186. ShK-186 also reduced total immune infiltrates in the bronchoalveolar lavage and number of infiltrating lymphocytes, eosinophils, and neutrophils assessed by differential counts. Rats with the ovalbumin-induced model of asthma had elevated levels of the Th2 cytokines IL-4, IL-5, and IL-13 measured by ELISA in their bronchoalveolar lavage fluids. ShK-186 administration reduced levels of IL-4 and IL-5 and induced an increase in the production of IL-10. Finally, ShK-186 inhibited the proliferation of lung-infiltrating ovalbumin-specific T cells. Our results suggest that K_V1.3 channels represent effective targets for the treatment of allergic asthma.     

Molecular aspects of allergy

Atopic diseases such as asthma, rhinitis, eczema and food allergies have increased in most industrialised countries of the world during the last 20 years. The reasons for this increase are not known and different hypotheses have been assessed including increased exposure to sensitising allergens or decreased stimulation of the immune system during critical periods of development.

In allergic diseases there is a polarisation of the Th2 response and an increase in the production of type 2 cytokines which are involved in the production of immunoglobulin E and the development of mast cells, basophils and eosinophils leading to inflammation and disease. The effector phase of atopy is initiated by interaction with FcRI expressed on effector cells such as mast cells and basophils but also found on an ever increasing list of cells. Binding of a polyvalent allergen to the variable part of IgE leads to a cross-link of the receptor that triggers the cell to release histamine and pharmacological mediators of the symptomatic allergic response. Cross-linking of FcRI by autoantibodies against the -chain of the FcRI, causing subsequent histamine release is thought to be involved in the pathogenesis of other diseases such as chronic idiopathic urticaria (CIU).

To date, most therapeutic strategies are aimed at inhibiting and controlling components of the inflammatory response. Recently, new treatment strategies have emerged that focus on the development of preventive and even curative treatments. The most promising therapeutic approaches are aimed at inhibiting the IgE–FcRI interaction with the use of non-anaphylactogenic anti-IgE or anti-FcRI autoantibodies. Clinical trials in humans using an humanised anti-IgE antibody showed that this antibody was well tolerated and reduced both symptoms and use of medication in asthma and allergic rhinitis. Thus interruption of the atopic cascade at the level of the IgE–FcRI interaction with the use of non-anaphylactogenic antibodies is effective and represents an attractive therapy for the treatment of atopic disease.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.