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Abstract: Mast cells are well known as versatile cells capable of releasing and producing a variety of inflammatory mediators upon activation and are often found in close proximity of neurons. In addition, inflammation leads to local activation of neurons resulting in the release neuropeptides, which also play an important immune modulatory role by stimulation of immune cells. In intestinal disorders like inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), the number of mast cells is known to be much higher than in the normal intestine. Moreover, both these disorders are also reported to be associated with alterations in neuropeptide content and in neural innervation. Mutual association between mast cells and enteric nerves has been demonstrated to be increased in pathophysiological conditions and contribute to spreading and amplification of the response in IBD and IBS. In this review the focus lies on studies appointed to the direct interaction between mast cells and nerves in IBD, IBS, and animal models for these disorders so far. [Copyright &y& Elsevier]

Airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are characterised by airway hyperresponsiveness, epithelial damage, oxidative stress and airway inflammation. Inflammatory cells, including macrophages, neutrophils, eosinophils and lymphocytes, are crucial in the pathogenesis of asthma and COPD. The prevalence of asthma and COPD is increasing, especially in Western countries. Symptomatic treatment is based on reduction of airway obstruction by inhalation of β2-receptor agonists and attenuation of the underlying inflammatory reactions by inhalation of corticosteroids. Because these agents only suppress symptoms, there is a need for medicines that remove the cause of these airway diseases. [Copyright &y& Elsevier]

Immunoglobulin (Ig) free light (L)-chains have long been considered as the meaningless remnants of a spillover in the regular Ig production by B cells. The recently discovered role for Ig free L-chains in mediating hypersensitivity-like responses sheds new light on their potential role in immune responses. Ig free L-chains can sensitize mast cells, such that a second encounter with the appropriate antigen results in mast-cell activation. The possible importance of this reaction for the induction of T-cell-mediated immune reactions, leading to contact sensitivity, multiple sclerosis and rheumatoid arthritis is discussed. [Copyright &y& Elsevier]

Describes the pharmacological relevance of the putative epithelium-derived relaxing factors (EpiDRF), prostaglandin E2 and nitric oxide (NO), in the modulation of airway tone under basal conditions in vitro and in vivo. Ways wherein damage to the airway epithelium can affect airway responsiveness; Information on tachyphylaxis; Information on airway hyper-responsiveness.

Abstract: Extracellular ATP is a signalling molecule that often serves as a danger signal to alert the immune system of tissue damage. This molecule activates P2 nucleotide receptors, that include the ionotropic P2X receptors and the metabotropic P2Y receptors. Several publications highlight the importance of purinergic signalling in the pathogenesis of chronic airway inflammation. Recently, it has been reported that ATP accumulates in the airways of both animal models and patients with asthma or chronic obstructive pulmonary diseases (COPD); however, the role and function of ATP in the diseases process of COPD are not well understood. In this perspective, a brief overview is given on the role of ATP and P2 receptors in the pathogenesis of lung emphysema and COPD with a focus on neutrophils as messengers in intercellular communication between epithelial cells and macrophages and the activation of inflammasome pathways. Finding the link between purinergic signalling with inflammasome pathways will be a challenge for the future and could lead to the discovery of new therapeutic drugs for suppressing inflammation in the lungs of COPD patients. [Copyright &y& Elsevier]

Background: Tobacco smoking irritates and damages the respiratory tract and contributes to a higher risk of developing lung emphysema. At present, smoking cessation is the only effective treatment for reducing the progression of lung emphysema, however, there is hardly anything known about the effects of smoking cessation on cytokine and chemokine levels in the airways. To the best of our knowledge, this is the first reported in vivo study in which cytokine profiles were determined after cessation of cigarette smoke exposure. Methods: The severity of airway remodeling and inflammation was studied by analyzing alveolar enlargement, heart hypertrophy, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and lung tissue and by determining the cytokine and chemokine profiles in the BALF of A/J mice exposed to cigarette smoke for 20 weeks and 8 weeks after smoking cessation. Results: The alveolar enlargement and right ventricle heart hypertrophy found in smoke-exposed mice remained unchanged after smoking cessation. Although the neutrophilic inflammation in the BALF of cigarette smokeexposed animals was reduced after smoking cessation, a sustained inflammation in the lung tissue was observed. The elevated cytokine (IL-1α and TNF-α) and chemokine (CCL2 and CCL3) levels in the BALF of smoke-exposed mice returned to basal levels after smoking cessation, while the increased IL-12 levels did not return to its basal level. The cigarette smoke-enhanced VEGF levels did not significantly change after smoking cessation. Moreover, IL- 10 levels were reduced in the BALF of smoke-exposed mice and these levels were still significantly decreased after smoking cessation compared to the control animals. Conclusion: The inflammatory changes in the airways caused by cigarette smoke exposure were only partially reversed after smoking cessation. Although smoking cessation should be the first step in reducing the progression of lung emphysema, additional medication could be provided to tackle the sustained airway inflammation. [ABSTRACT FROM AUTHOR]

Abstract: The gastro-intestinal tract is well known for its largest neural network outside the central nervous system and for the most extensive immune system in the body. Research in neurogastroenterology implicates the involvement of both enteric nervous system and immune system in symptoms of inflammatory bowel disease and irritable bowel syndrome. Since both disorders are associated with increased immune cell numbers, nerve growth and activation of both immune cells and nerves, we focus in this review on the involvement of immune cell–nerve interactions in inflammatory bowel disease and irritable bowel syndrome. Firstly, the possible effects of enteric nerves, especially of the nonadrenergic and noncholinergic nerves, on the intestinal immune system and their possible role in the pathogenesis of chronic intestinal inflammatory diseases are described. Secondly, the possible effects of immunological factors, from the innate (chemokines and Toll-like receptors) as well as the adaptive (cytokines and immunoglobulins) immune system, on gastro-intestinal nerves and its potential role in the development of inflammatory bowel disease and irritable bowel syndrome are reviewed. Investigations of receptor-mediated and intracellular signal pathways in neuro-immune interactions might help to develop more effective therapeutic approaches for chronic inflammatory intestinal diseases. [Copyright &y& Elsevier]

In recent years, novel therapeutic strategies have become available for the treatment of chronic inflammatory disease. Neutralizing proinflammatory mediators such as leukotrienes and TNF-α, in addition to anti-IgE therapies (Omaluzimab) that target higher in the inflammatory cascade, have shown success in the treatment of allergic or autoimmune disorders. Free immunoglobulin light chains, which are produced by B lymphocytes and secreted into serum, might play a crucial role in the pathogenesis of inflammatory disease. Concentrations of free light chains are significantly increased under diverse pathological conditions in humans, and have been linked to the progression and severity of immune diseases. Here we discuss the importance of free immunoglobulin light chains as a potential therapeutic target in the treatment of chronic inflammatory disease. [Copyright &y& Elsevier]

Chronic respiratory inflammation is caused by a sustained influx of macrophages and neutrophils, which leads to tissue remodeling and collagen breakdown. Recently, we have identified collagen-breakdown products that can activate and attract inflammatory cells via the CXC (two cysteines with an inverting amino acid)1 and CXC2 receptors (CXCR1 and CXCR2). By using a technique called ‘inverted hydropathy’, small peptides were synthesized that interact specifically with the responsible collagen-breakdown products and inactivate them. After inactivation, the collagen-breakdown products are no longer able to bind to their receptors. These neutralizing peptides inhibited neutrophil influx, heart hypertrophy and lung emphysema in animal models, and they are likely to be useful in other diseases that are characterized by a chronic inflammation, such as rheumatoid arthritis and inflammatory bowel diseases, where neutrophils are potential target cells. In this opinion article we will present a new hypothesis through which the chronicity and remodeling of tissue of several inflammatory diseases could be explained. [Copyright &y& Elsevier]

Inflammatory bowel disease (IBD) describes chronic inflammatory conditions of the gastrointestinal tract, and TNF-α plays a pivotal role in mediating the response. The proinflammatory cytokine TNF-α is rapidly released by mast cells after degranulation. In the present study, we hypothesized TNF-α to be an important player in our recently described mast cell-dependent murine model for IBD. The effect of neutralizing anti-TNF-α MAb was studied on colonic hypersensitivity in mice induced by a skin application of dinitrofluorobenzene (DNFB) followed by an intrarectal challenge with dintrobenzene sulfonic acid. Features of the colonic hypersensitivity response included diarrhea, mast cell infiltration and activation, infiltration of inflammatory cells in the colon, colonic patch hypertrophy, and increased mast cell-derived TNF-α levels in the colon. Anti-TNF-α MAb could effectively abrogate diarrhea in DNFB-sensitized mice 72 h after the challenge. The numbers of colonic patches and total tissue damage scores were reduced by anti-TNF-α MAb treatment in DNFB-sensitized mice 72 h after the challenge. Mast cell infiltration and activation remained unaffected by neutralizing anti-TNF-α MAb. Treatment with the corticosteroid dexamethasone, a frequently used therapeutic treatment in IBD, resulted in a reduction of diarrhea, cellular infiltration, and total tissue damage scores to the same extent as anti-TNF-α MAb. Additionally, dexamethasone treatment could also reduce total TNF-α levels in the colon, mast cell numbers, and mast cell activation in both vehicle- and DNFB-sensitized mice 72 h after the challenge. These findings suggest that TNF-α can play an instrumental role in causing inflammatory responses in the present murine model for IBD downstream from mast cell activation. [ABSTRACT FROM AUTHOR]

Abstract: Recently it has been shown that mast cells, through release of their pro-inflammatory mediators, are involved in aspirin attacks in aspirin-sensitive patients. To date, little information is available concerning 8-isoprostane (8-epi-prostaglandin F) production by mast cells. Therefore, we examined whether exposure of mast cells to aspirin can lead to isoprostane production. In this study we show that in mast cells, IgE and antigen stimulates an intracellular oxidative burst inducing H2O2 and 8-epi-PGF production. Moreover, we show that exposure of mast cells to aspirin directly induces the production of 8-epi-PGF. Our study suggests that production of 8-epi-PGF by mast cells could contribute to the inflammatory response in e.g. aspirin-sensitive asthma patients. [Copyright &y& Elsevier]

Abstract: Occupational exposure to low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter inhaled compounds. These cells can produce many different mediators that have a putative role in asthma. In this study, we examined the role of AMs in lung function and airway inflammation of rats exposed to TMA. Female Brown Norway rats were sensitized by dermal application of TMA or received vehicle alone on days 0 and 7. One day before challenge, rats received intratracheally either empty or clodronate-containing liposomes to deplete the lungs of AMs. On day 21, all rats were challenged by inhalation of TMA in air. Lung function parameters were measured before, during, within 1 h after, and 24 h after challenge. IgE levels and parameters of inflammation and tissue damage were assessed 24 h after challenge. Sensitization with TMA led to decreased lung function parameters during and within 1 h after challenge as compared to non-sensitized rats. AM depletion alleviated the TMA-induced drop in lung function parameters and induced a faster recovery compared to sham-depleted TMA-sensitized rats. It also decreased the levels of serum IgE 24 h after challenge, but did not affect the sensitization-dependent increase in lung lavage fluid IL-6 and tissue TNF-α levels. In contrast, AM depletion augmented the TMA-induced tissue damage and inflammation 24 h after challenge. AMs seem to have a dual role in this model for TMA-induced occupational asthma since they potentiate the immediate TMA-induced decrease in lung function but tended to dampen the TMA-induced inflammatory reaction 24 h later. [Copyright &y& Elsevier]

Objective: Mast cells are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat shock proteins (HSPs). It has been reported that HSPs are capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system. In the current study, we explored whether the stress response induces HSPs and affects the signaling pathways of mast cells. Methods: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1−/− mice, responsiveness to exogenous and endogenous HSP70 was monitored by measuring cytokine release. Results: Using BMMC, we show that treatment with heat shock or acetylsalicylic acid results in a selective induction of HSPs, and leads to release of HSP70 into the extracellular environment. The release of HSP70 from mast cells may be of functional importance. We found that after induction of HSP70, the production of TNF-α and IL-6 was increased. In a number of experiments, we demonstrated that exogenous/secreted HSP70 is most likely responsible for the activation of mast cells to produce cytokines. Extracellular HSP70 induced production of TNF-α and IL-6 through the activation of the TLR4 receptor pathway, which was evidenced by an abrogation of the response in mast cells cultured from TLR4null or HSF-1−/− mice. Conclusion: Our experiments suggest that stress conditions can induce pro-inflammatory cytokine production by mast cells through an autocrine or paracrine stimulation of TLR receptors after a heat shock response. The recognition that heat shock proteins induce mast cell activation suggests an involvement of these cells in the immunological processes induced by heat shock response. [Copyright &y& Elsevier]

Abstract: Several studies have demonstrated that nonsteroidal anti-inflammatory drugs, such as acetylsalicylic acid (ASA), can have inhibitory or enhancing effects on inflammatory cell function. These effects seem independent of cyclooxygenase activity and prostaglandin synthesis inhibition. Here, we examined the effect of ASA on bone marrow-derived mast cells in more detail. ASA blocked the expression of cyclooxygenase-2, the production of tumor necrosis factor-α and interleukin-6, and the release of granule mediators from mast cells in a concentration-dependent fashion. Concomitantly, ASA inhibited nuclear factor (NF)-κB activity, as well as the phosphorylation and breakdown of the inhibitory protein IκB-α. We thus propose that the anti-inflammatory effects of ASA in mast cells are due to suppression of IκB kinase activity, thereby inhibiting subsequent phosphorylation and degradation of IκB-α, activation of NF-κB, and transcription of proinflammatory cytokines. The inhibition of BMMC degranulation was independent of NF-κB activation, however. Interestingly, the expression of cyclooxygenase-2 was not inhibited at 1mM ASA, but was even enhanced significantly. The latter might contribute to the adverse effects of ASA in ASA-sensitive asthmatics. [Copyright &y& Elsevier]

The ‘hygiene hypothesis’ suggests that a lack of infections favours the development of allergic disease. Mycobacteria and helminths are important in the hygiene hypothesis as infections caused by these agents generate regulatory mechanisms that can restore the immune balance. The hygiene hypothesis may be viewed as a complex multifactorial interaction between the environment and the genetic background of the individual, both of which contribute to the development of allergic disease. For instance, the Nramp1 gene (recently designated Slc11a1), which encodes bacterial resistance and determines the level of macrophage activation, affects treatment of allergic asthma with mycobacteria. Thus, Nramp1, and other innate immunity genes, might provide a link between the genetic background, the bacterial environment and the development of allergic diseases. It is likely that the macrophage has a crucial role in this link, connecting innate immunity with adaptive immunity in allergic disease. [Copyright &y& Elsevier]

Mast cells play a central role in immediate type hypersensitivity and inflammatory events. Activation of mast cells not only can result in the release of preformed granule-associated mediators generally followed by de novo synthesis of lipid-derived substances. In the present study, we show that mast cell can be activated to release lipid mediators in absence of granule exocytosis. Primary cultured murine mast cells were stimulated with substance P and produced leukotriene C4, and prostaglandin D2 without the release of the granule-associated enzyme β-hexosaminidase. Indomethacin and nordihydroguaiaretic acid caused complete inhibition of arachidonic metabolite generation. Leukotriene C4 and prostaglandin D2 production was blocked by genistein, a specific inhibitor of tyrosine kinases, and bisindolylmaleimide, a protein kinase C inhibitor, indicating a role for both phosphorylation pathways in the substance P-stimulated lipid mediator production. We suggest that the cytokine microenvironment of the mast cell determines whether mast cell stimulation leads to only lipid mediator release or full activation. Analysis of granule-associated mediators only might underestimate the role of mast cell activation under (patho)physiological conditions. [Copyright &y& Elsevier]

Eotaxin is a chemokine implicated in eosinophil trafficking and may be involved in the development of airway hyperresponsiveness. The role of eotaxin in a mouse model for allergic asthma was investigated. Challenging ovalbumin-sensitised mice with ovalbumin aerosol leads to airway hyperresponsiveness and airway eosinophilia 24 h after the last challenge. Furthermore, eotaxin concentrations were markedly increased in lungs and broncho-alveolar lavage fluid of ovalbumin-challenged mice compared to vehicle treated mice. This could mean that eotaxin is implicated in the pathology of this model. To further investigate the role of eotaxin in this murine model for allergic asthma, the ovalbumin response was modulated by either treatment with eotaxin antibodies or additional eotaxin, to suppress or promote the development of airway hyperresponsiveness and inflammation. Administration of eotaxin antibodies or an additional intravenous eotaxin injection did not alter the development of ovalbumin-induced airway hyperresponsiveness and eosinophilia. In conclusion, eotaxin concentrations were increased in a murine model for allergic airway inflammation. However, anti-eotaxin antibodies or additive intravenous murine eotaxin did not influence airway inflammation and hyperresponsiveness in this mouse model for allergic asthma. [Copyright &y& Elsevier]

Hypersensitivity reactions induced by low molecular weight compounds are investigated extensively in the skin. However, these reactions can also occur in the lungs of previously sensitized individuals after local airway challenge. This hapten-induced pulmonary hypersensitivity reaction resembles features observed in asthmatic patients, such as bronchial hyperreactivity, accumulation of inflammatory cells, and airway edema. We review data that hapten-induced hypersensitivity reactions in mouse airways can be models for both atopic and non-atopic asthma associated with low molecular weight compounds. [Copyright &y& Elsevier]

Abstract: To investigate the possible role of the vanilloid receptor-1 (TRPV1) in allergic airway responses, the effect of the specific TRPV1 receptor antagonist capsazepine was examined. Capsazepine significantly decreased the ovalbumin-induced contraction of isolated tracheal rings from ovalbumin-sensitized guinea pigs. This is the first report directly showing the involvement of the TRPV1 in experimental allergic airway responses. [Copyright &y& Elsevier]

Reports on the identification of novel potential target molecules that modulate or inactivate disease-inducing, allergen-specific T-helper type 2 (Th2) lymphocytes. Analysis of expression of genes in the draining lymph node in a mouse model of allergic asthma using representational difference analysis of complementary DNAs.

Beside the production of complete immunoglobulins IgG, IgE, IgA, IgM and IgD, consisting of tetrameric heterodimers of immunoglobulin heavy and light chains, B cells also secrete immunoglobulin free light chains (Ig-fLC). Previous studies showed that Ig-fLCs are able to induce immediate hypersensitivity reactions. It is apparent that recognition and binding of antigen are crucial steps in the onset of these inflammatory responses. In this study, the binding characteristics of Ig-fLC to antigen were further investigated using various biochemical approaches. In addition, we investigated whether antigenmediated crosslinking of Ig-fLC is required to initiate allergic skin inflammation in vivo. Our study shows that binding of IgfLCs to antigen can be measured with different experimental setups. Surface plasmon resonance analysis showed real-time antigen binding characteristics. Specific antigen binding by Ig-fLCs was further detected using immunoblotting and ELISA. Using the ELISA-based assay, a binding affinity of 76.9±3.8 nM was determined for TNP-specific Ig-fLC. Antigen-induced ear swelling in mice passively sensitized with trinitrophenol-specific Ig-fLC was inhibited when multivalent antigen was combined with excess of monovalent antigen during challenge. We conclude that Ig-fLCs are able to interact with antigen, a prerequisite for antigen-specific cellular activation. In analogy to antigen-specific Fc receptor-induced mast cell activation, crosslinking of Ig-fLCs is necessary to initiate a local allergic response. [ABSTRACT FROM AUTHOR]

Abstract: Tobacco smoke is the main factor in the etiology of lung emphysema. Generally prolonged, substantial exposure is required to develop the disease. Humic acid is a major component of cigarette smoke that accumulates in smokers’ lungs over time and induces tissue damage. Objectives: To investigate whether humic acid pre-loading potentiates the development of cigarette smoke-induced lung emphysema in mice and increases IL-8 release by human monocytes. Methods: C57BL/6J mice received humic acid or aqueous vehicle by tracheal installation on day 0 and day 7. From day 21 to day 84, the mice were exposed to cigarette smoke or clean air for 5 days/week. Twenty-four hours after the last exposure we determined leukocytes in lung lavage, heart hypertrophy and alveolar wall destruction. Human monocytes were incubated with cigarette smoke extract (CSE), humic acid or the combination overnight. Results: Humic acid nor cigarette smoke caused alveolar wall destruction within two months. Interestingly, the combination did induce lung emphysema. Humic acid, cigarette smoke or the combination did not change leukocyte types and numbers in lung lavage fluid, but the combination caused peribronchiolar and perivascular lymphocyte infiltration. Humic acid treatment resulted in a high proportion of alveolar macrophages heavily loaded with intracellular granula. Humic acid also induces the release of IL-8 from human monocytes and enhances the CSE-induced IL-8 release. Conclusions: Humic acid deposition in the lungs potentiates the development of cigarette smoke-induced interstitial inflammation and lung emphysema. Moreover, humic acid promotes IL-8 release from human monocytes. Since humic acid accumulates steadily in the lungs of smokers, this may provide an explanation for the natural history on late onset of this disease. The model described here offers a novel way to study emphysema and may direct the search for new therapeutic approaches. [Copyright &y& Elsevier]

Abstract: Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline–Glycine–Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation. [Copyright &y& Elsevier]

There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide l-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema. [ABSTRACT FROM AUTHOR]

van Esch BCAM, Schouten B, Hofman GA, van Baalen T, Nijkamp FP, Knippels LMJ, Willemsen LEM, Garssen J. Acute allergic skin response as a new tool to evaluate the allergenicity of whey hydrolysates in a mouse model of orally induced cow’s milk allergy. Pediatr Allergy Immunol 2010: 21: e780–e786. © 2009 John Wiley & Sons A/S Hypoallergenic milk formulae are used for cow’s milk allergic infants and may be a good option for infants at risk. Clinical studies have shown that the protein source or the hydrolysis methodology used may influence the effectiveness in infants stressing the importance of adequate pre-clinical testing of hypoallergenic formulae in an in vivo model of orally induced cow’s milk allergy. This study was undertaken to introduce a new read-out system to measure the residual allergenicity of whey hydrolysates on both the sensitization and challenge phase of orally induced cow’s milk allergy in mice. Mice were sensitized orally to whey or a partial whey hydrolysate (pWH) to measure the residual sensitizing capacity. To predict the residual allergenicity of hydrolysates, whey allergic mice were challenged in the ear with pWH, extensive whey hydrolysate or an amino acid-based formula. An acute allergic skin response (ear swelling at 1 h), whey-specific serum antibodies, and local MCP-1 concentrations were measured. In contrast to whey, oral sensitization with pWH did not result in the induction of whey-specific antibodies, although a minor residual skin response to whey was observed after challenge. Skin exposure to whey hydrolysates showed a hydrolysation dependent reduction of the acute allergic skin response in whey allergic mice. In contrast to whey, skin exposure to pWH did not enhance tissue MCP-1 levels. The acute allergic skin response in mice orally sensitized to cow’s milk proteins reveals a new pre-clinical tool which might provide information about the residual sensitizing capacity of hydrolysates supporting the discussion on the use of hypoallergenic formulae in high risk children. This mouse model might be a relevant model for the screening of new hypoallergenic formulae aimed to prevent or treat cow’s milk allergy. [ABSTRACT FROM AUTHOR]

Abstract: Macrophages are key inflammatory cells in chronic obstructive pulmonary disease (COPD). The pathophysiology of cigarette smoke-induced lung emphysema is complex but there is a clear role for reactive oxygen species (ROS, such as peroxynitrite), tumor necrosis factor (TNF-α) and interleukin (IL)-8. We investigated whether TNF-α or cigarette smoke medium (CSM) alone or in combination induces the production of IL-8 by human macrophages or monocyte lymphoma U937. CSM and TNF-α induce a dose- and time-dependent increase in IL-8 production. Interestingly, when sub-threshold concentrations of CSM and TNF-α were co-incubated, a 1500% increase in IL-8 production was observed compared to either of the compounds alone. Similar results were obtained with TNF-α and the peroxynitrite donor SIN-1. Moreover, the overproduction of IL-8 was associated with an enhanced increase in the translocation of NF-κB and an enhanced decrease in glutathione levels. Preincubation of the cells with antioxidants, such as N-acetyl-l-cysteine (NAC), prevented the overproduction of IL-8 and activation of NF-κB. In conclusion, CSM exposure of macrophages up-regulates the expression and the production of IL-8 via reactive oxygen species and NF-κB activation. Moreover, CSM dramatically enhances the production of IL-8 in combination with TNF-α. Based upon the strong synergistic action, a combination therapy directed against ROS and TNF-α could be a new approach to stop the progression in lung damage during emphysema. [Copyright &y& Elsevier]

Abstract: Extracellular ATP is a signaling molecule that often serves as a danger signal to alert the immune system of tissue damage. This molecule activates P2 nucleotide receptors, that include the ionotropic P2X receptors and metabotropic P2Y receptors. Recently, it has been reported that ATP accumulates in the airways of both asthmatic patients and sensitized mice after allergen challenge. The role and function of ATP in the pathogenesis of chronic obstructive pulmonary diseases (COPD) are not well understood. In this study we investigated the effect of cigarette smoke on purinergic receptors and ATP release by neutrophils. Neutrophils and their mediators are key players in the pathogenesis of lung emphysema. Here we demonstrated that in an in vivo model of cigarette smoke-induced lung emphysema, the amount of ATP was increased in the bronchoalveolar lavage fluid. Moreover, activation of neutrophils with cigarette smoke extract induced ATP release. Treatment of neutrophils with apyrase (catalyses the hydrolysis of ATP to yield AMP) and suramin (P2-receptor antagonist) abrogated the release of CXCL8 and elastase induced by cigarette smoke extract and exogenous ATP. These observations indicate that activation of purinergic signaling by cigarette smoke may take part in the pathogenesis of lung emphysema. [Copyright &y& Elsevier]

Abstract: Immunoglobulin-free light chains (IgLC) secreted by B lymphocytes, have been shown to mediate hypersensitivity by inducing antigen-specific mast cell activation. Although both mast cells and sensory neurons contribute to the hypersensitivity response, the role of IgLC in relation to sensory neurons is unknown. We therefore aimed to investigate the effects of IgLC on cultures of murine dorsal root ganglion (DRG) neurons. Immunohistochemistry demonstrated that IgLC and IgE could specifically bind to DRG neurons, on which the presence of FcεRI, the specific receptor for IgE, was demonstrated by western blotting. Further, optical recordings with Fluo-4 showed that application of the corresponding antigen to IgLC- or IgE-sensitized DRG neurons induces a sustained increase in intracellular Ca2+ in about half of these neurons. These results show that IgLC and IgE can mediate antigen-specific responses in murine neurons. Our findings present a novel way of antigen-specific neuronal activation. [Copyright &y& Elsevier]

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Cigarette smoke has been considered a major player in the pathogenesis of COPD. The inflamed airways of COPD patients contain several inflammatory cells including neutrophils, macrophages,T lymphocytes, and dendritic cells (DCs). The relative contributions of these various inflammatory cells to airway injury and remodeling are not well documented. In particular, the potential role of DCs as mediators of inflammation in the smoker's airways and COPD patients is poorly understood. In the current study we analyzed the effects of cigarette smoke extract on mouse bone marrow derived DC and the production of chemokines and cytokines were studied. In addition, we assessed CSE-induced changes in cDC function in the mixed lymphocyte reaction (MLR) examining CD4+ and CD8+ T cell proliferation. Cigarette smoke extract induces the release of the chemokines CCL3 and CXCL2 (but not cytokines), via the generation of reactive oxygen species (ROS). In a mixedleukocyte reaction assay, cigarette smoke-primed DCs potentiate CD8+T cell proliferation via CCL3. In contrast, proliferation of CD4+T cells is suppressed via an unknown mechanism. The cigarette smoke-induced release of CCL3 and CXCL2 by DCs may contribute to the influx of CD8+T cells and neutrophils into the airways, respectively. [ABSTRACT FROM AUTHOR]

The combination of inhaled corticosteroids and long-acting β2-adrenoceptor agonists is increasingly used in chronic obstructive pulmonary disease (COPD). Recently, we have demonstrated that combination of salmeterol and fluticasone propionate (FP) additionally suppress the production of IL-8 by human monocyte. In this study, the molecular mechanism behind the effectiveness of this combination therapy is investigated in human neutrophils. Human neutrophils were preincubated with salmeterol or FP or the combination. The amount of interleukin-8 (IL-8), elastase and matrix metalloproteinases (MMP)-2 and -9 releases, and reactive oxygen species (ROS) generation and expression of MAP kinase phosphatase (MKP-1) and glucocorticoid receptor (GR) were determined. Cigarette smoke medium (CSM) induces an increased expression of CXC receptors and the production of ROS that may explain the strong production of IL-8 by neutrophils. The expression of CXC receptors, the production of ROS, and the release of elastase and MMP-2 and -9 were not influenced by salmeterol, FP, or the combination. Interestingly, the combination therapy had an additive suppressive effect on the CSM-induced production of IL-8. The latter could be explained by an increased mRNA expression of MKP-1, the GR and an increased translocation of the GR to the nucleus. This leads eventually to suppression of both the NF-κB and MAPK pathways and, hence, to less IL-8 production by the neutrophil. These data are in support for the use of a combination therapy in COPD patients. [ABSTRACT FROM AUTHOR]

In patients with chronic obstructive pulmonary disease (COPD), an inflammatory process is ongoing in the lungs, with concomitant damage of the alveolar structures and loss of airway function. In this inflammatory process, extracellular matrix degradation is observed. During this lung matrix degradation, small peptide fragments consisting of proline and glycine repeats generated from collagen fibers are liberated from the matrix by matrix metalloproteinases. Chemotactic activities of these collagen-derived peptides such as N-acetyl-proline-glycine-proline (PGP) via CXCR1 and CXCR2 have been reported. We show here that PGP induces neutrophil migration in vivo, which is dose dependent. Moreover, PGP is involved in the development of emphysema-like changes in the airways. The complementary peptide, L-arginine-threonine-arginine (RTR), has been shown to bind to PGP sequences and inhibit neutrophil infiltration. We show that RTR impedes both PGPo and interleukin-8-induced chemotaxis in vitro. In vivo, RTR prevents both migration and activation of neutrophils induced by PGP. Furthermore, RTR completely inhibits PGP-induced lung emphysema, assessed by changes in alveolar enlargement and right ventricular hypertrophy. In conclusion, these data indicate that collagen breakdown products, especially PGP, are important in the pathogenesis of COPD and that PGP antagonism via RTR ameliorates lung emphysema. [ABSTRACT FROM AUTHOR]

Abstract: Allergies are the result of an inappropriate reaction against innocuous environmental proteins. The prevalence and severity of allergic diseases has increased dramatically during the last decade in developed countries. Allergen-specific T helper (Th) cells play a pivotal role in the pathogenesis of allergic hypersensitivity reactions. These Th cells activate a complex immune reaction that triggers the release of potent mediators and enhances the recruitment of inflammatory cells, which in turn elicit an inflammatory response that leads to the clinical symptoms of allergic disease. The current therapies for allergic diseases focus primarily on control of symptoms and suppression of inflammation, without affecting the underlying cause. However, the knowledge about the pathophysiology of allergic diseases has substantially increased, offering new opportunities for therapeutic intervention. In this review, we will focus on current insights into the mechanism of allergic reactions. [Copyright &y& Elsevier]

Abstract: Chronic obstructive pulmonary disease (COPD) is a global health problem. Being a progressive disease characterized by inflammation, it deteriorates pulmonary functioning. Research has focused on airway inflammation, oxidative stress, and remodelling of the airways. Macrophages, neutrophils and T cells are thought to be important key players. A number of new research topics received special attention in the last years. The combined use of inhaled corticosteroids and long-acting β2-adrenoceptor agonists produces better control of symptoms and lung function than that of the use of either compound alone. Furthermore, collagen breakdown products might be involved in the recruitment and activation of inflammatory cells by which the process of airway remodelling becomes self-sustaining. Also, TLR (Toll-like receptor)-based signalling pathways seem to be involved in the pathogenesis of COPD. These new findings may lead to new therapeutic strategies to stop the process of inflammation and self-destruction in the airways of COPD patients. [Copyright &y& Elsevier]

Abstract: Cigarette smoke is the major risk factor for the development of chronic obstructive pulmonary disease (COPD). Macrophages are suggested to orchestrate the chronic inflammatory response and tissue destruction associated with COPD by secreting interleukin (IL)-8, a major neutrophil chemoattractant. The combination of inhaled corticosteroids and long-acting β2-adrenoceptor agonists are increasingly used as maintenance therapy in patients with COPD. The aim of this study was to determine whether combined fluticasone propionate, a corticosteroid, and salmeterol, a long-acting β2-adrenoceptor agonist, can suppress IL-8 production by human macrophages. To mimic resident macrophages in the lung, human monocytes were cultured for 5 days in medium containing Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF). In human Monocyte-Derived Macrophages, we found that cigarette smoke medium strongly enhanced IL-8 release in a time- and concentration-dependent manner. IL-8 release by cigarette smoke was significantly suppressed in a concentration-dependent manner by fluticasone and salmeterol. Coincubation of the drugs potentiated the inhibitory effect on cigarette smoke medium-induced IL-8 production and longer preincubation times resulted in more IL-8 inhibition. Interestingly, preincubation of cells with suboptimal concentration of salmeterol for 4 h before fluticasone administration for 30 min potentiates the inhibitory effect of fluticasone on IL-8 release. In conclusion, combination therapy may provide benefits over monotherapy for the treatment of COPD patients. [Copyright &y& Elsevier]

Abstract: Immunoglobulin (Ig) E-dependent activation of mast cells is central to the allergic response. The engagement of IgE-occupied receptors initiates a series of molecular events that causes the release of preformed, and de novo synthesis of, allergic mediators. Cysteinyl leukotrienes are able to contract airway smooth muscle and increase mucus secretion and vascular permeability and recruit eosinophils. Mast cells have also recently been recognized as active participants in innate immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat-shock proteins (HSPs). We previously demonstrated that treatment of mast cells with heat shock or acetylsalicylic acid results in an increase of TNF-α and IL-6 release. This effect was paralleled by expression of HSP70. In the current study, we further investigated the effects of heat shock and acetylsalicylic acid on the activation of mast cells and the release of cysteinyl leukotrienes. In mouse mast cells, derived from a culture of bone marrow cells, responsiveness to heat shock, acetylsalicylic acid and exogenous or endogenous HSP70 was monitored by measuring leukotriene C4 release. We show that after heat shock treatment and exposure to acetylsalicylic acid leukotriene production was increased. Moreover, exogenous rHSP70 also induced leukotriene production. Because it has been reported that leukotriene production in mast cells may be mediated by Toll like receptor (TLR) activation, and HSP70 also activates TLRs signaling, we further explored these issues by using mast cells that are not able to produce HSP70, i.e. heat shock factor-1 (HSF-1) knockout cells. We found that in HSF-1 knockout bone marrow derived mast cells, heat shock and acetylsalicylic acid failed to induce release of leukotrienes. Moreover, in wild type cells the surface expression of TLR4 was attenuated, whereas the intracellular expression was up-regulated. We conclude that heat shock and acetylsalicylic acid induce the production and release of heat shock proteins from mast cells, which in turn stimulate leukotriene synthesis through activation of TLR4. [Copyright &y& Elsevier]

Background: It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated. Methods: In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21. Results: We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-γ (IFN-γ) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-α (TNF-α) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness. Conclusion: Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD. [ABSTRACT FROM AUTHOR]

Abstract: The gastro-intestinal tract is highly innervated by both intrinsic and extrinsic sensory nerves and this neuronal component is thought to play a role in local inflammatory responses. This in vivo study was designed to determine the function of substance P and the tachykinin NK1 receptor in the pathogenesis of inflammatory bowel disease by the use of the specific antagonist RP 67580. The dinitrofluorobenzene (DNFB)-induced colonic hypersensitivity model is associated with increased levels of substance P in the colon. The tachykinin NK1 receptor antagonist RP 67580 was used to investigate the role of substance P on the development of diarrhea, mast cell infiltration and activation, colonic tissue damage, hypertrophy of colonic lymphoid structures and leukocyte infiltration. The formation of watery diarrhea could completely be abrogated by treatment with RP 67580 in DNFB-sensitized animals 72 h after challenge. Antagonizing the tachykinin NK1 receptor in these animals also resulted in significantly reduced colonic patch hypertrophy, leukocyte recruitment and tissue damage. Total levels of substance P in the colon of DNFB-sensitized mice treated with the inactive enantiomer of the tachykinin NK1 receptor antagonist were significantly higher compared to DNFB-sensitized mice treated with RP 67580 72 h after challenge. Although RP 67580 was capable of reducing the total number of mast cells present in the colon, mast cell activation was not affected by this treatment. In conclusion, in this chemically-induced immunological model for inflammatory bowel disease we demonstrated an important role for tachykinin NK1 receptors, and its ligand substance P, in the development of colitis downstream from mast cell activation. [Copyright &y& Elsevier]

Occupational exposure to low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter these inhaled compounds and were previously shown to affect TMA-induced asthma-like symptoms in the Brown Norway rat (Valstar et al., Toxicol. Appl. Pharmacology 211:20–29, 2006). TMA is a hapten that will bind to endogenous proteins upon entrance of the body. Therefore, in the present study we determined if TMA conjugated to albumin is able to induce asthma-like symptoms and if these are affected by AM depletion. Female Brown Norway rats were sensitized by dermal application of TMA or received vehicle alone on days 0 and 7. One day prior to the inhalation challenge the rats were treated intratracheally with either empty liposomes or liposomes containing clodronate (dichloromethylene diphosphonate) to specifically deplete the lungs of AMs. On day 21, all groups of rats were challenged by inhalation of TMA-BSA. Breathing frequency, tidal volume, and minute ventilation were measured before, during, within 1 h, and 24 h after challenge and the gross respiratory rate score was determined during challenge. Total and TMA-specific IgE levels were determined in serum and lung lavage fluid and parameters of inflammation and tissue damage were assessed in lung lavage fluid and/or lung tissue 24 h after challenge. Sensitization with TMA had no effect on the lung function before challenge, but TMA-BSA challenge resulted in an early asthmatic response as compared to the non-sensitized rats, irrespective of AM depletion. AM depletion had no effect on the sensitization-induced serum and lung lavage fluid IgE levels. TMA-BSA inhalation did not induce airway inflammation and tissue damage irrespective of sensitization, unless AM were depleted. Data indicate that AMs inhibit immunologically non-specific damage and inflammatory cell influx into the lungs as caused by TMA-BSA inhalation. Since effects of inhalation challenge with TMA-BSA are partly different from those of TMA, challenge with the latter is to be preferred for hazard identification. [ABSTRACT FROM AUTHOR]

Objective: It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid, ibuprofen, and indomethacin, induce anti-inflammatory effects through inhibition of cyclooxygenase enzyme activity. However, it has also been established that a variety of their anti-inflammatory effects are independent of cyclooxygenase. In the search for alternative modes of action, it was found that NSAIDs share some cellular effects with heat shock treatment. This prompted us to investigate whether NSAIDs modulate production of proinflammatory cytokines by mast cells through the heat shock response. Materials and Methods: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1−/− mice, responsiveness to heat shock and NSAIDs was monitored by measuring tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and signaling pathways. Results: In bone marrow-derived mast cells (BMMC), we found that heat shock and a number of NSAIDs induced heat shock protein 70 (HSP70), which was closely paralleled with inhibition of IL-6 and TNF-α production. Surprisingly, in BMMC from HSF-1−/−mice, heat shock and selected NSAIDs were still able to suppress cytokine production in the absence of HSP70 induction. Conclusion: In this article, we provide evidence that inhibition of release of proinflammatory cytokines by NSAIDs and heat shock may be attributed to inhibition of the inhibitory nuclear factor κB (NF-κB) kinase activity, extracellular signal-regulated kinases 1/2, and p38 pathways, resulting in decreased transcriptional activity of the NF-κB pathway. [Copyright &y& Elsevier]

We describe the tripeptide neutrophil chemoattractant N-acetyl Pro-Gly-Pro (PGP), derived from the breakdown of extracellular matrix (ECM), which shares sequence and structural homology with an important domain on alpha chemokines. PGP caused chemotaxis and production of superoxide through CXC receptors, and administration of peptide caused recruitment of neutrophils (PMNs) into lungs of control, but not CXCR2-deficient mice. PGP was generated in mouse lung after exposure to lipopolysaccharide, and in vivo and in vitro blockade of PGP with monoclonal antibody suppressed PMN responses as much as chemokine-specific monoclonal antibody. Extended PGP treatment caused alveolar enlargement and right ventricular hypertrophy in mice. PGP was detectable in substantial concentrations in a majority of bronchoalveolar lavage samples from individuals with chronic obstructive pulmonary disease, but not control individuals. Thus, PGP's activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: Occupational exposure to airborne low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter these inhaled compounds and were previously shown to influence TMA-induced asthma-like symptoms in the Brown Norway rat. TMA is a hapten that will bind to endogenous proteins upon entrance of the body. Therefore, in the present study we determined if TMA and TMA conjugated to serum albumin induced the production of the macrophage mediators nitric oxide (NO), tumour necrosis factor (TNF), and interleukin 6 (IL-6) in vitro using the rat AM cell line NR8383 and primary AMs derived from TMA-sensitized and naïve Brown Norway rats. Methods: Cells were incubated with different concentrations of TMA, TMA conjugated to bovine serum albumin (BSA), and BSA as a control for 24 h and the culture supernatant was analyzed for mediator content. Results: TMA alone was not able to induce the production of mediators by NR8383 cells and primary AMs from sensitized and sham-treated rats. TMA-BSA, on the contrary, dose-dependently stimulated the production of NO, TNF, and IL-6 by NR8383 cells and of NO and TNF, but not IL-6, by primary AMs independent of sensitization. Conclusion: Results suggest that although TMA is a highly reactive compound, conjugation to a suitable protein is necessary to induce mediator production by AMs. Furthermore, the observation that effects of TMA-BSA were independent of sensitization suggests involvement of an immunologically non-specific receptor. In the discussion it is argued that a macrophage scavenger receptor is a likely candidate. [ABSTRACT FROM AUTHOR]

Background: The major risk factor for the development of COPD is cigarette smoking. Smoking causes activation of resident cells and the recruitment of inflammatory cells into the lungs, which leads to release of pro-inflammatory cytokines, chemotactic factors, oxygen radicals and proteases. In the present study evidence is found for a new cellular mechanism that refers to a link between smoking and inflammation in lungs. Methods: Employing human monocyte-derived macrophages, different techniques including FACS analysis, Cytometric Bead Array Assay and ELISA were achieved to evaluate the effects of CS on pro-inflammatory cytokine secretion including IL-8. Then, Toll-like receptor neutralization was performed to study the involvement of Toll-like receptor-4 in IL-8 production. Finally, signaling pathways in macrophages after exposure to CS medium were investigated performing ELISA and Western analysis. Results: We demonstrate that especially human monocytes are sensitive to produce IL-8 upon cigarette smoke stimulation compared to lymphocytes or neutrophils. Moreover, monocyte-derived macrophages produce high amounts of the cytokine. The IL-8 production is dependent on Toll-like receptor 4 stimulation and LPS is not involved. Further research resolved the cellular mechanism by which cigarette smoke induces cytokine production in monocyte-derived macrophages. Cigarette smoke causes subsequently a concentration-dependent phosphorylation of IRAK and degradation of TRAF6. Moreover, IκBα was phosphorylated which suggests involvement of NF-κB. In addition, NFκB -inhibitor blocked cigarette smoke-induced IL-8 production. Conclusion: These findings link cigarette smoke to inflammation and lead to new insights/therapeutic strategies in the pathogenesis of lung emphysema. [ABSTRACT FROM AUTHOR]

Background: Asthma is associated with airway hyperresponsiveness and enhanced T-cell number/activity on one hand and increased levels of exhaled nitric oxide (NO) with expression of inducible NO synthase (iNOS) on the other hand. These findings are in paradox, as NO also relaxes airway smooth muscle and has immunosuppressive properties. The exact role of the endothelial NOS (eNOS) isoform in asthma is still unknown. We hypothezised that a delicate regulation in the production of NO and its bioactive forms by eNOS might be the key to the pathogenesis of asthma. Methods: The contribution of eNOS on the development of asthmatic features was examined. We used transgenic mice that overexpress eNOS and measured characteristic features of allergic asthma after sensitisation and challenge of these mice with the allergen ovalbumin. Results: eNOS overexpression resulted in both increased eNOS activity and NO production in the lungs. Isolated thoracic lymph nodes cells from eNOS overexpressing mice that have been sensitized and challenged with ovalbumin produced significantly less of the cytokines IFN-γ, IL-5 and IL-10. No difference in serum IgE levels could be found. Further, there was a 50% reduction in the number of lymphocytes and eosinophils in the lung lavage fluid of these animals. Finally, airway hyperresponsiveness to methacholine was abolished in eNOS overexpressing mice. Conclusion: These findings demonstrate that eNOS overexpression attenuates both airway inflammation and airway hyperresponsiveness in a model of allergic asthma. We suggest that a delicate balance in the production of bioactive forms of NO derived from eNOS might be essential in the pathophysiology of asthma. [ABSTRACT FROM AUTHOR]

Objective: Mast cells play pivotal roles in immediate-type and inflammatory allergic and nonallergic reactions. Cross-linking of the high-affinity receptor for IgE (FcεRI) on mast cells activates a signaling pathway leading to Ca2+ mobilization and is followed by degranulation and the release of histamine and other preformed mediators, as well as de novo synthesis of arachidonic acid metabolites. In a previous study, we have demonstrated that heat shock activates heat shock transcription factor-1 (HSF-1), induces heat shock protein 70 (HSP70), and suppresses cytokine production in bone marrow–derived mast cells (BMMC). In this study, we further investigated the effects of heat shock on the activation of mast cells and the release of mast cell mediators. Methods: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1−/−mice, responsiveness to heat shock was monitored by measuring β-hexosaminidase and leukotriene C4 (LTC4) release. Results: Using BMMC, we found that heat shock inhibits degranulation of BMMC without affecting leukotriene production. To further elucidate the mechanism of suppression of degranulation, we studied the effects of heat shock on the regulation of signal transduction in more detail. We found that heat shock inhibits calcium mobilization and tyrosine phosphorylation of Syk and SHIP upon IgE receptor activation, but increases the phosphorylation of SHP-1 and -2. Moreover, our results revealed that suppression of tyrosine phosphorylation of Syk and SHIP coincided with an increased tyrosine phosphatase activity. Conclusion: The inhibitory action of heat shock toward mast cell degranulation is likely due to shifting the balance between kinase and phosphatase activity. [Copyright &y& Elsevier]

The observation that only 50% of patients with adult asthma manifest atopy indicates that other inflammatory mechanisms are likely involved in producing the characteristic features of this disorder; namely reversible airway obstruction, hyperresponsiveness, and pulmonary inflammation. Our recent discovery that antigen-specific lg free light chains (LCs) mediate hypersensitivity- like responses suggests that these molecules may be of import in the pathophysiology of asthma. Using a murine experimental model of nonatopic asthma, we now have shown that an IC antagonist, the 9-mer peptide F991, can abrogate the development of airway obstruction, hyperresponsiveness, and pulmonary inflammation. Further, passive immunization with antigen-specific LCs and subsequent airway challenge can elicit a mast cell-dependent reaction leading to acute bronchoconstriction. These findings, and the demonstration that the concentration of free k LCs in the sera of patients with adult asthma were significantly increased (as compared with age-matched non asthmatic individuals), provide previously undescribed insight into the pathogenesis of asthma. In addition, the ability to inhibit pharmacologically IC-induced mast cell activation provides a therapeutic means to prevent or ameliorate the adverse bronchopulmonary manifestations of this incapacitating disorder. [ABSTRACT FROM AUTHOR]

Mast cell activation, bronchoconstriction, inflammation and airway hyperreactivity are prominent features of non-atopic hypersensitivity reactions in mouse airways. We studied the role of tachykinin receptors in mice that were skin-sensitized with dinitrofluorobenzene (or vehicle) and challenged intranasally with dinitrobenzene sulfonic acid. Tachykinin NK1 receptor blockade, by treatment with the antagonist RP67580, or absence of the tachykinin NK1 receptor resulted in a strong reduction in the accumulation of neutrophils in the bronchoalveolar lavage fluid, and in the development of tracheal hyperreactivity in mice 48 h after challenge. In contrast, treatment with the tachykinin NK2 receptor antagonist SR48968 did not affect the dinitrofluorobenzene-induced hypersensitivity reaction. We have previously shown that mast cells play a crucial role in the development of non-atopic asthma. However, we did not observe an inhibitory effect of the tachykinin receptor antagonists or the genetic absence of tachykinin NK1 receptors on mast cell protease release. In conclusion, distal from mast cell activation, the tachykinin NK1 receptor is crucial for the infiltration of pulmonary neutrophils and the development of tracheal hyperreactivity in non-atopic asthma. [Copyright &y& Elsevier]

We previously described two novel peptides, Ca2+-like peptide (CALP) 1 and CALP2, which interact with Ca2+-binding EF hand motifs, and therefore have the characteristics to define the role of the Ca2+-sensing regulatory protein calmodulin in asthma. In the present study, the effects of the calcium-like peptides were investigated in an animal model for allergic asthma. For that purpose, sensitized guinea pigs were intratracheally pretreated with CALP1 or CALP2. Thirty minutes later, the animals were challenged with aerosolized ovalbumin. Acute bronchoconstriction was measured as well as characteristic features of asthma 6 and 24 hours (h) after challenge. Neither CALP1 nor CALP2 prevented the anaphylactic response elicited by ovalbumin challenge. However, CALP1 pretreatment attenuated the influx of inflammatory cells in the lungs 6 h after challenge. Furthermore, radical production by these cells was diminished both 6 and 24 h after challenge. Moreover, CALP1 completely inhibited airway hyperresponsiveness in vitro 24 h after challenge. We conclude that CALP1, as a selective calmodulin agonist, inhibits the development of asthmatic features probably via the attenuation of mast cell degranulation and radical production. Specific modulation of calmodulin activity might therefore be a potential new target for the treatment of allergic asthma. [Copyright &y& Elsevier]

The hypothesis that a lack of early childhood bacterial infections would favor the development of allergic disease suggests that bacteria can be used as a potential treatment for allergic asthma. Therefore, in this study, we investigated the therapeutic potential of heat-killed Mycobacterium vaccae in two mouse models of allergic asthma. For this purpose, mice were sensitized i.p. with ovalbumin/alum (severe model) or ovalbumin alone (mild model) and challenged on days 77, 80 and 83 by inhalation of either ovalbumin or saline aerosols. Treatment of mice with M. vaccae (s.c. 107 or 108 colony-forming units) on days 56 and 63, however, did not reduce airway hyperresponsiveness and eosinophilia, IgE and interleukin-5 production 24 h after ovalbumin challenge in either mouse model. We therefore conclude that treatment of sensitized mice with M. vaccae before allergen exposure is not able to reduce the allergic and asthma-like response in a mild and a severe model of allergic asthma. [Copyright &y& Elsevier]