René Moser

Toll-like receptor and tumour necrosis factor dependent endotoxin-induced acute lung injury

Recent studies on endotoxin/lipopolysaccharide (LPS)‐induced acute inflammatory response in the lung are reviewed. The acute airway inflammatory response to inhaled endotoxin is mediated through Toll‐like receptor 4 (TLR4) and CD14 signalling as mice deficient for TLR4 or CD14 are unresponsive to endotoxin. Acute bronchoconstriction, tumour necrosis factor (TNF), interleukin (IL)‐12 and keratinocyte‐derived chemokine (KC) production, protein leak and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecules myeloid differentiation factor 88 (MyD88) and Toll/Interleukin‐1 receptor (TIR)‐domain‐containing adaptor protein (TIRAP), but independent of TIR‐domain‐containing adaptor‐inducing interferon‐beta (TRIF). In particular, LPS‐induced TNF is required for bronchoconstriction, but dispensable for inflammatory cell recruitment. Lipopolysaccharide induces activation of the p38 mitogen‐activated protein kinase (MAPK). Inhibition of pulmonary MAPK activity abrogates LPS‐induced TNF production, bronchoconstriction, neutrophil recruitment into the lungs and broncho‐alveolar space. In conclusion, TLR4‐mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin are dependent on TLR4/CD14/MD2 expression using the adapter proteins TIRAP and MyD88, while TRIF, IL‐1R1 or IL‐18R signalling pathways are dispensable. Further downstream in this axis of signalling, TNF blockade reduces only acute bronchoconstriction, while MAPK inhibition abrogates completely endotoxin‐induced inflammation.

Dual Effects of p38 MAPK on TNF-Dependent Bronchoconstriction and TNF-Independent Neutrophil Recruitment in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome

The administration of endotoxins from Gram-negative bacteria induces manifestations reminding of acute respiratory distress syndrome. p38 MAPKs have been implicated in this pathology. In this study, we show that the specific p38 α,β MAPK inhibitor, compound 37, prevents LPS-induced bronchoconstriction and neutrophil recruitment into the lungs and bronchoalveolar space in a dose-dependent manner in C57BL/6 mice. Furthermore, TNF induction and TNF signals were blocked. In TNF-deficient mice, bronchoconstriction, but not neutrophil sequestration, in the lung was abrogated after LPS administration. Therefore, TNF inhibition does not explain all of the effects of the p38 MAPK inhibitor. The p38 α,β MAPK inhibitor also prevented LPS-induced neutrophilia in TNF-deficient mice. In conclusion, LPS provokes acute bronchoconstriction that is TNF dependent and p38 MAPK mediated, whereas the neutrophil recruitment is independent of TNF but depends on LPS/TLR4-induced signals mediated by p38 MAPK.

IL-1R1/MyD88 Signaling Is Critical for Elastase-Induced Lung Inflammation and Emphysema

Lung emphysema and fibrosis are severe complications of chronic obstructive pulmonary disease, and uncontrolled protease activation may be involved in the pathogenesis. Using experimental elastase-induced acute inflammation, we demonstrate here that inflammation and development of emphysema is IL-1R1 and Toll/IL-1R signal transduction adaptor MyD88 dependent; however, TLR recognition is dispensable in this model. Elastase induces IL-1β, TNF-α, keratinocyte-derived chemokine, and IL-6 secretion and neutrophil recruitment in the lung, which is drastically reduced in the absence of IL-1R1 or MyD88. Further, tissue destruction with emphysema and fibrosis is attenuated in the lungs of IL-1R1- and MyD88-deficient mice. Specific blockade of IL-1 by IL-1R antagonist diminishes acute inflammation and emphysema. Finally, IL-1β production and inflammation are reduced in mice deficient for the NALP3 inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and we identified uric acid, which is produced upon elastase-induced lung injury, as an activator of the NALP3/ASC inflammasome. In conclusion, elastase-mediated lung pathology depends on inflammasome activation with IL-1β production. IL-1β therefore represents a critical mediator and a possible therapeutic target of lung inflammation leading to emphysema.

Arthropod-derived histamine-binding protein prevents murine allergic asthma

Because histamine receptor type I blockade attenuates allergic asthma, we asked whether complete neutralization of histamine by an arthropod-derived, high affinity histamine-binding protein (EV131) would prevent allergic asthma. Intranasal administration of EV131 given before Ag challenge in immunized mice prevented airway hyperreactivity by 70%, and abrogated peribronchial inflammation, pulmonary eosinophilia, mucus hypersecretion, and IL-4 and IL-5 secretion. Saturation with histamine abrogated the inhibitory effect of EV131 on bronchial hyperreactivity. The inhibitory effect of EV131 on bronchial hyperreactivity was comparable to that of glucocorticosteroids. These results demonstrate that histamine is a critical mediator of allergic asthma. Therefore, complete neutralization of histamine, rather than specific histamine receptor blockade, may have a profound effect on allergic asthma.

Tumor Necrosis Factor α Enhances the Expression of the Interleukin (IL)-4 Receptor α-Chain on Endothelial Cells Increasing IL-4 or IL-13-induced Stat6 Activation

Functional receptors for interleukin (IL)-4 and IL-13 on endothelial cells consist of the 130-kDa IL-4 receptor α-chain (IL-4Rα) and a 65-75-kDa IL-13 binding subunit that are expressed in a ratio of about 1:3, respectively. The restricted number of IL-4Rα limits subunit heterodimerization and in turn receptor-mediated signaling. We report here, the effects of tumor necrosis factor α (TNF-α) on the expression of the receptor subunits for IL-4 and IL-13. By flow cytofluorometry and receptor-binding analysis of iodinated IL-4 and IL-13, stimulation with TNF-α-induced a 2-3-fold increase of the IL-4Rα expression. The up-regulation was also confirmed at the transcriptional level by reverse transcription-polymerase chain reaction. Radioligand cross-linking experiments revealed no change in the subunit composition of the TNF-α-induced receptor complex. Nevertheless, TNF-α stimulation led to increased activation of the IL-4-specific signal transducers and activators of transcription protein (Stat6) by IL-4 and IL-13. Thus, TNF-α corrects the subunit imbalance of the endothelial IL-4·;IL-13 receptor complex thereby increasing receptor heterodimerization and in turn the signaling capability by IL-4 and IL-13.

Allergic Lung Inflammation Is Mediated by Soluble Tumor Necrosis Factor (TNF) and Attenuated by Dominant-Negative TNF Biologics

Tumor Necrosis Factor (TNF) is a pleiotropic cytokine consisting of soluble and transmembrane forms, with distinct roles in inflammation and immunity. TNF is an important factor in allergic airway inflammation. However, the disparate functions of soluble (sol) and transmembrane (tm) TNF in lung pathology are not well understood. Our aim was to assess the activities of solTNF and tmTNF in murine models of allergic airway disease, and to evaluate the efficacy of solTNF-selective inhibition. We used ovalbumin sensitization and challenge of TNF knockout, tmTNF knockin, and wild-type C57BL/6 mice to distinguish differences in airway inflammation and hyperreactivity mediated by solTNF and tmTNF. Functions of solTNF and tmTNF in hyperresponsive, wild-type Balb/c mice were assessed by comparing dominant-negative anti-TNF biologics, which antagonize solTNF yet spare tmTNF, to etanercept, a nonselective inhibitor of both TNF forms. Responses in transgenic C57BL/6 mice demonstrated that solTNF, and not tmTNF, is necessary to drive airway inflammation. In Balb/c mice, dominant-negative TNF biologics administered during immunization decreased the recruitment of eosinophils and lymphocytes into the bronchoalveolar space and lung parenchyma, reduced specific serum IgE, goblet-cell hyperplasia, and eosinophilic inflammation, and suppressed methacholine-induced airway hyperreactivity. Concentrations of IL-5, CCL5/RANTES, CCL11/eotaxin, and CCL17/TARC were also reduced in bronchoalveolar lavage. Dominant-negative TNFs reduced lung eosinophilia, even when given only during antigen challenge. The selective inhibition of soluble TNF suppresses inflammation, hyperreactivity, and remodeling in transgenic and wild-type murine models of allergic airway disease, and may offer safety advantages in therapies that preserve the immunoprotective functions of transmembrane TNF.

Petasites extract Ze 339 (PET) inhibits allergen‐induced Th2 responses, airway inflammation and airway hyperreactivity in mice

Background: The herbal Petasites hybridus (butterbur) extract (Ze 339, PET) is known to have leukotriene inhibiting properties, and therefore might inhibit allergic diseases.

Use of transgenic animals to investigate drug hypersensitivity

Hypersensitivity reactions to drugs and environmental agents are often due to exaggerated humoral (Th(2)) or cell mediated (Th(1)) immune responses with typical cytokine profiles. Overexpression of Th(2) cytokines, such as IL-4, IL-5 or IL-13 in mice, enhances an IgE antibody mediated response, while deletion of these cytokines attenuates and/or prevents allergic responses. Conversely, modulation of Th(1) cytokine gene expression may affect cell-mediated immune responses. Therefore, cytokine transgenic mice are used as investigative tools to study potential chemicals and/or drug allergies. In addition to cytokines and chemokines, other factors are important for the development of allergic responses, such as IgE, Fc receptors, vasopressin and several other factors, which can be tested in transgenic mice.

The plant extract Isatis tinctoria L. extract (ITE) inhibits allergen-induced airway inflammation and hyperreactivity in mice.

BACKGROUND The herbal Isatis tinctoria extract (ITE) inhibits the inducible isoform of cyclooxygenase (COX-2) as well as lipoxygenase (5-LOX) and therefore possesses anti-inflammatory properties. The extract might also be useful in allergic airway diseases which are characterized by chronic inflammation. METHODS ITE obtained from leaves by supercritical carbon dioxide extraction was investigated in ovalbumin (OVA) immunised BALB/c mice given intranasally together with antigen challenge in the murine model of allergic airway disease (asthma) with the analysis of the inflammatory and immune parameters in the lung. RESULTS ITE given with the antigen challenge inhibited in a dose related manner the allergic response. ITE diminished airway hyperresponsiveness (AHR) and eosinophil recruitment into the bronchoalveolar lavage (BAL) fluid upon allergen challenge, but had no effect in the saline control mice. Eosinophil recruitment was further assessed in the lung by eosinophil peroxidase (EPO) activity at a dose of 30 microg ITE per mouse. Microscopic investigations revealed less inflammation, eosinophil recruitment and mucus hyperproduction in the lung in a dose related manner. Diminution of AHR and inflammation was associated with reduced IL-4, IL-5, and RANTES production in the BAL fluid at the 30 microg ITE dose, while OVA specific IgE and eotaxin serum levels remained unchanged. CONCLUSION ITE, which has been reported inhibiting COX-2 and 5-LOX, reduced allergic airway inflammation and AHR by inhibiting the production of the Th2 cytokines IL-4 and IL-5, and RANTES.

BINDING OF INTERLEUKIN-13 AND INTERLEUKIN-4 TO THE INTERLEUKIN (IL)-4/IL-13 RECEPTOR OF HUMAN SYNOVIAL FIBROBLASTS

Synovial fibroblasts expressed transcripts for IL-4R alpha, and IL-13R alpha 1 and IL-13R alpha 2. Using weighted nonlinear computer modeling of the data from equilibrium binding studies, a 2 bindings sites model fitted the data best. After occupation of the shared high affinity receptors by the non-signaling, double mutant IL-4(121)R-->D, 124Y-->D (RY-IL-4) the high affinity binding of IL-13 could be abolished. A 2 binding site model still could be fitted, however the improvement in fit over a onesite model was not statistically significant. Using affinity spectra, at least 2 binding sites are apparent. After treatment with RY-IL-4, some of the high affinity binding was abolished, however not completely. A correlation between the number of binding sites and the affinity is apparent, which seriously casts doubt on the classical evaluation of binding isotherms, where the parameters are assumed to be independent. In a previous study we suggested that the large number of IL-13R alpha 2 monomers are silent receptors, likely representing a decoy target for IL-13. The high affinity binding therefore most likely represents the binding to the heterodimer consisting of IL-4R alpha and IL-13R alpha 1 or IL-13R alpha 2. The low affinity binding may represent the IL-13R alpha 2.