Gert Folkerts

The ENFUMOSA cross-sectional European multicentre study of the clinical phenotype of chronic severe asthma

Since severe asthma is a poorly understood, major health problem, 12 clinical specialist centres in nine European countries formed a European Network For Understanding Mechanisms Of Severe Asthma (ENFUMOSA). In a cross-sectional observational study, a total of 163 subjects with severe asthma were compared with 158 subjects whose asthma was controlled by low doses of inhaled corticosteroids (median dose of beclomethasone equivalents 666 µg). Despite being treated with higher doses of inhaled corticosteroids (median dose 1773 µg) and for a third of the severe asthmatics also being treated with regular, oral-steroid therapy (median daily dose 19 mg), the subjects with severe asthma met the inclusion criteria. The criteria required subjects to have undergone at least one asthma exacerbation in the past year requiring oral steroid treatment. Females dominated the severe asthma group (female/male ratio 4.4:1 versus 1.6:1 in the controlled asthmatics), and compared with controlled asthmatics, they had a predominantly neutrophilic inflammation (sputum neutrophils, 36 versus 28%) and evidence of ongoing mediator release but less atopy. From these findings and other physiological and clinical data reported in this paper, it is suggested that severe asthma might be a different form of asthma rather than an increase in asthma symptoms. The findings prompt for longitudinal studies and interventions to define the mechanisms in severe asthma.

A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation

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, PGPs activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases.

Inflammatory Disease Processes and Interactions with Nutrition

Inflammation is a stereotypical physiological response to infections and tissue injury; it initiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. Acute inflammatory reactions are usually self-limiting and resolve rapidly, due to the involvement of negative feedback mechanisms. Thus, regulated inflammatory responses are essential to remain healthy and maintain homeostasis. However, inflammatory responses that fail to regulate themselves can become chronic and contribute to the perpetuation and progression of disease. Characteristics typical of chronic inflammatory responses underlying the pathophysiology of several disorders include loss of barrier function, responsiveness to a normally benign stimulus, infiltration of inflammatory cells into compartments where they are not normally found in such high numbers, and overproduction of oxidants, cytokines, chemokines, eicosanoids and matrix metalloproteinases. The levels of these mediators amplify the inflammatory response, are destructive and contribute to the clinical symptoms. Various dietary components including long chain omega-3 fatty acids, antioxidant vitamins, plant flavonoids, prebiotics and probiotics have the potential to modulate predisposition to chronic inflammatory conditions and may have a role in their therapy. These components act through a variety of mechanisms including decreasing inflammatory mediator production through effects on cell signaling and gene expression (omega-3 fatty acids, vitamin E, plant flavonoids), reducing the production of damaging oxidants (vitamin E and other antioxidants), and promoting gut barrier function and anti-inflammatory responses (prebiotics and probiotics). However, in general really strong evidence of benefit to human health through anti-inflammatory actions is lacking for most of these dietary components. Thus, further studies addressing efficacy in humans linked to studies providing greater understanding of the mechanisms of action involved are required.

Peroxynitrite: a two-faced metabolite of nitric oxide.

The discovery that nitric oxide (NO) reacts with superoxide (O2.-) forming peroxynitrite (ONOO-) (1) and the proof that this reaction occurs in vivo (2,3) holds enormous implications for the understanding of free radicals in biological systems. Not only in mammalian defense mechanisms against microorganisms, but also in pathophysiology during overexposure of tissues to radicals or other highly reactive species. Peroxynitrite is a highly reactive compound with harmful effects on cells and could therefore be an important microbicidal compound. Furthermore, the reaction of superoxide with NO interferes with NO signalling mechanisms. NO is not only released in response to inflammatory agents by inflammatory cells, but is also an important messenger molecule in paracrine mechanisms and neurotransmission. Whether peroxynitrite formation is a negative side effect of NO and superoxide release, or a functional characteristic is yet to be determined, and will be discussed in this review.

Apocynin inhibits peroxynitrite formation by murine macrophages

Peroxynitrite (ONOO−) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathogenesis of an increasing number of (inflammatory) diseases. At present, however, selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH‐oxidase inhibitor apocynin (4‐hydroxy‐3‐methoxy‐acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro The murine macrophage cell‐line J774A.1, stimulated with IFNγ/LPS, was used as a model. Conversion of 123‐dihydrorhodamine (123‐DHR) to its oxidation product 123‐rhodamine was used to measure peroxynitrite production. Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation. It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH‐oxidase dependent superoxide formation. The NADPH‐oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.

Toll-like receptor-4 mediates cigarette smoke-induced cytokine production by human macrophages

BackgroundThe 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.MethodsEmploying 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.ResultsWe 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.ConclusionThese findings link cigarette smoke to inflammation and lead to new insights/therapeutic strategies in the pathogenesis of lung emphysema.

Reactive nitrogen and oxygen species in airway inflammation

The free radical nitric oxide (NO) is an important mediator of many biological processes. Interestingly, the molecule appears to be a two-edged sword. Apart from NO having a function as a paracrine messenger, NO-derived oxidants are important weapons against invading pathogens. The role of NO in the airways is similarly ambiguous. Besides the task as a bronchodilator, NO and its derivatives play a role in the pathophysiology of asthma via their putative damaging effects on the airways. This deleterious effect can be increased by a nitrosative response to respiratory tract infections, since both the infectious agent and the host may suffer from the consequent nitrosative stress. Interestingly, respiratory infections can also compromise the beneficial (bronchodilator) effects of NO. This paper gives an overview on NO and its derivatives in the pathophysiology of airway inflammation.

Nitric Oxide and Bronchial Hyperresponsiveness

Increasing evidence points to an important role for nitric oxide in the regulation of pulmonary functions and in pulmonary disease. In the respiratory tract, sensory nerves, endothelial cells, vascular and airway smooth muscle cells, inflammatory cells and the airway epithelium are sources of nitric oxide. Different nitric oxide synthases have been isolated, cloned and sequenced. Functionally, there are constitutive and inducible forms of nitric oxide synthase. A number of cytokines have been shown to inhibit or induce the expression of the inducible nitric oxide synthase. In human airways, endogenous nitric oxide appears to account for the bronchodilator nonadrenergic and noncholinergic response. Nitric oxide-containing vasodilators, such as glyceryl trinitrate and sodium nitroprusside, induce relaxation of the isolated airway smooth muscle, activate guanylate cyclase and raise c-GMP levels. Nitric oxide (constitutive), produced by the epithelial layer, appears to be important in blunting the histamine contractile response of the airway tissue. Furthermore, tracheal relaxation by, e.g., bradykinin or potassium chloride, is mediated by the release of nitric oxide. The virus (Parainfluenza type 3)-induced airway hyperreactivity in guinea-pigs is correlated with a deficiency in endogenous constitutive nitric oxide production by the airways and can be blocked by low doses of L-arginine. In inflamed tissue, nitric oxide quickly reacts with superoxide anion, resulting in the formation of the toxic peroxynitrite which promotes lipid and sulfhydryl oxidation. Asthmatic patients have higher amounts of nitric oxide in the expired air, possibly due to the inflammation. This increased nitric oxide production can be inhibited by inhaled corticosteroids. The effect of inhaled nitric oxide on the lung function of asthmatic patients is variable. In contrast, low doses of inhaled nitric oxide are effective in reversing the pulmonary vasoconstriction. These results point to an important role for nitric oxide in modulating airway reactivity.

Virus-induced airway hyperresponsiveness in guinea pigs is related to a deficiency in nitric oxide.

Intratracheal inoculation of parainfluenza type 3 virus to guinea pigs induces a marked increase in airway responsiveness in vivo and in vitro. In spontaneously breathing anesthetized guinea pigs inhalation of an aerosol containing the nitric oxide (NO) precursor L-arginine (2.0 mM) completely prevented the virus-induced airway hyperresponsiveness to histamine. In addition, perfusion of L-arginine (200 microM) or the direct NO-donor S-nitroso-N-acetyl-penicillamine (SNAP, 1 microM) through the lumen of tracheal tubes from infected animals prevented the increase in airway responsiveness to histamine or the cholinoceptor agonist methacholine. The NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 120 microM) did not further increase the virus-induced airway hyperresponsiveness. In additional experiments, NO was measured with an Iso-NO nitric oxide meter and sensor. Stimulation of control tissues in vitro with histamine (10(-3) M) resulted in a contraction with a simultaneous release of NO (44.5 +/- 5.4 nM). The release of NO was markedly reduced by 75% (P < 0.01, 11.4 +/- 3.1 nM) in tracheas from virus-infected animals that demonstrated enhanced contractile responses. Preincubation of tissues from virus-treated guinea pigs with L-arginine (200 microM) completely prevented the enhanced contraction and simultaneously returned the NO production to control values (51.2 +/- 3.4 nM). An NO deficiency might be causally related to the development of airway hyperresponsiveness after a viral respiratory infection.

Targeting chemokine receptors in chronic inflammatory diseases: An extensive review

The traffic of the different types of immune cells is an important aspect in the immune response. Chemokines are soluble peptides that are able to attract cells by interaction with chemokine receptors on their target cells. Several different chemokines and receptors exist enabling the specific trafficking of different immune cells. In chronic inflammatory disorders there is abundance of immune cells present at the inflammatory site. This review focuses on the role of chemokine receptors in chronic inflammatory disorders of the lungs, intestine, joints, skin and nervous system and the potential of targeting these receptors as therapeutic intervention in these disorders.