P. A. J. Henricks

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.

1 alpha,25-dihydroxyvitamin D(3) potentiates the beneficial effects of allergen immunotherapy in a mouse model of allergic asthma: Role for IL-10 and TGF-beta

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a potent inhibitor of NF-κB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)2D3 potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-β are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 μg. Interestingly, coadministration of 10 ng of 1,25(OH)2D3 with 100 μg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-β and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-κB pathway using the selective IκB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-β before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)2D3 potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-β play a crucial role in the effector phase of this mouse model.

Adhesion molecules: a new target for immunoliposome-mediated drug delivery

The anti‐ICAM‐1 monoclonal antibody F10.2 was conjugated to liposomes to target to cells expressing the cell adhesion molecule ICAM‐1. We demonstrate that F10.2 immunoliposomes bind to human bronchial epithelial cells (BEAS‐2B) and human umbilical vein endothelial cells (HUVEC) in a specific, dose‐ and time‐dependent manner. It appears that the degree of ICAM‐1 expression is the limiting factor in the degree of immunoliposome binding to the cells. These results are a first step in the strategy for specific drug delivery to target sites characterised by increased expression of adhesion molecules.

Inflammatory changes in the airways of mice caused by cigarette smoke exposure are only partially reversed after smoking cessation

BackgroundTobacco 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.MethodsThe 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.ResultsThe 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 smoke-exposed 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.ConclusionThe 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.

Modulation of airway hyperresponsiveness and eosinophilia by selective histamine and 5‐HT receptor antagonists in a mouse model of allergic asthma

Since both histamine and 5‐hydroxytryptamine (5‐HT) can be released by murine mast cells, we investigated the possible role of these autacoids on airway hyperresponsiveness (AHR), eosinophil infiltration and serum‐IgE levels in a murine model of allergic asthma. Ovalbumin‐sensitized mice were exposed to either ovalbumin (2 mg ml−1) or saline aerosols on 8 consecutive days. Starting one day before the challenge, animals were injected i.p. twice a day with a 5‐HT‐type 1 (5‐HT1) or type 2 (5‐HT2) receptor antagonist (methiotepine, 1.25 or 2.0 mg kg−1 and ketanserin, 12 mg kg−1, respectively) or a histamine‐type 1 (H1) or type 2 (H2) receptor antagonist (mepyramine, 12 or 20 mg kg−1 and cimetidine, 10 or 25 mg kg−1, respectively). Furthermore, animals were injected with a combination of cimetidine and ketanserin or with an α‐adrenoceptor antagonist (phentolamine, 5 mg kg−1). In vehicle‐treated ovalbumin‐challenged animals airway responsiveness to intravenous injections of methacholine in vivo was significantly (9 fold increase, P<0.01) increased when compared to vehicle‐treated saline‐challenged animals. Furthermore, ovalbumin challenge of vehicle‐treated animals induced a significant increase in both eosinophil numbers in bronchoalveolar lavage (BAL) fluid (0±0, vehicle/saline and 15.0±5.9×104 cells vehicle/ovalbumin, P<0.05) and ovalbumin‐specific IgE levels in serum (157±69 and 617±171 units ml−1, respectively, P<0.05) compared to saline‐challenged mice. Virtually no eosinophils could be detected in saline‐challenged animals after all different treatments. Treatment with ketanserin or cimetidine resulted in a partial but significant decrease of the ovalbumin‐induced AHR compared to ovalbumin‐challenged controls (P<0.05) and reduced eosinophil infiltration after ovalbumin challenge by 60% and 58%, respectively. The combination of cimetidine and ketanserin almost completely abolished AHR whereas eosinophilia was decreased by 49%. No effects of these antagonists were observed on IL‐16 levels in BAL fluid or on serum antigen‐specific IgE levels. Treatment with either the H1‐receptor, the 5‐HT1‐receptor or the α‐adrenoceptor antagonist, did not decrease the observed ovalbumin‐induced airway responsiveness or eosinophilia in vehicle‐treated animals. Higher doses of either methiotepine (2.0 mg kg−1) or mepyramine (20 mg kg−1) did decrease ovalbumin‐induced eosinophil infiltration (by 67%, P<0.05 and 73%, respectively), whereas no effects of these antagonists were observed on ovalbumin‐specific IgE levels in serum. From these data it can be concluded that both histamine and 5‐HT play a role in antigen‐induced AHR and eosinophilia in the mouse.

Airway hyper‐responsiveness in allergic asthma in guinea‐pigs is mediated by nerve growth factor via the induction of substance P: a potential role for trkA

Background: The neurotrophin nerve growth factor (NGF) has been implicated as a mediator in allergic asthma. Direct evidence that inhibition of NGF‐induced activation of neurotrophin receptors leads to improvement of airway symptoms is lacking. We therefore studied the effects of inhibitors of NGF signal transduction on the development of airway hyper‐responsiveness (AHR) and pulmonary inflammation in a guinea‐pig model for allergic asthma.

Indoleamine 2,3-dioxygenase–dependent tryptophan metabolites contribute to tolerance induction during allergen immunotherapy in a mouse model

BACKGROUND The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. OBJECTIVE We examined (1) whether IDO activity is required during tolerance induction by allergen immunotherapy or for the subsequent suppressive effects on asthma manifestations and (2) whether tryptophan depletion or generation of its downstream metabolites is involved. METHODS Ovalbumin (OVA)-sensitized and OVA-challenged BALB/c mice that display increased airway responsiveness to methacholine, serum OVA-specific IgE levels, bronchoalveolar eosinophilia, and TH2 cytokine levels were used as a model of allergic asthma. Sensitized mice received subcutaneous optimal (1 mg) or suboptimal (100 microg) OVA immunotherapy. RESULTS Inhibition of IDO by 1-methyl-DL-tryptophan during immunotherapy, but not during inhalation challenge, partially reversed the suppressive effects of immunotherapy on airway eosinophilia and TH2 cytokine levels, whereas airway hyperresponsiveness and serum OVA-specific IgE levels remained suppressed. Administration of tryptophan during immunotherapy failed to abrogate its beneficial effects toward allergic airway inflammation. Interestingly, administration of tryptophan or its metabolites, kynurenine, 3-hydroxykynurenine, and xanthurenic acid, but not 3-hydroxyanthranilinic acid, quinolinic acid, and kynurenic acid, during suboptimal immunotherapy potentiated the reduction of eosinophilia. These effects coincided with reduced TH2 cytokine levels in bronchoalveolar lavage fluid, but no effects on IgE levels were detected. CONCLUSION During immunotherapy, the tryptophan metabolites kynurenine, 3-hydroxykynurenine, and xanthurenic acid generated through IDO contribute to tolerance induction regarding TH2-dependent allergic airway inflammation.

Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease

Objective Proline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD). Design In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies. Results In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine. Conclusions The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

Cells, mediators and Toll-like receptors in COPD

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 beta(2)-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.

Cellular uptake of liposomes targeted to intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelial cells

Previously, it was demonstrated that immunoliposomes, bearing anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (mAb F10.2), can specifically bind to different cell types expressing ICAM-1. In this study, we have quantified the amount of immunoliposomes binding to IFN-gamma activated human bronchial epithelial cells (BEAS-2B) in vitro and studied the subsequent fate of cell-bound anti-ICAM-1 immunoliposomes. We demonstrate that binding of the immunoliposomes to the epithelial cells depends on the liposome concentration used. After binding to the cell surface, the anti-ICAM-1 immunoliposomes are rapidly internalised by the epithelial cells. Sixty percent of cell-bound immunoliposomes were internalised by the epithelial cells within 1 h of incubation at 37 degrees C. The results indicate that ICAM-1 targeted immunoliposomes may be used as carriers for the intracellular delivery of anti-inflammatory drugs to sites of inflammation characterised by an increased expression of ICAM-1.