Humic acid enhances cigarette smoke-induced lung emphysema in mice and IL-8 release of human monocytes.

UNLABELLED: 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.

The contact allergen dinitrochlorobenzene (DNCB) and respiratory allergy in the Th2-prone Brown Norway rat.

All LMW respiratory allergens known to date can also induce skin allergy in test animals. The question here was if in turn skin allergens can induce allergy in the respiratory tract. Respiratory allergy was tested in Th2-prone Brown Norway (BN) rats by dermal sensitization with the contact allergen dinitrochlorobenzene (DNCB; 1%, day 0; 0.5%, day 7) and a head/nose-only inhalation challenge of 27mg/m3 of DNCB (15 min, day 21), using a protocol that successfully identified chemical respiratory allergens. Skin allergy to DNCB was examined in BN rats and Th1-prone Wistar rats in a local lymph node assay followed by a topical patch challenge of 0.1% DNCB. Sensitization of BN rats via the skin induced DNCB-specific IgG in serum, but not in all animals, and an increased number of CD4+ cells in the lung parenchyma. Subsequent inhalation challenge with DNCB did not provoke apneas or allergic inflammation (signs of respiratory allergy) in the BN rats. However, microarray analysis of mRNA isolated from the lung revealed upregulation of the genes for Ccl2 (MCP-1), Ccl4 (MIP-1beta), Ccl7 and Ccl17. Skin challenge induced considerably less skin irritation and allergic dermatitis in the BN rat than in the Wistar rat. In conclusion, the Th2-prone BN rat appeared less sensitive to DNCB than the Wistar rat; nevertheless, DNCB induced allergic inflammation in the skin of BN rats but even a relatively high challenge concentration did not induce allergy in the respiratory tract, although genes associated with allergy were upregulated in lung tissue.

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.

Trimellitic anhydride-conjugated serum albumin activates rat alveolar macrophages in vitro.

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.

Induction of HSP70 is dispensable for anti-inflammatory action of heat shock or NSAIDs in mast cells.

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-alpha (TNF-alpha) 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-alpha 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 kappaB (NF-kappaB) kinase activity, extracellular signal-regulated kinases 1/2, and p38 pathways, resulting in decreased transcriptional activity of the NF-kappaB pathway.

Alveolar macrophages suppress non-specific inflammation caused by inhalation challenge with trimellitic anhydride conjugated to albumin.

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.

Alveolar macrophages have a dual role in a rat model for trimellitic anhydride-induced occupational asthma.

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-alpha 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.

Respiratory allergy to trimellitic anhydride in rats: concentration-response relationships during elicitation.

The present study investigated whether airway responses of sensitized rats to trimellitic anhydride (TMA) were concentration dependent and whether these were related to irritation by TMA. Groups of BN and Wistar rats were sensitized by two dermal applications of TMA (50% w/v, followed by 25% w/v in vehicle). Controls received vehicle (acetone-olive oil 4:1, v/v). All animals were challenged 3 wk after the first sensitization by inhalation of one of a range of concentrations of TMA (0.2-61 mg/m3 for BN rats, 15-250 mg/m3 for Wistar rats). Breathing pattern, breathing frequency, and tidal volume were measured before, during, and after challenge to assess allergic and irritative airway responses. One day after challenge, nonspecific airway responsiveness to a range of concentrations of methacholine was measured. At necropsy on the same day, blood was withdrawn for measuring total serum immunoglobulin E (IgE) and organs were weighed. Larynx, trachea and lungs were examined histopathologically. In BN rats, TMA sensitization elevated total IgE levels; subsequent inhalation challenge with 2 mg/m3 of TMA and higher caused laryngeal inflammation with squamous epithelial metaplasia, and pulmonary hemorrhages. Concentration-related decreases in breathing frequency and alterations in breathing pattern, which differed from the irritation-induced pattern, were also observed at these levels. Inhalation challenge with TMA concentrations of 12 mg/m3 and higher increased lung weight. Increased nonspecific airway responsiveness was observed at the 2 next higher tested concentrations of 46 and 61 mg/m3. In unsensitized BN rats, only laryngeal squamous metaplasia was observed, albeit at higher challenge concentrations of TMA, and decreased breathing frequency, a typical breathing pattern characteristic of irritation. Identically sensitized Wistar rats showed airway inflammation and pulmonary hemorrhages upon challenge with TMA, but no functional changes, even at distinctly irritating concentrations of TMA up to 250 mg/m3. In conclusion, TMA challenge of sensitized BN rats caused challenge concentration-related allergic airway inflammation, asthmalike changes in breathing pattern, and increased nonspecific airway responsiveness. The lowest no-observed-effect level (NOEL) based on the most sensitive endpoint investigated was 0.2 mg/m3, a value that is well below the irritation concentration. The presence of a NOEL in the sensitized BN rat suggests that assessment of safe human exposure levels is feasible.

Influence of the macrophage bacterial resistance gene, Nramp1 (Slc11a1), on the induction of allergic asthma in the mouse.

Based on the hygiene hypothesis that lack of early childhood bacterial infections would favor development of allergic disease, we hypothesize that genes controlling antibacterial resistance may be important as well. We, therefore, studied whether Nramp1 alleles that determine resistance (Nramp1r) or susceptibility (Nramp1s) to intracellular bacteria at the macrophage level affect sensitivity to induction of allergic asthma. Nramp1s and congenic Nramp1r mice were sensitized with ovalbumin/alum on days 0 and 14 and challenged with ovalbumin or saline aerosols on days 42, 45, and 48. On day 49, airway responsiveness was assessed, blood was withdrawn, and lung lavage was performed. We demonstrated that ovalbumin sensitization and challenge of Nramp1s and Nramp1r mice caused comparable airway hyperreactivity and airway eosinophilia and a similar increase in serum levels of ovalbumin-specific IgG1 and IgG2a. Ovalbumin challenge, however, induced significantly lower serum levels of total and ovalbumin-specific IgE and significantly lower mast cell degranulation in Nramp1r mice as compared with Nramp1s mice. In addition, ovalbumin challenge of Nramp1r mice led to significantly less release of Th2 cytokines into the airways. Results show that Nramp1 can affect the development of allergy but not the development of airway hyperresponsiveness in the mouse.

Respiratory allergy and pulmonary irritation to trimellitic anhydride in Brown Norway rats.

Occupational exposure to low-molecular-weight (LMW) allergens such as acid anhydrides can result in occupational asthma, an allergic disease characterized by episodic airway obstruction, airways inflammation, and non specific airways hyperresponsiveness. Since LMW irritants can provoke rather similar effects and since most, if not all, LMW allergens have irritant properties, this study addressed the distinction between allergenic and irritant effects of the respiratory allergen trimellitic anhydride (TMA). BN rats were sensitized by dermal application of TMA or vehicle alone and 3 weeks later were challenged by inhalation of a slightly irritating concentration of TMA or the vehicle. Lung function was measured before, during, and shortly after challenge. One day after challenge, in vivo and in vitro nonspecific airways hyperresponsiveness to methacholine was measured, and bronchoalveolar lavage was performed to measure total protein, lactate dehydrogenase, N-acetyl-glucosaminidase, and total and differential leukocyte numbers in the fluid. In addition, IgE measurements and histopathological examinations of the respiratory tract were carried out. TMA challenge of sensitized, but not sham-sensitized, BN rats reduced breathing frequency during challenge, elevated total and TMA-specific serum IgE levels, and caused a typical allergic asthma-associated airway pathology, as observed earlier. Vehicle challenge did not cause these effects, irrespective of sensitization. Hyperresponsiveness to methacholine was only seen in TMA-sensitized and -challenged rats. These rats also showed increased levels of the biochemical parameters and increased numbers of eosinophils and neutrophils in the lung lavage fluid; TMA challenge of sham-sensitized rats caused similar but markedly less pronounced effects. During TMA challenge of sham-sensitized rats, a breathing pattern typical of irritation was noticed but a clearly distinct pattern was seen upon TMA challenge of sensitized rats. In conclusion, TMA challenge of sensitized rats caused sensitization-dependent asthma-like early changes in breathing pattern that clearly could be distinguished from irritant-induced changes and non-specific airways hyperresponsiveness 24 h after challenge. Sensitization-dependent functional changes were accompanied by inflammatory changes characteristic of asthma and biochemical evidence of airway damage.

Glutathione and other low-molecular-weight thiols relax guinea pig trachea ex vivo: interactions with nitric oxide?

The aim of this study was to determine the effects of glutathione (GSH) on trachea smooth muscle tension in view of previously reported interactions between GSH and nitric oxide (NO) (Gaston B. Biochim Biophys Acta 1411: 323-333, 1999; Kelm M. Biochim Biophys Acta 1411: 273-289, 1999; and Kharitonov VG, Sundquist AR, and Sharma VS. J Biol Chem 270: 28158-28164, 1995) and the high (millimolar) concentrations of GSH in trachea epithelium (Rahman I, Li XY, Donaldson K, Harrison DJ, and MacNee W. Am J Physiol Lung Cell Mol Physiol 269: L285-L292, 1995). GSH and other thiols (1.0-10 mM) dose dependently decreased the tension in isolated guinea pig tracheas. Relaxations by GSH were paralleled with sevenfold increased nitrite levels (P < 0.05) in the tracheal effluent, suggesting an interaction between GSH and NO. However, preincubation with a NO scavenger did not reduce the relaxations by GSH or its NO adduct, S-nitrosoglutathione (GSNO). Inhibition of guanylyl cyclase inhibited the relaxations induced by GSNO, but not by GSH. Blocking potassium channels, however, completely abolished the relaxing effects of GSH (P < 0.05). Preincubation of tracheas with GSH significantly (P < 0.05) suppressed hyperreactivity to histamine as caused by removal of tracheal epithelium. These data indicate that GSH plays a role in maintaining tracheal tone. The mechanism is probably an antioxidative action of GSH itself rather than an action of NO or GSNO.

Differential responsiveness of proximal and distal parts of isolated guinea pig trachea.

This study addressed the question whether proximal and distal guinea pig tracheal segments respond differently to contractile agents. Using a perfused trachea set-up, histamine, KCl or the cyclo-oxygenase inhibitor, indomethacin, could be administered selectively to the mucosa (at the inside) or the serosa (at the outside) of the tracheal segments. Proximal parts contracted significantly more (40-60%) than distal parts when 1 mM histamine was administered to the mucosal or serosal side or when KCl (50 mM) was added to the serosal side. When histamine was administered to the mucosal side of epithelium-denuded segments, the contractions were twice as high in proximal than in distal parts (3057 vs. 1526 mg). Inhibition of tracheal cyclo-oxygenase with indomethacin at the mucosal side increased proximal and distal reactivity to mucosally administered histamine to the same extent. Serosal administration of indomethacin, however, increased histamine reactivity only in proximal segments (from 2690 to 5180 mg). In the latter segments, subsequent administration of histamine to the serosal side further increased the contraction, while serosal histamine in the absence of serosal indomethacin produced a relaxation (net difference of 4672 mg). In conclusion, the higher intrinsic contractility of proximal tracheal segments is counteracted by serosal cyclo-oxygenase products.