Iron administration reduces airway hyperreactivity and eosinophilia in a mouse model of allergic asthma.

The prevalence of allergic diseases has increased dramatically during the last four decades and is paralleled by a striking increase in iron intake by infants in affluent societies. Several studies have suggested a link between increased iron intake and the marked increase in prevalence of allergic diseases. We hypothesized that the increased iron intake by infants offers an explanation for the increased prevalence of allergic disease in industrialized societies during the past four decades. A well-established mouse model of ovalbumin (OVA)-driven allergic asthma was used to test the effects of differences in iron intake and systemic iron levels on the manifestations of allergic asthma. Surprisingly, iron supplementation resulted in a significant decrease in airway eosinophilia, while systemic iron injections lead to a significant suppression of both allergen-induced airway eosinophilia and hyperreactivity compared to placebo. In contrast, mice fed on an iron-deprived diet did not show any difference in developing experimentally induced allergic asthma when compared to those fed on an iron-sufficient control diet. In contrast to our hypothesis, airway manifestations of allergic asthma are suppressed by both increased levels of iron intake and systemic iron administrations in the mouse model.

A low vitamin A status increases the susceptibility to cigarette smoke-induced lung emphysema in C57BL/6J mice.

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. Vitamin A metabolites have been implicated in the repair of lung damage. Exposure to cigarette smoke has been shown to depress levels of retinol in lungs of rats. The purpose of this study was to investigate if a low, but not deficient, vitamin A status potentiated susceptibility to the development of cigarette smoke-induced lung emphysema in mice. Mice were bred that were the offspring's of 3 generations of mice that were fed a purified diet containing low levels of vitamin A and exposed to cigarette smoke for 3 months, every weekday. Then, levels of 9-cis, 13-cis, and all-trans retinoic acid, retinol and retinyl palmitate were measured in plasma, liver and right lung lobe. The left lung lobe was used to assess mean linear intercept (Lm), as a measure of smoke-induced lung damage. Average feed intakes were not different between treatment groups. We show that both retinol and retinyl palmitate levels were dramatically decreased in the storage organs of mice on the low vitamin A diet (retinol 2-fold in both lung and liver, and retinyl palmitate 5- fold in lung) which shows that the depletion was successful. However, this treatment did not result in the development of lung emphysema. However, smoke exposure led to a significant increase in Lm in mice with a low vitamin A status compared to the room air-breathing controls. Lung levels of acid retinoids were similar in all mice, irrespective of diet or smoke exposure. Concluding, a low vitamin A status increases the susceptibility to the development of cigarette smoke-induced lung emphysema, possibly because of decreased anti-oxidant capacity in the lungs due to locally reduced retinol and retinyl palmitate levels. These observations indicate that human populations with a low vitamin A status and a high prevalence of smoking may be at increased risk of developing lung emphysema.

A whey-based glutathione-enhancing diet decreases allergen-induced airway contraction in a guinea-pig model of asthma.

Since an allergen-induced early asthmatic reaction is likely to be accompanied by oxidative stress and since levels of the endogenous antioxidant glutathione can be enhanced by a whey-based diet (undenatured whey protein concentrate, UWPC), it was investigated whether UWPC could alleviate allergen-induced lung contractions. Guinea pigs were fed water or UWPC twice a day starting at day - 3 up to day 20. The animals were sensitised to ovalbumin or received saline on day 0. Serum samples were taken at several days after sensitisation to measure allergen-specific IgG. On day 20, lungs were isolated and perfused with buffer containing the allergen ovalbumin. Airway contractions were assessed, and mediators and indicators for oxidative stress were measured in the lung effluent. Moreover, glutathione levels were determined in the liver. The indicator of oxidative stress and airway contractile mediator, 8-iso-PGF(2α), was increased upon ovalbumin challenge in ovalbumin-sensitised groups. Furthermore, thiobarbituric acid-reactive substances (TBARS) were increased as well. Sensitisation with ovalbumin increased IgG levels from day 12 up to day 20, which were not influenced by the UWPC diet. In contrast, the UWPC diet significantly enhanced glutathione levels in the liver. Moreover, the UWPC diet significantly reduced the ovalbumin-induced anaphylactic response by 45 % and decreased PGE2 levels by 55 % in the effluent fluid. We show for the first time that during anaphylaxis, there is acute oxidative stress in the respiratory tract. The UWPC diet did not influence the sensitisation response to the allergen but did increase endogenous glutathione levels. The UWPC diet profoundly reduces allergen-induced airway constrictions, which opens new avenues for dietary management of allergic diseases.

Bacille-Calmette-Guerin vaccination and the development of allergic disease in children: a randomized, prospective, single-blind study.

BACKGROUND: The increase in the prevalence of allergic diseases in countries with a so-called western lifestyle may be due to a decrease in exposure to infectious agents in early life. OBJECTIVE: To establish the effect of Bacille-Calmette-Guerin (BCG) vaccination in 6-week-old high-risk infants in a prospective single-blind, randomized, placebo-controlled trial on the prevalence of allergic disease at the age of 4 and 18 months. METHODS: Subjects were 121 predominantly Caucasian high-risk newborns, having either a mother, or both a father and at least one sibling with past or present allergic disease. BCG or placebo was administered at the age of 6 weeks, and repeated once when both a post-vaccination scar and a positive TB skin test were absent at the age of 4 months. RESULTS: At the age of 18 months, the prevalence of allergic disease was not significantly different between the two groups. A trend towards less eczema (P=0.07) and significantly less use of medication for eczema was shown in the BCG group compared with the placebo group (P=0.04). CONCLUSION: A single (or once repeated) BCG vaccination in 6-week-old high-risk Caucasian infants was not associated with a 50% reduction in the prevalence of allergic disease. However, there could be a smaller beneficial effect of BCG, especially because a trend towards less eczema and significantly less use of medication for eczema was shown. For definite proof, a larger study should be carried out.

Stress and hypothermia in mice in a nose-only cigarette smoke exposure system.

In nose-only exposure systems, animals need to be restrained inside a tube, which leads to stress. Stress is known to cause hyperthermia in rodents. Chronically repeated episodes of hyperthermia could be detrimental to animal health and influence results of nose-only exposure studies. Therefore we investigated whether hyperthermia occurred in male C57BL/6J mice that were restrained for increasing lengths of time, using nosepieces held at room temperature, preheated at 37 degrees C, or thermostat controlled at different temperatures, with and without exposure to different concentrations of cigarette smoke. Body temperature, body weight, plasma corticosterone levels, and adrenal weights were recorded. Restraint using nosepieces at room temperature caused a time-dependent decrease in body temperature, which could be reversed by preheating the nosepieces to 37 degrees C. Cigarette smoke dose-dependently caused an additional decrease, which was counteracted by controlling nosepiece temperature at 38 degrees C. During 3 mo of exposure using heated nosepieces, Delta body temperature remained constant. Body weight gain did not differ between smoke-exposed and room air-breathing animals exposed using either heated or room-temperature nosepieces, but both groups gained significantly less weight, while adrenal weights were significantly and similarly increased, when compared to unrestrained littermates. Plasma corticosterone levels did not differ between the three groups. In conclusion, during restraint in nose-only exposure tubes with room temperature metal nosepieces, mice suffer a pronounced hypothermia. Preventing this by heating the nosepieces does not reduce the stress experienced by the animals.

Regulatory T-lymphocytes in asthma.

T-helper cell type (Th)2 lymphocytes play an important role in the initiation, progression and persistence of allergic diseases, including asthma. However, little is known about immunoregulatory mechanisms that determine susceptibility to, severity of, or persistence of asthma. The concept of a disturbed Th1/Th2 balance, although having furthered the present understanding of immunoregulation in asthma, has recently been named a "procrustean paradigm", because of its failure to adequately explain many (pre)clinical observations. In recent years, the general knowledge regarding the regulation of infectious, autoimmune diseases, asthma and allergen immunotherapy by T-regulatory (Treg) cells, has rapidly increased. Many different Treg subsets have been described, including CD8+ Treg cells, natural killer (NK) cells and several different CD4+ Treg cell subsets. In this review, the authors will focus on two major and well-described CD4+ Treg cell subsets. These consist of naturally occurring CD25+ Treg cells and adaptive Treg cells that are postulated to prevent immune responses against self-antigens and adaptive immune responses, respectively. The adaptive T-regulatory cells are further subdivided into T-regulatory cells type 1 and T-helper cell type 3 that mediate suppression exclusively via the cytokines interleukin-10 and transforming growth factor-beta, respectively.

Glutathione-conjugated toluene diisocyanate causes airway inflammation in sensitised mice.

Toluene diisocyanate (TDI) is a highly volatile compound that reacts readily with nucleophilic compounds, sulfhydryl groups in particular. Since the epithelial lining fluid of the airways contains high levels of the sulfhydryl, glutathione (GSH), inhalation of TDI is likely to result in the formation of GS-TDI conjugates. We therefore investigated whether GS-TDI is capable of provoking irritant and/or allergic reactions. Irritant effects of GS-TDI were studied after intratracheal administration of a range of doses of GS-TDI in saline to naive BALB/c mice. GS-TDI caused a dose-dependent increase in neutrophils in the lungs 24 h after instillation. A dose equivalent to 150 microg of TDI or lower had no effect. For provocation of allergic reactions, mice were sensitised by application of 1% TDI onto the skin on days 0 and 1, and challenged intratracheally with a sub-irritant dose of GS-TDI on day 8. GS-TDI did not induce non-specific tracheal hyperreactivity to carbachol 24 and 48 h after challenge in TDI-sensitised mice. However, it increased the numbers of neutrophils in the lungs as compared with the control mice. These findings suggest that GSH conjugation does not diminish the capacity of TDI to elicit irritant-induced inflammation in the lungs of mice at doses above 150 microg of TDI in the conjugate. Moreover, the capacity to induce allergic-specific inflammation was retained at concentrations of GS-TDI being devoid of irritant activity. However, the GS-TDI conjugate failed to induce non-specific tracheal hyperreactivity. This may be the consequence of the deposition of excess of GSH upon local dissociation of the conjugate.

Modulation of airway hyperresponsiveness by thiols in a murine in vivo model of allergic asthma.

OBJECTIVE AND DESIGN: Since oxidative stress contributes to the pathogenesis of asthma, this study addressed the question whether supplementing the endogenous antioxidant, glutathione (GSH), would alleviate features of allergic asthma in the mouse. MATERIAL AND METHODS: Ovalbumin-sensitized mice received aerosols of the GSH-donors, glutathione-ethyl ester (GSEt) or N-acetylcysteine, before or during respiratory allergen challenges, or during methacholine challenges given one day after the last allergen challenge. Lung GSH levels were measured shortly after allergen or methacholine challenge. In addition, the effect of GSH supplements on airway hyperresponsiveness and inflammatory cell numbers in the airway lumen was assessed. RESULTS: GSEt decreased allergen-induced airway hyperresponsiveness when given in combination with methacholine. However, when given before or during allergen challenge, both GSH-donors failed to decrease the methacholine-induced airway contractility, change cell numbers in the airway lumen, or increase lung GSH levels. In addition, allergen challenges of sensitized mice did not decrease lung GSH levels. CONCLUSION: In contrast to guinea pigs and humans, allergen challenges in mice does not lead to acute oxidative stress.

The environmental pollutant hexachlorobenzene causes eosinophilic and granulomatous inflammation and in vitro airways hyperreactivity in the Brown Norway rat.

Based on observations that the persistent environmental pollutant hexachlorobenzene (HCB) induces inflammatory skin lesions and eosinophilic and granulomatous lung pathology as well as in vivo airways hyperresponsiveness to methacholine in the BN/SsNOlaHsd rat (Michielsen et al., Toxicol Appl Pharmacol 172:11-20, 2001), which are features of human Churg-Strauss syndrome (CSS), we have investigated whether HCB induced other features of CSS such as asthma and systemic vasculitis involving the heart and kidneys in this strain of rat. To this end, BN/SsNOlaHsd rats received control feed or feed supplemented with 450 mg/kg HCB. On days 6, 14 or 21, tracheas were isolated to assess non-specific in vitro airways hyperresponsiveness (AHR) to cumulative concentrations of arecoline and serotonin. In addition, lungs were lavaged to count and differentiate lavage cells, and skin, lungs, heart, kidneys, and lymph nodes were processed for histopathological investigation. HCB induced eosinophilic and granulomatous lung pathology in the BN/SsNOlaHsd rat, which became more severe with time and was associated with significant in vitro AHR to arecoline. Moreover, as in CSS-patients, systemic effects on spleen and lymph nodes were observed in HCB-fed BN/SsNOlaHsd rats, as well as development of skin lesions with vascular changes and eosinophilic infiltrates. In contrast, cardiac or renal involvement, frequently seen in CSS-patients, was not seen in HCB-fed rats. More importantly, there were no indications of necrotizing vasculitis, a hallmark feature of CSS, in the lungs and skin of BN/SsNOlaHsd rats. Thus, it is concluded that the persistent environmental pollutant HCB possibly induces a mild or early stage of CSS in the BN/SsNOlaHsd rat that may evolve into fully developed CSS after prolonged exposure to HCB.

Respiratory irritation by trimellitic anhydride in Brown Norway and Wistar rats.

Several acid anhydrides are known for their sensitizing and irritative properties. Since both irritation and respiratory allergy can cause changes of lung function, proper testing of allergen-dependent effects on the respiratory tract requires knowledge of the respiratory irritant effects. To study the latter effects, groups of female Brown Norway (BN) and Wistar rats were exposed for 30 min to a range of concentrations (10 to 300 mg/m(3)) of the well-known respiratory allergen trimellitic anhydride (TMA). Breathing pattern and frequency were monitored before, during, and after exposure. Animals were necropsied and lung weights were determined 1 day after exposure. In BN rats, changes in breathing pattern were seen at levels of 29 mg/m(3) and higher and decreases in frequency at 60 mg/m(3) and higher, whereas in Wistar rats changes in both pattern and frequency (increases followed by decreases) were seen at levels of 34 mg/m(3) and higher. Changes in breathing pattern consisted of a spiked form instead of a wave form of the respiratory cycle, with a pause between breaths at the end of expiration. The length of the pause increased with increasing concentrations of TMA while the duration of the respiratory cycle decreased slightly, implying that breathing frequency was mainly determined by the magnitude of the increase in pause. These reversible changes in breathing pattern and frequency were considered to be suggestive of lower airway irritation, rather than upper airway irritation. No concentration-related changes in lung weights were observed. The highest level at which no acute airway irritation as based on both breathing pattern and frequency was observed in both rat strains was 14 mg/m(3).

Hexachlorobenzene-induced eosinophilic and granulomatous lung inflammation is associated with in vivo airways hyperresponsiveness in the Brown Norway rat.

We investigated whether the eosinophilic and granulomatous lung pathology that develops in Brown Norway (BN/SsNOlaHsd) rats upon feeding hexachlorobenzene (HCB) is associated with nonspecific in vivo airways hyperresponsiveness (AHR) to methacholine. To this end, female BN/SsNOlaHsd rats were exposed to diets with no supplementation or diets supplemented with 450 mg HCB per kg feed. On days 7 or 21 of exposure in vivo airways hyperresponsiveness to increasing concentrations of methacholine was assessed both by whole body plethysmography and by visual scoring. In addition, lungs were lavaged to count and differentiate lavage cells, and skin and lungs were processed for histology. Lungs of the control rats showed some scattered microgranulomas and by 3 weeks of control diet some rats showed rather extensive granuloma formation and perivascular and peribronchiolar infiltration of eosinophils, as well as increased responsiveness to methacholine. Oral exposure to HCB for 7 days caused a moderate perivasculitis, but no increase of total serum IgE levels and no AHR to methacholine was found. Prolonged HCB exposure for 21 days resulted in severe and extensive eosinophilic and granulomatous lung inflammation, a threefold increase of total serum IgE levels, and marked cholinergic AHR in all rats. Correlation analysis revealed a significant relation between the AHR and lung inflammation, as judged by granuloma formation and increased numbers of eosinophilic granulocytes in the lung interstitium, particularly around the bronchi and bronchioli. No correlation was observed between serum IgE levels and AHR. Data suggest that HCB induces AHR by stimulating eosinophilic lung inflammation and that the preexistent microgranulomas may predispose to development of the HCB-induced lung pathology.

Predictive testing for pathogenic autoimmunity: the morphological approach.

The term autoimmunity refers to physiologically normal immune processes against self-antigens. In rare cases, the regulatory mechanisms become deflective and the uncontrolled production of autoantibodies or activation of autoreactive T-cells can subsequently cause disease. Substances may be capable of evoking autoimmune disease, and it is a challenge in routine toxicology to recognize such substances. In in vivo toxicity studies, uncommon inflammation in exposed animals should be discussed in terms of non-immune toxicity (e.g. irritation), infection, allergy and autoimmunity, taking into account that a response in even a few animals may be significant. Moreover, early morphological indicators of inflammation and lymphoid organ alterations can direct further investigation.

The mercapturic acid biotransformation pathway of hexachlorobenzene is not involved in the induction of splenomegaly, or skin and lung lesions in the Brown Norway rat.

Involvement of the mercapturic acid pathway in the induction of splenomegaly and skin and lung pathology by hexachlorobenzene (HCB) in the rat was investigated by seeking to determine whether pentachloronitrobenzene (PCNB) has the same inflammatory effects as HCB, since both compounds are directly conjugated to glutathione, and further processed into the same mercapturic acid metabolites which are excreted via the urine. Female Brown Norway (BN/SsNO1aHsd) rats at 3 to 4 weeks of age were orally exposed to diets with or without supplementation with 450 mg HCB or equimolar (467 mg) or higher (934 mg) amounts of PCNB per kilogram of diet over 4 weeks. Gross skin lesion development and body weight gains were assessed during exposure and spleen and liver weights as well as histopathologic changes in skin and lung were assessed after exposure. After 3 weeks of exposure, urinary metabolites of the mercapturic acid and oxidative biotransformation pathways were identified using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Oral exposure of the rats to 450 mg/kg HCB resulted in an increase in relative spleen and liver weights as well as in the development of skin and lung pathology in the absence of overall liver toxicity. Equimolar or higher concentrations of PCNB caused none of these effects. Urinary levels of the mercapturic acid N-acetyl-S-(pentachlorophenyl)-cysteine (PCP-NAC), were comparable in HCB- and PCNB-treated rats. Levels of closely related methylsulfide derivatives of PCP-NAC, also generated via the same mercapturic acid pathway, appeared to be significantly higher in PCNB- than in HCB-treated rats, whereas the reverse was true for the urinary levels of the oxidative metabolite pentachlorophenol (PCP). Thus, results indicate that metabolites of the mercapturic acid pathway are not involved in the induction of splenomegaly and skin and lung pathology caused by HCB exposure in BN rats and that the main urinary metabolite of HCB in these BN rats is PCP. Since PCP itself, as well as other cytochrome P450-derived metabolites from HCB, are not likely to be involved in the induction of splenomegaly and skin and lung pathology, it is suggested that either the parent compound HCB or as-yet-unidentified non-P450-generated metabolites are involved in these inflammatory effects of HCB.

The role of thymus-dependent T cells in hexachlorobenzene-induced inflammatory skin and lung lesions.

The involvement of thymus-dependent T cells in the inflammatory skin and lung lesions and spleen effects induced by hexachlorobenzene (HCB) was investigated by using genetically athymic and euthymic WAG/Rij rats and Brown Norway (BN) rats with or without depletion of T cells by adult thymectomy, lethal irradiation, and bone marrow reconstitution. Rats were exposed to diets with no supplementation or diets supplemented with 150 or 450 mg HCB per kg diet for 4 (BN) or 6 (WAG/Rij) weeks. Skin lesion development and body weight gains were assessed during exposure and spleen and liver weights as well as histopathologic changes in skin, lung, and spleen were assessed after exposure. Oral HCB exposure of athymic and euthymic rats of both rat strains resulted in a dose-dependent increase of relative liver weight at doses of 150 and 450 mg/kg HCB and increased relative spleen weights at a dose of 450 mg/kg. HCB exposure of both strains further resulted in inflammatory changes in skin, lungs, and splenic red pulp independent of the T cell status except for skin lesions in the BN strain. HCB-exposed T cell-competent BN rats showed faster skin lesion development than the T cell-depleted rats, although qualitatively and quantitatively similar skin pathology was observed at the end of the 4-week exposure in both groups. In the WAG/Rij strain skin lesions could not be comparatively assessed due to preexistent inflammatory skin pathology in the nude rats. This study showed that thymus-derived T cells are not required for the induction of skin and lung pathology and splenic changes by HCB and therefore it is suggested that HCB acts differently from many allergenic and autoimmunogenic low molecular weight compounds that trigger pathology via thymus-dependent mechanisms. A role for mononuclear phagocytes and, in BN rats, eosinophilic granulocytes, in the HCB-induced pathology is suggested since these cells were prominently present in the HCB-induced lesions.

Airway morphology and function of rats following dermal sensitization and respiratory challenge with low molecular weight chemicals.

Local lymph node activation and increased total serum IgE levels are suggested to be predictive parameters of airway hypersensitivity caused by low molecular weight (LMW) chemicals. Whether increases of total serum IgE are indicative of actual induction of specific airway reactions (morphological and functional) after inhalation challenge was examined in the present study. In Brown Norway (BN) and Wistar rats, serum IgE concentrations were examined following topical exposure of chemicals with known diverse sensitization potential in humans: trimellitic anhydride (TMA), a dermal and respiratory sensitizer; dinitrochlorobenzene (DNCB), a dermal sensitizer with no known potential to cause respiratory allergy; and methyl salicylate, a skin irritant devoid of sensitizing properties. Functional and histopathological changes in the respiratory tract were examined after subsequent inhalatory challenge with these chemicals. Of the three tested chemicals, only topical exposure to TMA resulted in a significant increase in total serum IgE concentrations in the high-IgE-responding BN rat. Upon subsequent inhalatory challenge of these rats, TMA induced specific airway reactions which included a sharp decrease in respiratory rate during challenge, followed by an increase in breathing rate with a concomitant decrease in tidal volume 24 and 48 h after inhalatory challenge, and histopathological changes in the larynx and lungs of animals necropsied 48 h after challenge. Interestingly, despite low IgE levels, TMA induced histopathological changes in the larynx and lungs of Wistar rats too. Laryngeal changes were also observed in Wistar rats upon sensitization and challenge with DNCB. These data suggest that increased total serum IgE after topical sensitization is associated with immediate-type specific airway reactivity after inhalation challenge in BN rats and thus may be a valuable parameter in testing for respiratory sensitization potential of LMW compounds. Histopathological examination upon subsequent inhalation challenge of sensitized low-IgE-responders may provide information on other allergic inflammatory airway reactions.

Hexachlorobenzene-induced immunomodulation and skin and lung lesions: a comparison between brown Norway, Lewis, and Wistar rats.

Strain dependence of the induction of skin and lung lesions by hexachlorobenzene (HCB) in the rat was studied to further the insight into the etiology of the lesions. To this end, 3- to 4-week-old female Brown Norway (BN), Lewis, and Wistar rats received diets supplemented with 150 mg (BN and Lewis), 450 mg (BN, Lewis, and Wistar) or 900 mg (BN and Wistar) HCB per kilogram diet for 4 weeks. Gross skin lesion development during exposure as well as pathologic changes in skin and lungs and various parameters of immunomodulation after exposure were assessed. General toxicity as judged by a slight increase in body weight gain and induction of liver cell hypertrophy was similar in BN and Lewis rats exposed to 450 mg/kg HCB and in Wistar rats exposed to 900 mg/kg HCB. Skin lesions ranged from redness to large exudating sores with crusts. With regard to dose, time of onset, incidence, and severity, skin lesions were very severe in BN, moderate in Lewis, and negligible in Wistar. Porphyrins could not be detected in the skin, whereas porphyrins in the liver were seen only in Lewis rats. Histology showed epidermal hyperplasia, deep dermal venules with activated endothelium, and deep dermal inflammatory infiltrates mainly consisting of eosinophilic granulocytes in BN and of mononuclear cells in Lewis and Wistar. Nonlesional skin of HCB-exposed rats showed very similar, though less prominent, changes. Lung pathology appeared negligibly strain-dependent; histology showed venules with an activated endothelium surrounded by a perivascular infiltrate as well as focal alveolar macrophage accumulations in all strains. Parameters of immunomodulation showed moderate strain dependence; relative spleen weights were dose-dependently increased in BN and Wistar and in the 450 mg/kg group in Lewis rats. BN rats showed a more marked splenomegaly than the other strains. Relative popliteal lymph node weights were increased significantly in BN and Lewis rats exposed to 450 mg/kg HCB. In all strains, HCB increased lymph node HEVs. Serum IgE and IgG levels were increased significantly in a dose-dependent way in BN rats only. Total serum IgM levels were elevated significantly in BN, Lewis, and Wistar rats that received 450 mg/kg and in Wistar rats that received 900 mg/kg HCB. Serum IgM levels against ssDNA were dose-dependently increased in all strains, being more marked in BN and Lewis than in Wistar rats. It is concluded that the HCB-induced inflammatory skin and lung pathologies have different etiology. Pronounced strain differences in the skin lesions suggest a specific involvement of the immune system. Skin lesions correlated significantly with all assessed parameters of immunomodulation in BN, with some in Lewis and with none in Wistar rats. No correlation was observed between the parameters of immunomodulation and lung lesions.

Local lymph node activation and IgE responses in brown Norway and Wistar rats after dermal application of sensitizing and non-sensitizing chemicals.

The local lymph node assay (LLNA) and the IgE test in the mouse are proposed models for predictive recognition of low molecular weight chemicals causing IgE-mediated allergic airway reactions in man. Since rats are commonly used in routine toxicity studies and a previous study (Arts et al. (1996) Food Chem. Toxicol. 34, 55-62) has shown that several rat strains were found appropriate for the LLNA, the suitability of the rat for the IgE test was examined in the present study. Serum IgE concentrations were examined following topical exposure of Brown Norway (BN) and Wistar rats to each of four chemicals with known diverse sensitization potential in humans: trimellitic anhydride (TMA), a dermal and respiratory sensitizer, dinitrochlorobenzene (DNCB), a dermal sensitizer with no or limited potential to cause respiratory allergy; formaldehyde (FA), a skin irritant and dermal sensitizer with equivocal evidence for respiratory sensitizing potential; methyl salicylate (MS), a skin irritant devoid of sensitizing properties. Of the four tested chemicals, only exposure to TMA resulted in a significant increase in serum IgE concentration and this response was only evoked in the high-IgE-responding BN rat. The latter two chemicals were also tested for lymph node activation, in casu the ear-draining lymph nodes. FA caused a dose-dependent activation of the draining lymph nodes whereas MS was inactive. The results as obtained with TMA, DNCB and MS in the rat are in agreement with human data. The results with FA though, indicate the need for further studies of chemicals that have both irritant and sensitizing properties at about similar concentrations or may act through non-IgE-mediated immune mechanisms.

The popliteal lymph node assay in mice: screening of drugs and other chemicals for immunotoxic hazard.

The popliteal lymph node assay (PLNA) in mice represents a predictive test for assessing the sensitizing (allergenic and autoimmunogenic) potential of drugs and low molecular weight chemicals. Measuring activation of the draining lymph node of the hind paw, the PLNA facilitates the detection and analysis of immunotoxic effects in a rapid and reproducible manner. An attractive feature of the PLNA is that it can be performed in combination with the routine toxicity testing required for new drugs. Thus, it is possible to investigate whether animals exposed by the oral, intravenous, or inhalative route have been sensitized to the test compound or a reactive metabolite of the test compound generated in vivo. PLNAs may be appropriate supplements to routine toxicity screening of chemicals, thereby enhancing chemical safety.

The autoimmunogenic chemicals HgCl2 and diphenylhydantoin stimulate IgG production to TNP-Ficoll and TNP-OVA, supporting and extending the graft-versus-host hypothesis for chemical induction of autoimmunity.

Bypass of T-cell tolerance via non-cognate graft-versus-host (GVH)-like help from T-helper (Th) cells activated by chemically altered or induced epitopes, has been postulated as a mechanism underlying chemical induction of autoimmunity. To functionally test this hypothesis, we assessed whether the autoimmunogenic chemicals HgCl2 and diphenylhydantoin (DPH), like GVH reactions, stimulate specific immunoglobulin G (IgG) responses to trinitrophenyl (TNP)-Ficoll but not to TNP-ovalbumin. IgG responses were quantified in the popliteal lymph node by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) assays 7 days after s.c. injection of antigens, parental cells, chemicals or combinations thereof into the footpad of semi-allogeneic F1 mice. Antigens, chemicals, or cells alone induced few TNP-specific IgG antibody-forming cell (AFC) compared with untreated mice. Co-injection of parental cells or chemicals with TNP-Ficoll stimulated the TNP-specific response per lymph node approximately 50- and approximately 40-fold, respectively. In contrast, the IgG response to TNP-ovalbumin could not be stimulated by GVH reactions, whereas HgCl2 and DPH dose-dependently increased this response up to approximately 25- and approximately 250-fold, respectively. However, responses to TNP-ovalbumin pre-incubated with HgCl2 or DPH could be stimulated approximately 6-8 fold by GVH reactions. Observed similar adjuvanticity of chemicals and parental cells for TNP-Ficoll support a GVH-like action of autoimmunogenic chemicals. In addition, the chemicals modify TNP-ovalbumin such that B cells recognizing this antigen become susceptible to non-cognate stimulation by GVH reactions.