Nanne Bloksma

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-β, respectively.

Predictive immunotoxicological test systems: suitability of the popliteal lymph node assay in mice and rats.

This article reviews results obtained with popliteal lymph node assays (PLNAs) in rodents and discusses their ability to detect and analyze immunotoxic effects of drugs and other low molecular weight (LMW) chemicals. In its basic form, the PLNA measures activation of the draining lymph node of the hind paw (i.e., the PLN) after injection of a test chemical into the hind foot pad. The assay appears to be appropriate to recognize sensitizing, that is, allergenic and autoimmunogenic, chemicals, as well as nonsensitizing immunostimulatory chemicals. With modifications, PLNAs can detect immunosuppressive chemicals and distinguish sensitizing from nonsensitizing chemicals. Furthermore, modified PLNAs enable detection of known as well as unknown sensitizing metabolites, and may assist in the identification of the self-molecules that act as carriers for chemical sensitization or as targets of chemical-induced autoimmune disease. Experience with PLNAs shows that they are rapid, reproducible, and objective tests for recognition of sensitizing or otherwise immunomodulating chemicals. Because current protocols of toxicity testing are insensitive in predicting a chemicals potential to result in immunomodulation, PLNAs, when further validated, may provide welcome supplements to routine toxicity screening of chemicals, thus enhancing chemical safety.

The popliteal lymph node assay in mice to screen for the immune disregulating potential of chemicals — A preliminary study

Low mol. wt compounds were tested in the popliteal lymph node (PLN) assay to study whether PLN reactivity could be related to the ability of the compounds to induce autoimmune disorders in man. PLN reactions were measured 7 days after a single subcutaneous (s.c.) injection of dissolved compounds in amounts of 0.3-2.0 mg into one hind footpad of mice and assessed as the weight increase of the draining PLN relative to the PLN weight of the untreated contralateral paw. Hydralazine, chlorpromazine, diphenylhydantoin, carbamazepine, phenylbutazone and nitrofurantoin, all being drugs with a documented potential to induce systemic immunological disorders in man, caused marked PLN reactions. False negative PLN responses were observed following injection of procainamide and isoniazid. Among systemic drugs without known potential to induce autoimmune reactions in humans, quinacrine, denzimol and niridazole significantly increased PLN weights, while phenobarbital, levamisole and disulfiram had no effect. Chemicals with a well-known capacity to induce contact dermatitis in man like 2,4-dinitro-1-chlorobenzene, alpha-methylene-gamma-butyrolactone, p-phenylenediamine, 5-nitro-2-furaldehyde semicarbazone, 2-mercapto-benzothiazol and 1,3-dibutyl-2-thiourea caused marked PLN reactions, while the non-sensitizer 2,4-dichloro-1-nitrobenzene failed to do so. It is concluded that the PLN assay as applied in this study may give a rapid first indication of immunomodulating potential of low mol. wt compounds, but it does not discriminate as to the kind of immunomodulation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

Local lymph node activation in rats after dermal application of the sensitizers 2,4-dinitrochlorobenzene and trimellitic anhydride

Five rat strains were compared for their performance in the local lymph node assay (LLNA), a promising test system for the identification of the skin-sensitizing potential of chemicals in the mouse. The contract sensitizer 2,4-dinitrochlorobenzene (DNCB) and the contact and respiratory sensitizer trimellitic anhydride (TMA) were used as model chemicals and responses in rats were compared with those in BALB/c mice. The chemicals were applied to the dorsum of both ears, once daily for three consecutive days; 2 days (mice) or 3 days (rats) thereafter, proliferating cells were labelled by i.p. injection of BrdU 2 hr before the animals were killed. Systemic effects were subsequently assessed by determination of spleen, liver and kidney weights, skin effects by determination of swelling and inflammatory cell infiltration of the ears, and immune effects by determination of weight and proliferative activity of the local lymph nodes (LLN). Following application (x 3) of DNCB or TMA, minor systemic effects were observed, as indicated by slightly elevated spleen and liver weights in a few rat strains and the mice. Skin effects, consisting of increased ear thickness and presence of mononuclear inflammatory cell infiltrates, were observed in all rat strains treated with DNCB or TMA, LLN weights had increased, as had the proliferative activity in these nodes. It was concluded that effects induced by DNCB and TMA in all five rat strains were comparable with those in mice.

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).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 .

Endotoxin-induced antitumor activity in the mouse is highly potentiated by muramyl dipeptide

The ability of aqueous solutions of various endotoxin preparations, muramyl dipeptide (MDP) and combinations of endotoxin and MDP, to induce necrosis and regression of subcutaneous Meth A transplants in mice and their toxicity were studied. While intravenously injected toxic endotoxins, in contrast to a detoxified preparation and to MDP, induced considerable necrosis and regression of their own, addition of MDP potentiated the antitumor potential of both toxic and detoxified endotoxins to the same high degree. Detoxified endotoxin combined with MDP, however, was far less toxic than toxic preparations alone or combined with MDP. This indicates that toxicity is not directly related to therapeutic potential.