RESUMO
Idiosyncratic drug-induced liver injury (iDILI) has a poorly understood pathogenesis. However, iDILI is often associated with inflammatory stress signals in human patients as well as animal models. Tumor necrosis factor (TNF) and neutrophils play a key role in onset of trovafloxacin (TVX)-induced iDILI, but the exact role of neutrophils and other leukocytes remains to be defined. We therefore set out to study the kinetics of immunological changes during the development of TVX-induced iDILI in the established murine model of acute liver injury induced by administration of TVX and TNF. Initially, TNF stimulated the appearance of leukocytes, in particular neutrophils, into the liver of TVX-treated mice, but even more so in control mice treated with the non-DILI inducing analogue levofloxacin (LVX) or saline as vehicle (Veh). This difference was apparent at 2 hours after TNF administration, but at 4 hours, the relative neutrophil amounts were reduced again in Veh- and LVX-treated mice whereas the amounts in TVX-treated mice remained at the same increased level as at 2 hours. The influx of monocytes/macrophages, which was unaffected in Veh- and LVX-treated mice was markedly reduced or even absent in TVX-treated mice. Unlike controls, mice receiving TVX + TNF display severe hepatotoxicity with clear pathology and apoptosis, coagulated hepatic vessels and increased alanine aminotransferase levels and interleukin 6/10 ratios. Findings indicate that TVX delays the acute influx of neutrophils and monocytes/macrophages. Considering their known anti-inflammatory functions, the disruption of influx of these innate immune cells may hamper the resolution of initial cytotoxic effects of TVX and thus contribute to liver injury development.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fluoroquinolonas/toxicidade , Monócitos/efeitos dos fármacos , Naftiridinas/toxicidade , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/toxicidade , Alanina Transaminase/sangue , Animais , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citocinas/sangue , Citometria de Fluxo , Leucócitos/efeitos dos fármacos , Levofloxacino/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The fluoroquinolone trovafloxacin (TVX) is associated with a high risk of drug-induced liver injury (DILI). Although part of the liver damage by TVX+TNF relies on neutrophils, we have recently demonstrated that liver recruitment of monocytes and neutrophils is delayed by TVX. Here we show that the delayed leukocyte recruitment is caused by a combination of effects which are linked to the capacity of TVX to block the hemichannel pannexin 1. TVX inhibited find-me signal release in apoptotic HepG2 hepatocytes, decelerated freshly isolated human neutrophils toward IL-8 and f-MLF, and decreased the liver expression of ICAM-1. In blood of TVX+TNF-treated mice, we observed an accumulation of activated neutrophils despite an increased MIP-2 release by the liver. Depletion of monocytes and neutrophils caused increased serum concentrations of TNF, IL-6, and MIP-2 in TVX-treated mice as well as in mice treated with the fluoroquinolone levofloxacin, known to have a lower DILI-inducing profile. This supports the idea that early leukocyte recruitment regulates inflammation. In conclusion, disrupted regulation by leukocytes appears to constitute a fundamental step in the onset of TVX-induced liver injury, acting in concert with the capability of TVX to induce hepatocyte cell death. Interference of leukocyte-mediated regulation of inflammation represents a novel mechanism to explain the onset of DILI.
Assuntos
Anti-Infecciosos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Fluoroquinolonas/toxicidade , Naftiridinas/toxicidade , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Nucleotídeos/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Conexinas/metabolismo , Células Hep G2 , Humanos , Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologiaRESUMO
The development of immune-dependent drug hypersensitivity reactions (IDHR) is likely to involve activation of the innate immune system to stimulate neo-antigen specific T-cells. Previously it has been shown that, upon oral exposure to several drugs with immune-adjuvant capacity, mice developed T-cell-dependent responses to TNP-OVA. These results were indicative of the adjuvant potential of these drugs. The present study set out to evaluate the nature of this adjuvant potential by focusing on early immune changes in the spleen, by testing several drugs in the same experimental model. Mice were exposed to one or multiple oral doses of previously-tested drugs: the non-steroidal-anti-inflammatory drug (NSAID) diclofenac (DF), the analgesic acetaminophen (APAP), the anti-epileptic drug carbamazepine (CMZ) or the antibiotic ofloxacin (OFLX). Within 24 h after the final dosing, early innate and also adaptive immune parameters in the spleen were examined. In addition, liver tissue was also evaluated for damage. Exposure to APAP resulted in severe liver damage, increased levels of serum alanine aminotransferase (ALT) and local MIP-2 expression. DF exposure did not cause visible liver damage, but did increase liver weight. DF also elicited clear effects on splenic innate and adaptive immune cells, i.e. increased levels of NK cells and memory T-cells. Furthermore, an increase in plasma MIP-2 levels combined with an influx of neutrophils into the spleen was observed. OFLX and CMZ exposure resulted in increased liver weights, MIP-2 expression and up-regulation of co-stimulatory molecules on antigen-presenting cells (APC). The data suggested that multiple immune parameters were altered upon exposure to drugs known to elicit immunosensitization and that broad evaluation of immune changes in straightforward short-term animal models is needed to determine whether a drug may harbor the hazard to induce IDHR. The oral exposure approach as used here may be applied in the future as an immunotoxicological research tool in this type of evaluation.
Assuntos
Hipersensibilidade a Drogas/imunologia , Imunidade Inata , Fatores Imunológicos/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Fígado/efeitos dos fármacos , Baço/efeitos dos fármacos , Linfócitos T/imunologia , Acetaminofen/administração & dosagem , Acetaminofen/efeitos adversos , Imunidade Adaptativa/efeitos dos fármacos , Administração Oral , Animais , Carbamazepina/administração & dosagem , Carbamazepina/efeitos adversos , Células Cultivadas , Diclofenaco/administração & dosagem , Diclofenaco/efeitos adversos , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/efeitos adversos , Memória Imunológica , Células Matadoras Naturais/imunologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos C3H , Ofloxacino/administração & dosagem , Ofloxacino/efeitos adversos , Baço/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
Immune-mediated drug hypersensitivity reactions (IDHR) may result from immuno-sensitization to a drug-induced neo-antigen. They rarely occur in patients and are usually not predicted preclinically using standard toxicity studies. To assess the potential of a drug to induce T-cell sensitization, trinitrophenyl (TNP)-Ficoll was used here as a bystander antigen in animal experiments. TNP-Ficoll will only elicit TNP-specific IgG antibodies in the presence of non-cognate T-cell help. Therefore, the presence of TNP-specific IgG antibodies after co-injection of drug and TNP-Ficoll was indicative of T-cell sensitization potential. This TNP-Ficoll-approach was used here to characterize T-cell help induced by oral exposure to diclofenac (DF) or carbamazepine (CMZ). DF or CMZ was administered orally to BALB/c mice and after 3 w, the mice were challenged in a hind paw with TNP-Ficoll and a dose of the drug that by itself does only elicit a sub-optimal popliteal lymph node assay (PLNA) response. T-cell-dependent responses were then evaluated in paw-draining popliteal lymph nodes (PLN). Also, shortly after oral exposure, mesenteric lymph nodes (MLN) were excised for evaluation of local responses. Both drugs were able to increase PLN cellularity and TNP-specific IgG1 production after challenge. Both DF and CMZ stimulated CD4+ and CD8+ T-cells and caused shifts of the subsets toward an effector phenotype. DF, but not CMZ, appeared to stimulate interferon (IFN)-γ production. Remarkably, depletion of CD8+, but not CD4+, T-cells reduced TNP-specific IgG1 production, and was more pronounced in CMZ- than in DF-exposed animals. Local responses in the MLN caused by DF or CMZ also showed shifts of CD4+ and CD8+-cells toward a memory phenotype. Together, the data indicate that oral exposure to CMZ and DF differentially induced neo-antigen-specific T-cell reactions in the PLNA.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Carbamazepina/efeitos adversos , Diclofenaco/efeitos adversos , Hipersensibilidade a Drogas/imunologia , Linfócitos T/imunologia , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Antígenos/imunologia , Carbamazepina/uso terapêutico , Diclofenaco/uso terapêutico , Ficoll/análogos & derivados , Ficoll/imunologia , Imunoglobulina G/metabolismo , Interferon gama/metabolismo , Linfonodos/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Trinitrobenzenos/imunologiaRESUMO
To gain more insight into the mechanisms of particulate matter (PM)-induced adjuvant activity, we studied the kinetics of airway toxicity/inflammation and allergic sensitization to ovalbumin (OVA) in response to ultrafine carbon black particles (CBP). Mice were exposed intranasally to OVA alone or in combination with different concentrations of CBP. Airway toxicity and inflammation were assessed at days 4 and 8. Immune adjuvant effects were studied in the lung draining peribronchial lymph nodes (PBLN) at day 8. Antigen-specific IgE was measured at days 21 and 28, whereas allergic airway inflammation was studied after OVA challenges (day 28). Results show that a total dose of 200 microg CBP per mouse, but not 20 microg or 2 microg, induced immediate airway inflammation. This 200 microg CBP was the only dose that had immune adjuvant activity, by inducing enlargement of the PBLN and increasing OVA-specific production of Th2 cytokines (IL-4, IL-5, and IL-10). The immune adjuvant activity of 200 microg CBP dosing was further examined. Whereas increased OVA-specific IgE levels in serum on day 21 confirms systemic sensitization, this was further supported by allergic airway inflammation after challenges with OVA. Our data show a link between early airway toxicity and adjuvant effects of CBP. In addition, results indicate that local cytokine production early after exposure to CBP is predictive of allergic airway inflammation. In addition this model appears suitable for studying the role of airway toxicity, inflammation and other mechanisms of particle adjuvant activity, and predicting the adjuvant potential of different particles.
Assuntos
Carbono/toxicidade , Inflamação/induzido quimicamente , Hipersensibilidade Respiratória/induzido quimicamente , Doenças Respiratórias/induzido quimicamente , Administração Intranasal , Animais , Asma/imunologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Carbono/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Imunoglobulina E/imunologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/patologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Hipersensibilidade Respiratória/patologia , Doenças Respiratórias/patologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
In the RAIAP (respiratory allergy and inflammation due to ambient particles) project, qualitative properties of ambient air particles from Amsterdam, Oslo, Lodz and Rome were investigated in relation to inflammation and allergy. Most collected particle fractions were found to increase the allergen-specific IgE and IgG2a responses after subcutaneous injection of particles with allergen in mice. However, some fractions appeared to skew the antibody response towards more Th1- or Th2-associated antibody isotypes, and the fine fractions were found to be more potent than the coarse fractions with regard to IgE adjuvant activity. In the present study we investigated the cellular response in the draining lymph node 5 days after a subcutaneous injection of selected RAIAP particle fractions. The particles (100 microg) were injected into both hind footpads of BALB/cA mice, in the presence or absence of the allergen ovalbumin (OVA, 50 microg). We also studied if the coarse and fine RAIAP particle fractions affected the cellular responses to OVA differently. The number of lymph node cells, as well as the relative number of B and T lymphocytes and T helper cells were determined. Expression of cell surface molecules (MHC class II, CD86 and CD23) and ex vivo cytokine production (IL-4, IL-10 and IFN-gamma) by the lymph node cells were measured. Overall, particles in the presence of allergen enhanced the levels of the various cellular parameters compared to allergen alone or particles alone. In the absence of allergen, ambient air particles, in contrast to diesel exhaust particles, marginally affected some cellular parameters. By histological examination of the lymph node, the particles appeared to be scattered between the lymphocytes, often localised within macrophage-like (acid phosphatase positive) cells. The cell parameters measured could, for the individual sample, neither predict the degree of a Th2- or Th1-skewed antibody response, nor the stronger antibody adjuvant capacity of the fine than the coarse particle fractions. In conclusion, we have shown that coarse and fine ambient air particles from different European cities enhance the cellular response in the draining lymph node after injection with an allergen. In the absence of allergen, ambient particles only marginally affected the cellular parameters.
Assuntos
Poluentes Atmosféricos/toxicidade , Linfócitos B/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Emissões de Veículos/toxicidade , Animais , Formação de Anticorpos , Antígenos CD/análise , Linfócitos B/imunologia , Antígeno B7-2 , Antígenos CD4/análise , Células Cultivadas , Cidades , Europa (Continente) , Feminino , Genes MHC da Classe II/imunologia , Injeções Subcutâneas , Linfonodos/imunologia , Linfonodos/patologia , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Tamanho da Partícula , Receptores de IgE/análise , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Hexachlorobenzene (HCB) is a persistent environmental pollutant with (auto)immune effects in humans and rats. The Brown Norway (BN) rat is very susceptible to HCB-induced immunopathology, and oral exposure causes inflammatory skin and lung lesions, splenomegaly, lymph node (LN) enlargement, and increased serum levels of IgE and anti-ssDNA IgM. The role of T cells in HCB-induced immunopathology is unclear and to elucidate this Cyclosporin A (CsA) was used. BN rats were exposed to either a control diet or a diet supplemented with 450 mg/kg HCB for 21 days. CsA treatment started 2 days prior to HCB exposure and rats were injected daily with 20 mg/kg body weight CsA. Treatment with CsA prevented the HCB-induced immunopathology significantly. The onset of skin lesions was delayed and the severity was also strongly decreased. Furthermore, CsA prevented the HCB-induced increase in spleen weight partly and the increase in auricular LN weight completely. The increase in serum IgE and IgM against ssDNA levels was prevented completely. Macrophage infiltrations into the spleen and lung still occurred but infiltrations of eosinophilic granulocytes into the lung were prevented. Restimulation of spleen cells with the T-cell mitogen ConA and the macrophage activator LPS clearly showed that CsA inhibited T-cell activation, but not macrophage activation. Together, our results show that both T cells and macrophages play a prominent role in HCB-induced immunopathology.
Assuntos
Poluentes Ambientais/toxicidade , Hexaclorobenzeno/toxicidade , Sistema Imunitário/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Ciclosporina/farmacologia , Citocinas/biossíntese , DNA de Cadeia Simples/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunoglobulina E/biossíntese , Imunoglobulina M/biossíntese , Imuno-Histoquímica , Imunossupressores/farmacologia , Interleucina-2/biossíntese , Pulmão/patologia , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos BN , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/imunologia , Baço/citologia , Baço/imunologia , Baço/patologia , Linfócitos T/efeitos dos fármacos , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Aumento de Peso/efeitos dos fármacosRESUMO
OBJECTIVE: We compared the effects of supplementing with vitamins A, C, and E, selenium, and zinc on a range of innate and specific T-helper 1 (Th1) and Th2-driven adaptive immune responses. METHODS: BALB/c mice were fed semi-purified AIN93 diets and randomly assigned to receive a diet supplemented with 120 mg/kg of vitamin A, 2500 mg/kg of vitamin C, 1000 mg/kg of vitamin E, 2 mg/kg of selenium, and 500 mg/kg of zinc (n = 15/group). After 4 wk of supplementation, mice were sensitized by topical application of di-nitro-chlorobenzene (DNCB); 2 wk later mice were challenged; and 5 d later they were killed to assess the effect on a range of innate responses (phagocytic activity, oxidative burst and tumor necrosis factor-alpha), adaptive Th1-driven responses (delayed-type hypersensitivity, DNCB-specific immunoglobulin [Ig] G2a and IgG2b, and interferon-gamma [IFN-gamma]), and adaptive Th2-driven responses (DNCB-specific IgE and IgG1 and interleukin-4 [IL-4]). RESULTS: Immune function was affected only in the vitamin A group. These mice gained less weight and were less capable of resolving the inflammatory response elicited during sensitization. The oxidative burst of blood cells was increased, but production of IFN-gamma and IL-4 and the ratio of IFN-gamma to IL-4 were markedly depressed. In concordance with the latter result, production of Th1-driven IgG2a antibodies was decreased, whereas Th2-driven isotypes were not affected (IgG1, IgE) and mucosal IgA was increased. CONCLUSIONS: These findings confirmed that supplementary amounts of vitamin A above dietary requirements enhance inflammatory responses accompanied by decreased Th1 and increased mucosal responses. However, supplementation of these sufficiently fed, non-stressed, young adult mice with vitamins C and E, selenium, or zinc had no effect on immune function. We speculate that using this model in aged, physiologically, or nutritionally stressed mice may provide outcomes more similar to those in sensitive human populations. If so, this would improve the usefulness of the model to assess, characterize, and rank effects of foods or nutrients on a range of immune functions, including Th1/Th2 polarization.
Assuntos
Ácido Ascórbico/administração & dosagem , Imunidade/efeitos dos fármacos , Selênio/administração & dosagem , Vitamina A/administração & dosagem , Vitamina E/administração & dosagem , Zinco/administração & dosagem , Animais , Dieta , Suplementos Nutricionais , Dinitroclorobenzeno/imunologia , Feminino , Hipersensibilidade Tardia , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacos , Células Th1/imunologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/análiseRESUMO
Recently, we have shown that AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses sensitization to peanut at least in part by inducing a functional shift toward CD4(+)CD25(+)Foxp3(+) T cells. Next to TCDD, numerous other AhR ligands have been described. In this study, we investigated the effect of three structurally different non-dioxin-like AhR ligands, e.g., 6-formylindolo[3,2-b]carbazole (FICZ), ß-naphthoflavone (ß-NF), and 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), on peanut sensitization. Female C57BL/6 mice were sensitized by administering peanut extract (PE) by gavage in the presence of cholera toxin. Before and during peanut sensitization, mice were treated with FICZ, ß-NF, or 6-MCDF. AhR gene transcription in duodenum and liver was investigated on day 5, even as the effect of these AhR ligands on CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes (MLNs). Mice treated with TCDD were included as a positive control. Furthermore, the murine reporter cell line H1G1.1c3 (CAFLUX) was used to investigate the possible role of metabolism of TCDD, FICZ, ß-NF, and 6-MCDF on AhR activation in vitro. TCDD, but not FICZ, ß-NF, and 6-MCDF, suppressed sensitization to peanut (measured by PE-specific IgE, IgG1, IgG2a and PE-induced interleukin (IL)-5, IL-10, IL-13, IL-17a, IL-22, and interferon-γ). In addition, FICZ, ß-NF, and 6-MCDF treatments less effectively induced AhR gene transcription (measured by gene expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1) compared with TCDD-treated mice. Furthermore, FICZ, ß-NF and 6-MCDF did not increase the percentage of CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes compared with PE-sensitized mice, in contrast to TCDD. Inhibition of metabolism in vitro increased AhR activation. Together, these data shows that TCDD, but not FICZ, ß-NF, and 6-MCDF suppresses sensitization to peanut. Differences in metabolism, AhR binding and subsequent gene transcription might explain these findings and warrant further studies to investigate the role of the AhR in food allergic responses.
Assuntos
Modelos Animais de Doenças , Hipersensibilidade a Amendoim/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Sequência de Bases , Primers do DNA , Feminino , Citometria de Fluxo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Immune-mediated drug hypersensitivity reactions are important causes of black box warnings and drug withdrawals. Despite the high demand for preclinical screening tools, no validated in vitro or in vivo models are available. In the current study, we used a previously described oral administration model using trinitrophenyl-ovalbumin (TNP-OVA) as an antigen to report immuno-adjuvating effects of the analgesic drug acetaminophen (APAP) and its nonhepatotoxic regioisomer 3'-hydroxyacetanilide (AMAP), the antibiotic ofloxacin (OFLX), the antiepileptic drug carbamazepine (CMZ), and the antidiabetic drug metformin (MET). Furthermore, APAP and AMAP were tested in a popliteal lymph node assay (PLNA) combined with TNP-OVA as reporter antigen (RA). C3H/HeOuJ mice were dosed by oral gavage with diclofenac (DF), APAP, AMAP, OFLX, MET, or CMZ. On the first exposure day, the mice received an ip injection with TNP-OVA. Fifteen days later, they were ear challenged with TNP-OVA and delayed-type hypersensitivity (DTH) responses were assessed 24 h later. One week after challenge, the ear-draining lymph node was removed and TNP-specific antibody-secreting cells were determined. DF, APAP, CMZ, and OFLX showed a significant increase in DTH responses to ear injection with TNP-OVA, whereas AMAP and MET did not. C57BL/6 mice were slightly less responsive to APAP and DF after oral gavage, and importantly both AMAP and APAP were negative in the RA-PLNA. The present work shows that the oral exposure model using RA and the RA-PLNA may serve to screen the immune-adjuvant potential of new chemical entities during preclinical drug development.
Assuntos
Adjuvantes Imunológicos/farmacologia , Hipersensibilidade a Drogas/imunologia , Ovalbumina/imunologia , Acetaminofen/farmacologia , Acetanilidas/farmacologia , Administração Oral , Animais , Formação de Anticorpos/efeitos dos fármacos , Carbamazepina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Injeções Epidurais , Ensaio Local de Linfonodo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Ofloxacino/farmacologia , Ovalbumina/farmacologiaRESUMO
Inflammatory bowel disease is characterized by chronic inflammation of the intestine and is accompanied by damage of the epithelial lining and by undesired immune responses towards enteric bacteria. It has been demonstrated that intestinal alkaline phosphatase (iAP) protects against the induction of inflammation, possibly due to dephosphorylation of lipopolysaccharide (LPS). The present study investigated the therapeutic potential of iAP in intestinal inflammation and epithelial damage. Intestinal epithelial damage was induced in C57BL/6 mice using detran sulfate sodium (DSS) and iAP was administered 4days after initial DSS exposure. Loss in body weight was significantly less in iAP-treated mice and accompanied with reduced colon damage (determined by combination of crypt loss, loss of goblet cells, oedema and infiltrations of neutrophils). Treatment with iAP was more effective in case of severe inflammation compared to situations of mild to moderate inflammation. Rectal administration of LPS into a moderate inflamed colon did not aggravate inflammation. Furthermore, soluble iAP did not lower LPS-induced nuclear factor-kappaB activation in epithelial cells in vitro but induction of cellular AP expression by butyrate resulted in decreased LPS response. In conclusion, the present study shows that oral iAP administration has beneficial effects in situations of severe intestinal epithelial damage, whereas in moderate inflammation endogenous iAP may be sufficient to counteract disease-aggravating effects of LPS. An approach including iAP treatment holds a therapeutic promise in case of severe inflammatory bowel disease.
Assuntos
Fosfatase Alcalina/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Animais , Butiratos/farmacologia , Linhagem Celular Transformada , Células Cultivadas , Quimiocinas/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Peroxidase/metabolismoRESUMO
The antitumor drug streptozotocin (STZ) is commonly used as a diabetogenic compound in animal models. At relatively low doses, STZ-induced beta cell destruction is associated with Th1-driven type 1 immune reactions, including macrophages (MPhi) and IFN-gamma-producing CD8(+) T cells. STZ induces similar Th1-dependent effects in the popliteal lymph node assay (PLNA), and because this assay allows straightforward examination of early immunostimulating processes, the PLNA was used to further examine the importance of MPhi and structural properties of STZ in relation to the induction of type 1 immune responses. Results show that elimination of MPhi with clodronate-containing liposomes prior to exposure to STZ prevents the occurrence of some (CD8(+) T cell activation, IFN-gamma production, and tissue destruction) but not all (IgG2a formation) type 1 immune responses. It appeared that stimulation of MPhi depends on the d-glucopyranose moiety of STZ, as well as on the intact reactive N-methyl-N-nitrosourea (MNU) moiety. However, the MNU moiety suffices to induce IgG2a formation. In addition, STZ-derived nitric oxide may have modulating effects on the elicitation of STZ-induced immune responses. Present results support the idea that MPhi activation is indispensable for the STZ-induced tissue destructive type 1 responses and that various STZ-induced type 1 immune responses are differently regulated.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Glucose/química , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Estreptozocina/farmacologia , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Ácido Clodrônico/metabolismo , Citocinas/metabolismo , Testes Imunológicos de Citotoxicidade , Relação Dose-Resposta a Droga , Imunoglobulina G/metabolismo , Interferon gama/metabolismo , Lipossomos/metabolismo , Linfonodos/imunologia , Macrófagos/citologia , Metilnitrosoureia/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Estreptozocina/química , Estreptozocina/imunologiaRESUMO
Some drugs have the undesired side effect of systemically stimulating the immune system, which may eventually lead to the development of drug-induced allergy or autoimmunity. Unfortunately, validated predictive screening tools to assess the immune stimulatory potential of compounds are presently unavailable. The popliteal lymph node assay (PLNA) with reporter antigens (RA) seems a valuable candidate for this purpose. The aims of the present study were 1) to provide additional mechanistic information on the very early induction phase of drug-induced type 1 (T(H)1-associated) and type 2 (T(H)2-associated) immune reactions in the PLNA and 2) to explore the use of these mechanism-based parameters to predict the immune stimulating potential of drugs. Streptozotocin (STZ), a chemotherapeutic drug, and D-Penicillamine (D-Pen, anti-rheumatic drug) were used as model compounds as they respectively induce clearly differentiated type 1 and type 2 immune responses in the PLNA. Type 1 responses were characterized by the production of high levels of IFNgamma and IL-12 from day 2 after exposure. Expression of CD40 was absent, but CD54, CD80, and CD86 were present predominantly on non-B APC, presumably macrophages. Increased percentages of activated CD8(+) T-cells and macrophages accompanied these phenomena. Type 2 responses were clearly different and were characterized by an early influx of dendritic cells (DC) and B-cells that expressed CD40, CD54, and CD86, but no CD80. First DC, but later B-cells, appeared to function as APC. In addition, the production of IL-4 was elevated. Apparently, reactivity of these type 1- and type 2-inducing drugs produce drug-specific patterns of immune stimulating factors that determine very different, time-dependent profiles of activated cells, cytokine production, and expression of co-stimulatory molecules very shortly after the initial exposure to drugs. As such, these parameters obtained with the PLNA may help to provide information about the immunological mechanisms in the early induction phase of drug-induced immune stimulation and may be useful in the development of an appropriate screening test to predict the immune stimulatory potential of drugs in a preclinical phase.
RESUMO
The use of mAbs to abrogate costimulatory interactions has attracted much attention with regard to prevention and modulation of adverse (auto)immune-like reactions. However, the role of costimulatory molecules and possible therapeutic use of Ab-treatment in drug-induced immunostimulation is poorly elucidated. In the present studies, we show that CD28/CTLA-4-CD80/CD86 costimulatory interactions differently regulate drug-induced type 1 and type 2 responses to an identical bystander Ag, TNP-OVA, in BALB/c mice using the reporter Ag popliteal lymph node assay. The antirheumatic drug D-Penicillamine, which may induce lupus-like side-effects, stimulated type 2 responses against TNP-OVA, characterized by the production of IL-4 and TNP-specific IgG1 and IgE. These responses were abrogated in CD80/CD86-deficient mice and in wild-type mice that were treated with anti-CD80 and anti-CD86, or CTLA-4-Ig. Anti-CTLA-4 intensively enhanced the D-Penicillamine-induced effects. In contrast, the type 1 response (IFN-gamma, TNF-alpha, IgG2a) to TNP-OVA induced by the diabetogen streptozotocin still developed in the absence of CD80/CD86 costimulatory signaling. In addition, it was demonstrated that coadministration of anti-CD80 and anti-CD86 mAbs slightly enhanced streptozotocin-induced type 1 responses, whereas the CTLA-4-Ig fusion protein completely abrogated this response. In conclusion, different drugs may stimulate distinct types of immune responses against an identical bystander Ag, which are completely dependent on (type 2) or independent of (type 1) the CD28/CTLA-4-CD80/CD86 pathway. Importantly, the effects of treatment with anti-CD80/CD86 mAbs and CTLA-4-Ig may be considerably different in responses induced by distinct drugs.
Assuntos
Antígenos CD/metabolismo , Efeito Espectador/imunologia , Hipersensibilidade a Drogas/imunologia , Ovalbumina/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Anticorpos/farmacologia , Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Antígeno CTLA-4 , Hipersensibilidade a Drogas/etiologia , Feminino , Imunidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Penicilamina/imunologia , Transdução de Sinais/imunologia , Estreptozocina/imunologiaRESUMO
Hexachlorobenzene (HCB) is a persistent environmental pollutant that causes adverse immune effects in man and rat. The Brown Norway (BN) rat is very susceptible to HCB-induced immunopathology and oral exposure causes inflammatory skin and lung lesions, splenomegaly, lymph node (LN) enlargement, and increased serum levels of IgE and anti-ssDNA IgM. T cells play an important role but do not account for all adverse effects induced by HCB. Macrophages are probably also important and the relationship between macrophages and T cells was further investigated. To eliminate macrophages clodronate-liposomes were used. Furthermore, a kinetic study was performed to obtain insight in the early phase of the HCB-induced immune response. Also, experiments were performed to detect specific memory T cells. Therefore, an adoptive transfer study was performed. Our results indicate that macrophages are indeed involved in HCB-induced skin lesions, lung eosinophilia, and elevation of IgM against ssDNA. Kinetics showed that both skin and lung lesions appeared early after exposure. Moreover, immune effects could not be adaptively transferred. Thus, both macrophages and T cells are involved in HCB-induced immune effects but HCB exposure does not lead to specific T cell sensitization. Presumably, HCB exposure induces macrophage activation, thereby generating adjuvant signals that polyclonally stimulate T cells. Together, these events may lead to the observed immunopathology in BN rats.
Assuntos
Hexaclorobenzeno/toxicidade , Macrófagos/imunologia , Analgésicos não Narcóticos , Animais , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Ácido Clodrônico , DNA de Cadeia Simples/imunologia , Feminino , Citometria de Fluxo , Imunoglobulina M/análise , Imunoterapia Adotiva , Pulmão/patologia , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Contagem de Linfócitos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos BN , Pele/patologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Aumento de Peso/efeitos dos fármacosRESUMO
Hexachlorobenzene (HCB) is an environmental pollutant that causes autoimmune-like effects in humans and rats. It is not completely clear whether T cells are involved and, if so, how they are stimulated after oral exposure to HCB. HCB as a rather inert chemical is not likely to bind covalently to macromolecules. The oxidative metabolite of HCB, tetrachlorobenzoquinone (TCBQ), which is in a redox equilibrium with tetrachlorohydroquinone (TCHQ), can bind to macromolecules, hence may form hapten-carrier complexes in vivo. We have assessed in the reporter antigen-popliteal lymph node assay whether HCB or TCHQ and TCBQ are able to induce a 2,4,6-trinitrophenyl (TNP) specific IgG1 response to the T cell-independent antigen TNP-Ficoll, which is indicative of neoantigen specific T cell help. To this end, these compounds and silica were injected into the footpad of Balb/c mice. Silica was included as an inert model compound, which causes autoimmune-like effects by activating macrophages. Seven days later, cell number and TNP specific antibody-secreting cells (ASC) in the popliteal lymph node (PLN) were determined. Furthermore, a secondary PLNA was performed to find out if TCHQ was capable of eliciting a memory response. Silica, TCHQ, and TCBQ, but not HCB, increased PLN cellularity and the number of IgM-producing ASC by ELISPOT. Both oxidative metabolites were able to induce the formation of germinal centers as assessed by immunohistochemistry and an IgG1 response to TNP-Ficoll. In the secondary PLNA, only mice primed with TCHQ and challenged with TCHQ together with TNP-Ficoll showed a significant increase in TNP specific IgG1 ASC. Present data show that TCHQ and TCBQ are capable of inducing neoantigen specific T cell help and that TCHQ can induce a compound specific memory response.
Assuntos
Adjuvantes Imunológicos/toxicidade , Autoimunidade/efeitos dos fármacos , Cloranila/toxicidade , Fungicidas Industriais/toxicidade , Hexaclorobenzeno/metabolismo , Linfonodos/efeitos dos fármacos , Animais , Formação de Anticorpos/efeitos dos fármacos , Autoimunidade/imunologia , Contagem de Células , Feminino , Citometria de Fluxo , Hidroquinonas/toxicidade , Imuno-Histoquímica , Ensaio Local de Linfonodo , Linfonodos/imunologia , Linfonodos/patologia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We have tested the effect of dietary fatty acids on aspects of innate and specific adaptive T helper (Th) 1- and Th2-driven immune responses in a murine sensitisation model using dinitrochlorobenzene as sensitiser. Six groups of fifteen BALB/c mice were fed diets containing 30 % fat (by energy) for 8 weeks. Diets were rich in saturated fatty acids, n-6 polyunsaturated fatty acid (PUFA), or n-3 PUFA, each at a sufficient (11, 35 and 68 mg/kg) and a supplemented vitamin E level (1028, 1031 and 1030 mg/kg respectively). Feeding n-6 PUFA marginally decreased % phagocytosing cells at the low vitamin E level, but had no other effects on immune function. The n-3 PUFA diets decreased production of prostaglandin E2 while increasing oxidative burst and tumour necrosis factor alpha production. In addition adaptive Th1-driven responses (immunoglobulin, Ig)G2a, IgG2b, interferon-gamma:interleukin 4) were decreased, whereas Th2-driven and mucosal immune responses were increased (IgE) or unaffected (IgG1, IgA). Combination with high levels of alpha-tocopherol did not affect the reduced prostaglandin E2 production, augmented the increase of tumour necrosis factor alpha production and tended to ameliorate the selective suppressive effects of n-3 PUFA on certain Th1-driven effects (interferon-gamma:interleukin 4 ratio and IgG2a levels). We conclude that the sensitisation model appears useful for application in nutrition research. It allows a broad assessment of the effects of dietary intervention on various aspects of immune responsiveness, and as such provides a valuable model to assess, characterise and rank effects of foods and/or nutrients on a range of immune functions, including Th1-Th2 polarisation.
Assuntos
Gorduras na Dieta/farmacologia , Imunidade Celular/efeitos dos fármacos , Modelos Animais , Animais , Citocinas/biossíntese , Gorduras Insaturadas na Dieta/farmacologia , Dinitroclorobenzeno , Dinoprostona/biossíntese , Ácidos Graxos Ômega-3 , Ácidos Graxos Insaturados/farmacologia , Feminino , Imunidade nas Mucosas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Fosfolipídeos/metabolismo , Explosão Respiratória/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Triglicerídeos/farmacologia , Vitamina E/sangue , Vitamina E/farmacologia , Aumento de Peso/efeitos dos fármacosRESUMO
The thymotoxic organotin compounds di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) are known to induce apoptosis in vitro in rat thymocytes. They also affect macromolecular synthesis, inhibiting DNA synthesis and increasing RNA synthesis. Since these RNA molecules, likely to be involved in the initiation of the apoptotic process, have not been identified yet, the purpose of this research was to characterize by a cDNA macroarray the expression of genes involved in DBTC-induced apoptosis. We found that nur77 was rapidly transcripted in vitro following exposure of freshly isolated rat thymocytes to 3 microM DBTC. nur77 induction has also been observed in vivo after treatment of rats with apoptotic doses (60 mg/kg body wt) of DBTC. The products of nur77 are known to be involved in the apoptotic process, as nur77 is a transcription factor expressed in response to T-cell receptor-mediated apoptosis in immature T cells. Antisense oligonucleotide inhibition of nur77 expression prevented apoptosis induced by DBTC, supporting a role for nur77 in organotin-induced apoptotic cell death.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Orgânicos de Estanho/toxicidade , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Apoptose/genética , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Perfilação da Expressão Gênica , Hibridização In Situ , Masculino , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos Antissenso/farmacologia , RNA/biossíntese , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/efeitos dos fármacos , Timo/patologiaRESUMO
CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.