RESUMO
BACKGROUND: The adjuvant activity of air pollution particles on allergic airway sensitization is well known, but the cellular mechanisms underlying this adjuvant potential are not clear. OBJECTIVE: We sough to study the role of dendritic cells and the costimulatory molecules CD80 and CD86 in the adjuvant activity of ultrafine carbon black particles (CBP). METHODS: The proliferation of CFSE-labeled DO11.10 CD4 cells was studied after intranasal exposure to particles and ovalbumin (OVA). Next the frequency of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells and their expression of CD80 and CD86 were studied in the peribronchial lymph nodes (PBLNs). The expression of costimulatory molecules was also studied on bone marrow-derived mDCs after exposure to CBPs in vitro, and the importance of costimulation in CBP adjuvant activity was assessed by using CD80/CD86-deficient mice or cytotoxic T lymphocyte-associated antigen 4 (CTLA4)-Ig in vivo. RESULTS: Our data show that CBPs plus OVA caused proliferation of DO11.10 CD4 cells and high levels of cytokine production in the PBLNs. Furthermore, the combined CBP plus OVA exposure increased the number of mDCs and expression of costimulatory molecules in the PBLNs. In addition, CBPs upregulated the expression of CD80/CD86 molecules on dendritic cells in vitro, which are necessary for the particle adjuvant effects in vivo. CONCLUSION: Together this study shows the importance of dendritic cells and costimulation in particle adjuvant activity. Furthermore, we show for the first time that CBPs can also directly induce maturation of dendritic cells.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células Dendríticas/efeitos dos fármacos , Material Particulado/imunologia , Hipersensibilidade Respiratória/induzido quimicamente , Fuligem/farmacologia , Transferência Adotiva , Animais , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Fluoresceínas , Pulmão/citologia , Pulmão/imunologia , Linfonodos/imunologia , Camundongos , Ovalbumina/imunologia , Tamanho da Partícula , Hipersensibilidade Respiratória/imunologia , SuccinimidasRESUMO
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
The capability of certain drugs to cause immune-mediated drug hypersensitivity reactions in susceptible individuals has initiated a search for pre-clinical screening tools to identify immunosensitizing drugs. Since most drugs are taken orally, hazard assessment of their immunosensitizing potential should include oral exposure models. In this study, the predictive value of the reporter antigen (RA) approach was investigated in combination with oral or intraperitoneal (ip) exposure to a selection of allergenic drugs, i.e., D-penicillamine (D-Pen), Diclofenac (DF), or Nevirapine (Nevi). The RA trinitrophenyl-Ovalbumin (TNP-OVA) was used to assess the capacity of the drugs to stimulate systemic immune responses to a bystander antigen, whereas the RA TNP-Ficoll was used to indicate whether the drugs were able to induce specific anamnestic T-cell responses. TNP-OVA was injected (ip) in C3H/HeOuJ mice that were subsequently exposed (orally or ip) to one of the drugs via different exposure protocols. All three model drugs used resulted in delayed type hypersensitivity reactions to TNP-OVA after ip and oral exposure. In addition, TNP-specific serum antibody levels were increased after ip exposure to Nevi, and after both oral and ip exposure to D-Pen and DF. These data indicate that the present drugs are able to stimulate immune responses to bystander antigens. Responses to TNP-Ficoll were measured in the popliteal lymph node of BALB/c mice three weeks after they received a single oral dose of D-Pen or DF. Results of this approach show that orally pre-treated mice responded with enhanced responses (TNP-specific IgG1 and IFN-gamma production) to sub-optimal doses of D-Pen or DF in a drug-specific manner. Data with TNP-Ficoll indicate that these drugs stimulate systemic formation of specific T cells. Together, the RA-approach allows assessment of systemic sensitization upon oral and/or ip exposure to the selected drugs. To further evaluate the utility of these models, more drugs, including non-allergenic drugs and those that require metabolic conversion to become allergenic need to be studied in the present models.
Assuntos
Alérgenos/toxicidade , Reações Antígeno-Anticorpo/efeitos dos fármacos , Antígenos/farmacologia , Modelos Animais de Doenças , Hipersensibilidade a Drogas/etiologia , Administração Oral , Alérgenos/administração & dosagem , Alérgenos/classificação , Animais , Hipersensibilidade a Drogas/patologia , Feminino , Ficoll , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Injeções Intraperitoneais , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Ovalbumina/imunologia , Valor Preditivo dos TestesRESUMO
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
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
Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.
Assuntos
Anafilaxia/imunologia , Fenômenos do Sistema Imunitário/imunologia , Hipersensibilidade a Amendoim/imunologia , Animais , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Plaquetas/metabolismo , Degranulação Celular/imunologia , Orelha/patologia , Imunidade Humoral/imunologia , Imunização , Leucócitos/imunologia , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos , Mucosa/imunologia , Mucosa/patologia , Extratos Vegetais/efeitos adversos , Extratos Vegetais/imunologia , Fator de Ativação de Plaquetas/metabolismo , Linfócitos T/imunologiaRESUMO
IgE-mediated food allergies are an important cause of life-threatening hypersensitivity reactions. Orally administered peanut antigens mixed with the mucosal adjuvant cholera toxin (CT) induce a strong peanut extract (PE)-specific serum IgE response that is correlated with T-helper type 1 (Th1) and type 2 (Th2)-like T-cell responses. This study was conducted to determine if respiratory enteric orphan virus (reovirus), a non-pathogenic virus that induces robust Th1-mediated mucosal and systemic responses could modulate induction of PE-specific allergic responses when co-administered with PE. Young mice were orally exposed to PE mixed with CT, reovirus, or both CT and reovirus. As expected, CT promoted PE-specific serum IgE, IgG1, and IgG2a and intestinal IgA production as well as splenic Th1- and Th2-associated cytokine recall responses. Reovirus did not alter PE-specific serum IgE and IgG1 levels, but substantially increased the PE-specific IgG2a response when co-administered with PE with or without CT. Additionally, reovirus significantly decreased the percentage of the Peyer's patch CD8+ T-cells and Foxp3+CD4+ T-regulatory cells when co-administered with PE. These results demonstrate that an acute mucosal reovirus infection and subsequent Th1 immune response is capable of modulating the Th1/Th2 controlled humoral response to PE. The reovirus-mediated increase in the PE-specific IgG2a antibody response may have therapeutic implications as increased levels of non-allergenic PE-specific IgG2a could block PE antigens from binding to IgE-sensitized mast cells.
Assuntos
Arachis/imunologia , Imunoglobulina G/imunologia , Hipersensibilidade a Amendoim/imunologia , Infecções por Reoviridae/imunologia , Animais , Arachis/química , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Linhagem Celular , Células Cultivadas , Toxina da Cólera/imunologia , Toxina da Cólera/farmacologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina A/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Intestino Delgado/virologia , Camundongos , Camundongos Endogâmicos C3H , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/virologia , Extratos Vegetais/imunologia , Extratos Vegetais/farmacologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Técnicas de Cultura de TecidosRESUMO
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
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.