Lung dendritic cells are stimulated by ultrafine particles and play a key role in particle adjuvant activity.

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.

Differential requirement for CD28/CTLA-4-CD80/CD86 interactions in drug-induced type 1 and type 2 immune responses to trinitrophenyl-ovalbumin.

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.

Ultrafine carbon black particles cause early airway inflammation and have adjuvant activity in a mouse allergic airway disease model.

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.

The reactive D-glucopyranose moiety of streptozotocin is responsible for activation of macrophages and subsequent stimulation of CD8+ T cells.

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.

Development of an oral exposure mouse model to predict drug-induced hypersensitivity reactions by using reporter antigens.

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.

Effects of supplementation with vitamins A, C, and E, selenium, and zinc on immune function in a murine sensitization model.

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.

Effects of dietary lipids on immune function in a murine sensitisation model.

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.

Organotin-induced apoptosis occurs in small CD4(+)CD8(+) thymocytes and is accompanied by an increase in RNA synthesis.

The organotin compounds di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) induce thymus atrophy in rats. At low doses they inhibit immature thymocyte proliferation, whereas at higher doses in particular TBTC induces apoptotic cell death. In vitro, a similar concentration-effect relationship was observed, i.e. low concentrations inhibit DNA and protein synthesis and higher concentrations induce apoptosis. The mechanism of apoptosis by organotins has been partly investigated, but their capacity to inhibit protein synthesis seems to contradict with the idea that macromolecular synthesis is required for organotin-induced apoptosis. Therefore, we aimed to evaluate the relation between apoptosis and the synthesis of RNA and proteins, with a focus on the apoptosis-sensitive thymocyte subset. Results showed that DBTC increases RNA synthesis in particular in the subset of small CD4(+)CD8(+) thymocytes, which normally shows a high incidence of DNA fragmentation. Moreover, the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide protected cells from apoptosis by DBTC or TBTC. Although organotin compounds increase synthesis of the heat shock protein HSC73/HSP72, heat shock treatment did not initiate apoptosis in thymocytes, neither antagonized organotin-induced apoptosis. This indicates that synthesis of heat shock proteins is not related to organotin-induced increase of RNA synthesis, and that other RNA-molecules are probably involved.

Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin.

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.