Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS).

Occupational asthma can be induced by a number of chemicals at the workplace. Risk assessment of potential sensitizers is mostly performed in animal experiments. With increasing public demand for alternative methods, human precision-cut lung slices (PCLS) have been developed as an ex vivo model. Human PCLS were exposed to increasing concentrations of 20 industrial chemicals including 4 respiratory allergens, 11 contact allergens, and 5 non-sensitizing irritants. Local respiratory irritation was characterized and expressed as 75% (EC25) and 50% (EC50) cell viability with respect to controls. Dose-response curves of all chemicals except for phenol were generated. Local respiratory inflammation was quantified by measuring the production of cytokines and chemokines. TNF-α and IL-1α were increased significantly in human PCLS after exposure to the respiratory sensitizers trimellitic anhydride (TMA) and ammonium hexachloroplatinate (HClPt) at subtoxic concentrations, while contact sensitizers and non-sensitizing irritants failed to induce the release of these cytokines to the same extent. Interestingly, significant increases in T(H)1/T(H)2 cytokines could be detected only after exposure to HClPt at a subtoxic concentration. In conclusion, allergen-induced cytokines were observed but not considered as biomarkers for the differentiation between respiratory and contact sensitizers. Our preliminary results show an ex vivo model which might be used for prediction of chemical-induced toxicity, but is due to its complex three-dimensional structure not applicable for a simple screening of functional and behavior changes of certain cell populations such as dendritic cells and T-cells in response to allergens.

The neuropeptide calcitonin gene-related peptide affects allergic airway inflammation by modulating dendritic cell function.

BACKGROUND: The neuropeptide calcitonin gene-related peptide (CGRP) is released in the lung by sensory nerves during allergic airway responses. Pulmonary dendritic cells (DC) orchestrating the allergic inflammation could be affected by CGRP. OBJECTIVE: To determine the immunomodulatory effects of CGRP on DC function and its impact on the induction of allergic airway inflammation. METHODS: CGRP receptor expression on lung DC was determined by RT-PCR and immunofluorescence staining. The functional consequences of CGRP receptor triggering were evaluated in vitro using bone marrow-derived DC. DC maturation and the induction of ovalbumin (OVA)-specific T cell responses were analysed by flow cytometry. The in vivo relevance of the observed DC modulation was assessed in a DC-transfer model of experimental asthma. Mice were sensitized by an intrapharyngeal transfer of OVA-pulsed DC and challenged with OVA aerosol. The impact of CGRP pretreatment of DC on airway inflammation was characterized by analysing differential cell counts and cytokines in bronchoalveolar lavage fluid (BALF), lung histology and cytokine responses in mediastinal lymph nodes. RESULTS: RT-PCR, immunofluorescence and cAMP assay demonstrated the expression of functionally active CGRP receptors in lung DC. RT-PCR revealed a transcriptional CGRP receptor down-regulation during airway inflammation. CGRP specifically inhibited the maturation of in vitro generated DC. Maturation was restored by blocking with the specific antagonist CGRP(8-37) . Consequently, CGRP-pretreated DC reduced the activation and proliferation of antigen-specific T cells and induced increased the numbers of T regulatory cells. The transfer of CGRP-pretreated DC diminished allergic airway inflammation in vivo, shown by reduced eosinophil numbers and increased levels of IL-10 in BALF. CONCLUSIONS AND CLINICAL RELEVANCE: CGRP inhibits DC maturation and allergen-specific T cell responses, which affects the outcome of the allergic airway inflammation in vivo. This suggests an additional mechanism by which nerve-derived mediators interfere with local immune responses. Thus, CGRP as an anti-inflammatory mediator could represent a new therapeutic tool in asthma therapy.

Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices.

Prediction of lung innate immune responses is critical for developing new drugs. Well-established immune modulators like lipopolysaccharides (LPS) can elicit a wide range of immunological effects. They are involved in acute lung diseases such as infections or chronic airway diseases such as COPD. LPS has a strong adjuvant activity, but its pyrogenicity has precluded therapeutic use. The bacterial lipopeptide MALP-2 and its synthetic derivative BPPcysMPEG are better tolerated. We have compared the effects of LPS and BPPcysMPEG on the innate immune response in human precision-cut lung slices. Cytokine responses were quantified by ELISA, Luminex, and Meso Scale Discovery technology. The initial response to LPS and BPPcysMPEG was marked by coordinated and significant release of the mediators IL-1ß, MIP-1ß, and IL-10 in viable PCLS. Stimulation of lung tissue with BPPcysMPEG, however, induced a differential response. While LPS upregulated IFN-γ, BPPcysMPEG did not. This traces back to their signaling pathways via TLR4 and TLR2/6. The calculated exposure doses selected for LPS covered ranges occurring in clinical studies with human beings. Correlation of obtained data with data from human BAL fluid after segmental provocation with endotoxin showed highly comparable effects, resulting in a coefficient of correlation >0.9. Furthermore, we were interested in modulating the response to LPS. Using dexamethasone as an immunosuppressive drug for anti-inflammatory therapy, we found a significant reduction of GM-CSF, IL-1ß, and IFN-γ. The PCLS-model offers the unique opportunity to test the efficacy and toxicity of biological agents intended for use by inhalation in a complex setting in humans.