Interleukin-17A mRNA and protein expression within cells from the human bronchoalveolar space after exposure to organic dust

BACKGROUND: In mice, the cytokine interleukin (IL)-17A causes a local accumulation of neutrophils within the bronchoalveolar space. IL-17A may thereby also contribute to an increased local proteolytic burden. In the current study, we determined whether mRNA for IL-17A is elevated and protein expression of IL-17A occurs locally in inflammatory cells within the human bronchoalveolar space during severe inflammation caused by organic dust. We also assessed the expression of the elastinolytic protease MMP-9 in this airway compartment. METHODS: Six healthy, non-smoking human volunteers were exposed to organic dust in a swine confinement, a potent stimulus of neutrophil accumulation within the human bronchoalveolar space. Bronchoalveolar lavage (BAL) fluid was harvested 2 weeks before and 24 hours after the exposure and total and differential counts were conducted for inflammatory BAL cells. Messenger RNA for IL-17A was measured using reverse transcript polymerase chain reaction-enzyme linked immunoassay (RT-PCR-ELISA). Intracellular immunoreactivity (IR) for IL-17A and MMP-9, respectively, was determined in BAL cells. RESULTS: The exposure to organic dust caused more than a forty-fold increase of mRNA for IL-17A in BAL cells. IL-17A immunoreactivity was detected mainly in BAL lymphocytes, and the number of these IL-17A expressing lymphocytes displayed an eight-fold increase, even though not statistically significant. The increase in IL-17A mRNA was associated with a substantial increase of the number of BAL neutrophils expressing MMP-9 immunoreactivity. CONCLUSION: Exposure to organic dust increases local IL-17A mRNA and because there is intracellular expression in BAL lymphocytes, this suggests that IL-17A protein can originate from lymphocytes within the human bronchoalveolar space. The fact that the increased IL-17A mRNA is associated with an increased number of MMP-9-expressing neutrophils is compatible with IL-17A increasing the local proteolytic burden through its neutrophil-accumulating effect

Lanthanide-based time-resolved luminescence immunoassays

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized