Analysis of mechanisms of neutrophil accumulation in the lung ; research utilizing LTB_4 receptor-expressed CHO cells.

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1. Determination of optimal condition of labeling LTB_4 receptor-expressed CHO cells with fluorescent probes.CHO cells could be adequately labeled for visualization with fluorescent microscopy with 4 micro molar CMFDA or 2 micro molar CMTMR.LTB_4 receptor-expressed CHO cells labeled with this conditions showed the same chemotaxis against LTB_4 as unlabeled cells in chemotaxis assay using chemotaxis chamber.2. Evaluation of neutrophil accumulation in mice lung with intratracheal administration of various agents and its reproducibility.As models of neutrophil accumulation in mice lung, we administered LTB_4, bleomycin, and silica intratracheally. After 4 hours of LTB_4 administration, neutrophilia over 10% in bronchoalveolar lavage fluid (BALF) cells were observed in only about 20% of examined mice, and after 24 hours of bleomycin administration, neutrophilia over 50% were observed in only about 50% of examined mice, suggesting that the reproducibility of neutrophil accumulation in mice … More lung by these methods was not adequate. In case of administration of silica, when silica was recovered in BALF, there were consistent marked neutrophilia (40-80%) in BALF cells 24 hours after silica administration, but, when silica was not recovered in BALF, neutrophilia were not observed. This findings indicated that expectoration and inaccessibility to lung parenchyme of intratracheally administered agents were the main cause of inadequate reproducibility of the procedures. Silica was a suitable agent for intratracheal administration in that its deposition into lung parenchyme could be verified by BALF analysis.3. in vivo Chemotaxis assay in mice model of intratracheal administration of silica using CMFDA-labeled LTB_4 receptor-expressed CHO cells.CMFDA-labeled LTB_4 receptor-expressed CHO cells and CMTMR-labeled control CHO cells were injected to mice intravenously via tail veins after 20 hours of intratracheal administration of silica. However, in BALF obtained 4 hours later, neither CMTMR nor CMFDA-labeled cells could not be recovered. Because there was a possibility that LTB_4 receptor-expressed CHO cells was not present freely in alveolar space but attached to pulmonary capillary endothelium or alveolar epithelium, we made histological specimens of lung tissues and examined fluorescent probe-labeled cells, but, at present, we could not obtain any definite results. Less