The goal of this study was to determine the temporal and spatial sequence of events that accompany lung injury and repair after parenteral administration of the Clara cell-specific cytotoxicant, naphthalene. Changes in airway epithelial cells were evaluated by measuring alterations in the expression of markers for differentiated Clara cells (CYPIIF and Clara cell 10-kDa secretory protein, CC10), distal airway/alveolar type II cells (surfactant protein B; SP-B) and for cycling/proliferating cells (cyclin dependent kinase 1;CDK1). Naphthalene-induced Clara cell cytotoxicity resulted in the exfoliation of epithelial cells containing CC10 protein. This was accompanied by a dramatic reduction in the abundance of mRNA for CC10 and CYPIIF. Large numbers of CDK1 mRNA-positive cells were identified in and around bronchioles and terminal bronchioles 48 h after treatment. This cellular proliferation resulted in the population of airways by immature epithelial cells lacking normal levels of CC10 mRNA but overexpressing SP-B mRNA. Seventy-two hours after naphthalene treatment a reduction in CDK1 mRNA-positive cells was noted within bronchioles and terminal bronchioles at all locations, with the exception of airway bifurcations. At airway bifurcations CDK1 mRNA appeared to be more abundant at the 72-h time point than at 48 h. Comparison of these sections with serial sections probed for CC10 mRNA demonstrated a correlation between the expression of CDK1 and CC10 mRNA at bifurcations. Temporal increases in the abundance of CC10 mRNA observed at later time points were largely accounted for by the processive maturation of newly repopulated cells neighboring bifurcations in bronchioles. These studies identify spatially distinct populations of cells that act in concert to repopulate naphthalene-injured airways and support the notion that branch point cells play an important role in the maturation of newly regenerated airway epithelial cells after acute injury.

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Human lung lavage proteins were fractionated by centrifuganon and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a p1 of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, 5crme, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara

Clara cells are nonciliated, nonmucous, secretory cells of the pulmonary airways. These cells are known to secrete a variety of proteins, including Clara cell 10‐kDa protein/uteroglobin. This protein consists of a homodimer of 70–77 amino acid polypeptides arranged in antiparallel fashion. In vitro testing suggests that the protein suppresses inflammation. The physiologic role of the protein remains to be determined.

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Pulmonary surfactant is a lipid-protein complex involved in maintaining alveolar stability. SP-A is the major surfactant-associated protein of 26 to 38 kD. A human SP-A gene (SP-A I) and two distinct SP-A cDNAs, MPSAP 1A and MPSAP 6A, have been reported previously. We have isolated and characterized a second human SP-A gene (SP-A II), which appears to code for the mRNA corresponding to the previously described MPSAP-1A cDNA. Both genes consist of five exons, a consensus recognition sequence for initiation, TATAAA, and a polyadenylation signal sequence. Significant divergence in the two genes is observed throughout. The divergence is highest in the upstream region, intron I, exon III, and noncoding portion of exon V. The coding regions of all other exons and the introns show much lower divergence. Transcripts from both genes were found in adult human lung, using gene-specific oligonucleotide probes in Northern blot analysis.

Developmental expression of marker genes representative of different mature cell types can be used to study differentiation of cell lineages. We used immunohistochemistry to study expression in developing mouse lung of calcitonin gene-related peptide (CGRP), Clara cell 10-KD protein (CC10), and surfactant protein-A (SP-A), markers that are differentially expressed in neuroendocrine cells, Clara cells, and Type II alveolar cells. Two distinct developmental phases were revealed. The earlier phase (embryonic days 13-15; E13-E15) was characterized by CGRP, CC10, and SP-A immunostaining in all epithelial cells of the distal airways, with the three patterns being virtually identical in adjacent sections. The later phase (E16-E18) was characterized by emergence of staining of the differentiated cell types. These expression patterns were recapitulated in serumless organ culture, demonstrating that information necessary to generate both phases of gene expression is present within the lung analage by E11. We conclude that CGRP, CC10, and SP-A are co-expressed in most or all cells of the distal lung epithelium at E13-E15 and later become restricted to different cell lineages. This transient expression in progenitor cells of gene products characteristic of diverse differentiated cell types may reflect an underlying mechanism of gene regulation.