Expression of a 130-kDa mesothelial and ciliated cell Ag (MCp130) in normal and developing human and rat lung and its role as a diagnostic marker for mesotheliomas and tumors of the female reproductive system.

BACKGROUND: Proteins in human lung lavage were analyzed to identify cell-specific markers for potential use in the study of the biology and pathology of pulmonary cells. EXPERIMENTAL DESIGN: Proteins associated with pulmonary surfactant were used to raise mAb. An Ab to a 130-kDa protein (MCp130) was reactive with ciliated and mesothelial cells. Expression of this Ag in normal organs, developing lung, and tumors was investigated. RESULTS: By Western blotting, the Ab stained a protein of about 130 kDa. In formalin-fixed, paraffin-embedded tissues from adult human and rat organs, the Ab specifically stained the luminal/apical surfaces of pulmonary and nonpulmonary ciliated and mesothelial cells. Staining of fetal airway cells was independent of ciliation. Airway cell staining was detectable in human fetal lungs at 12 weeks of gestation and at Day 18 of gestation in fetal rat. The Ab reacted with human and rat fetal mesothelial cells at the gestational ages of 15 weeks and 17 days, respectively. It also stained ciliated cells in endosalpinx and endometrium. Human epithelial mesotheliomas and ovarian and endometrial carcinomas stained selectively, whereas other pulmonary tumors and tumors of other organs did not react with the Ab. CONCLUSIONS: This 130-kDa mesothelial and ciliated cell plasma membrane protein appears in developing lung at an earlier age than secretory proteins. The marker is of potential use in the study of development of the different cell lineages in the lung and female reproductive tract. The Ab is expected to be useful in the diagnosis of epithelial mesotheliomas and ovarian/endometrial carcinomas, because it selectively stains these tumors and is reactive with formalin-fixed, paraffin-embedded tissues.

Immunohistochemistry of a human type I pneumocyte-associated protein in lung.

A mouse monoclonal antibody to a human lung lavage protein was raised using proteins, with the potential ability to bind surfactant, as the immunogen. The proteins were isolated from cadaver lung lavage. The antibody was tested for its reactivity with lung and other organs. It reacted with type I pneumocytes and some of the nonciliated cells in the surface epithelium of distal bronchioles. Staining was also seen in the cells surrounding the glandular structures, superficial keratinocytes of the skin, endothelium, and nerve sheath cells. With the exception of bronchiolar cells, the stained cells have a squamous morphology, and this protein may serve as a marker or determinant of this characteristic of cells. In pathologic lungs some of the cells in air spaces with "bronchiolarization" of the epithelium exhibited staining for the protein. It could not be ascertained whether the stained cuboidal cells were reactive type II pneumocytes or distal bronchiolar cells. The intraalveolar material in pulmonary alveolar proteinosis did not show remarkable staining for the protein. Even though the protein is not unique to type I pneumocytes, it may serve as a marker for these cells in the study of their development and biology.

Identification, isolation, and partial characterization of a 7.5-kDa surfactant-associated protein.

Human surfactant was analyzed for proteins associated with the lipids. Surfactant was isolated from lung lavage by the salt gradient centrifugation method, and the soluble proteins binding to the lipids were recovered by extraction with a low pH buffer. Antiserum to this preparation reacted with surfactant apoprotein A and a 7.5-kDa protein. The 7.5-kDa protein was isolated from reduced and alkylated lung lavage pellet by chromatography. The N-terminal amino acid sequence of the protein indicates that it is a novel protein.

Clara cell 10 kDa protein (CC10): comparison of structure and function to uteroglobin.

The cellular localization, functional activities and structures of rat and human Clara cell 10 kDa proteins (CC10) are compared to rabbit uteroglobin. CC10 is present exclusively in the non-ciliated cells of the surface epithelium of the pulmonary airways, whereas uteroglobin is reported to be present in the lung and reproductive organs. There is about 55% identity between the amino acid sequences of rat CC10 and either rabbit uteroglobin or human CC10. The latter two have 61% identity. Using the known structure of uteroglobin as the model, correlations between the structure and function for this group of proteins are made. Substitution of the residues for the rat and human CC10 into the structure of uteroglobin suggests that these proteins may be members of a structurally homologous family. Some of the functional differences may be due to distortion of the hydrophobic pocket in the dimeric protein and a surface hypervariability located on one contiguous helix and beta turn. Rat CC10 and rabbit uteroglobin both, nearly equally, inhibit papain and bind progesterone. Human CC10 does not inhibit papain and has markedly lower progesterone binding (4.6% of rabbit uteroglobin). Antiinflammatory activity of synthetic peptides corresponding to a homologous sequence region of uteroglobin and the two Clara cell proteins was tested. The region chosen has sequence similarity to lipocortin I. The peptides not only failed to inhibit carrageenan-induced foot pad swelling but exacerbated it. All three proteins inhibit pancreatic phospholipase A2. The phospholipase A2 inhibitory effect of CC10 may be important in regulating the inflammatory responses in the lung.

Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoclonal antibodies.

Using a monoclonal antibody and affinity-purified polyclonal antiserum against a 10 KD protein isolated from rat pulmonary lavage, we have localized the protein within Clara cells by a post-embedment protein A-gold technique. The gold particles were localized over the secretory granules of rat Clara cells. Ultrastructural immunolocalization was abolished when the primary antibodies were previously absorbed with purified 10 KD protein. Other pulmonary cells, including type II pneumocytes and ciliated cells, were negative with this technique. These results demonstrate the presence of the 10 KD protein in the secretory granules of the Clara cell and support the concept that this protein constitutes a specific and unique secretory product of Clara cells.

A quantitative assay for a Clara cell-specific protein and its application in the study of development of pulmonary airways in the rat.

Rat lung lavage contains a 10 kDa protein that has been shown by immunocytochemistry to be specific for Clara cells. An inhibition enzyme-linked immunosorbent assay was established for this protein using rabbit antibody to the 10 kDa Clara cell protein. The assay has a sensitivity of about 3.0 ng/ml and a working range of about 5 to 50 ng/ml. Quantitation of the 10 kDa protein in amniotic fluid revealed an increase of about 4-fold at day 20 of gestation. The 10 kDa protein content of lung homogenate increased steadily from day 18 of gestation to 1 wk after birth, after which a decline was observed. Nearly 60-fold increase in the concentration of the 10 kDa Clara cell protein in lungs was noted from day 18 of gestation to birth and a further about 7-fold increase was noted from the day of birth to 1 wk of age. A progressive increase in the 10 kDa protein, with increasing age, was also noted on immunoblot analysis of lung homogenates. As judged from the immunoblots of lung homogenates, stained with rabbit antirat 10 kDa protein antiserum, the content of an antigenically similar 200 kDa Clara cell protein was negligible. The quantitative results for 10 kDa Clara cell protein parallel the results of immunocytochemistry and quantitation of the volume density of Clara cell granules indicating that quantitation for the 10 kDa protein could be used to monitor the development of Clara cells and that of the pulmonary airways.

Secretory proteins of the lung in rodents: immunocytochemistry.

The reactivity of rabbit antisera to rat lung secretory proteins with other rodent species was evaluated by immunocytochemistry. Rabbit anti-rat surfactant apoprotein antiserum reacts with the cytoplasm of rat, mouse, and hamster type II pneumocytes and is specific for these cells. Rabbit antiserum to rat Clara cell secretory proteins stains rat, mouse, and hamster Clara cells. Rabbit antisera specific to the two antigenic types of rat Clara cell antigens were also both reactive with rat, mouse, and hamster Clara cells. An antiserum to the non-serum proteins of hamster lung lavage was also prepared and shown to be specifically reactive with hamster Clara cells. The availability of specific reagents for secretory proteins of rodent lungs is expected to facilitate studies of the respective cell types in various pathologic states.