Clara cell cultures from the mouse and their reaction to bronchiolar toxins.

Abstract

The major aim of this study was to determine if small numbers of freshly isolated mouse Clara cells could be used to rapidly screen the toxic effects of a number of diverse pulmonary toxins. A short-term (20 hr) culture of functionally competent (nitotetrazolium reductase positive) Clara cells was developed. In this culture the Clara cells were allowed to attach to an extracellular matrix in 96-well multiwell plates containing a culture medium of DCCM 1 and Ultroser G (0.4%). Pulmonary toxins (a total of 26 agents with concentrations ranging from 10(-7) M to 10(-3) M) were examined for their ability to reduce the attachment efficiency of functionally competent Clara cells and TD50 values (the amount of toxin required to reduce normal attachment efficiency by 50%) were calculated. With the possible exception of some halogenated hydrocarbons, the simple toxicity test in vitro correlated well with the known effects of the bronchiolar necrotic agents in vivo. For 13 compounds studied there was a direct correlation between TD50 values in vitro and LD50 values (mostly oral) in rodents in vivo, the correlation coefficient of the regression line being 0.783.

Clara cell adhesion and migration to extracellular matrix

“…Clara cells preferentially adhered to Fn (73 +/- 21) and Col I (46 +/- 11), but adhesion to Lm 332 (16 +/- 6), Col IV (16 +/- 5), Lm 511 (7 +/- 3) and Lm 111 (6 +/- 7) was not significantly above uncoated controls. These findings are consistent with prior reports that Clara cell adhesion was enhanced by Fn or Col I coating of substratum [ 31 ]. These data suggest that freshly isolated Clara cells preferentially bind to components of the provisional matrix (Fn, Col I) compared with basement membrane components (Col IV and laminins).…”

“…Similar to prior publications of Clara cell isolation techniques the primary contaminating cell type was ciliated (~20–30%) (Fig. 1E , white arrows) [ 25 , 31 ]. Likely this is due to the clumping of isolated cells that contain some ciliated cells still adherent to Clara cells.…”

“…Clara cells or trypsinized C22 cells (2 × 10 6 cells in 50 μl) were placed in the upper well of modified Boyden chambers. Clara cells require 2% serum for adhesion [ 31 ], so serum was added to the upper well only (serum was not added in migration experiments of C22 cells). Chambers were placed in an incubator at 37°C and 5% CO 2 and cells were allowed to migrate for 8, 16 or 24 hrs.…”

PARTITIONING LUNG AND PLASMA PROTEINS: CIRCULATING SURFACTANT PROTEINS AS BIOMARKERS OF ALVEOLOCAPILLARY PERMEABILITY‡

“…59−62 However, it does not appear to be a particularly useful marker of alveolocapillary injury per se . Not only is the protein synthesized and secreted into the airway rather than into the alveolus, but Clara cells are particularly susceptible to lung injury 63 . Acute exposure to various toxicants causes rapid destruction of the cells and decreased production of CC16.…”

“…Thus, the circulating levels of CC16 depend not only on the degree of lung damage, but also on the status of the Clara cells themselves. For example, whereas increased circulating levels have been found in patients with pulmonary fibrosis 61 and sarcoidosis, 62 decreased levels have been found in patients with bleomycin lung injury, 61 chronic obstructive pulmonary disease 62 and asbestosis 63 …”