Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A.

Abstract

Primary cultures of rat alveolar type II cells bind radiolabeled pulmonary surfactant protein A (SP-A) with high affinity. The binding of '25I-labeled SP-A is time-and temperature-dependent and is not accompanied by significant degradation. The binding process is saturable at low concentrations of SP-A (5 jsg/ml), and unlabeled SP-A readily competes with labeled SP-A for cellular binding sites. Subsequent to binding, two pools of cell-associated "MI-labeled SP-A can be identified based upon sensitivity to trypsin at 00C. It is likely that the trypsin-sensitive pool comprises '251-labeled SP-A bound to the cell surface and the trypsin-insensitive pool comprises the internalized protein. Scatchard analysis of cell surface binding of SP-A at 0.1-10 jag/ml shows positive cooperativity at concentrations between 0.1 and 1 jag/ml. Hill plots give nH = 1.34 ± 0.08 with an apparent dissociation constant Kd = 1.02 ± 0.32 gg/ml (which is 0.64 ± 0.19 nM if the native molecular mass of oligomeric SP-A is assumed to be 1.6 MDa). (18). Release of lactate dehydrogenase into the medium (19) was measured to determine nonspecific secretion. Alveolar macrophages were isolated by lavaging lungs with isotonic saline and centrifuging the recovered solution to sediment the cells. The macrophages were cultured at 0.5 x 106 cells per 35-mm dish. Binding of '25I-SP-A to alveolar type II cells and macrophages was assayed after cultivation of the freshly isolated cells for 20 hr. 1251I-SP-A binding to L2 cells and adult rat lung fibroblasts was assayed in confluent dishes of these cells.Purification of SP-A. Surfactant was isolated from 200-g rats given intratracheal instillation of 40 mg of silica in phosphatebuffered isotonic saline 4 weeks before lung lavage (20). The surfactant was purified as described by Hawgood et al. (21) and delipidated by extraction with 1-butanol (98 ml of butanol per 2 ml of pellet). The butanol-insoluble protein precipitate was isolated by centrifugation at 2000 x g for 30 min. Residual solvent was removed by evaporation under a stream of nitrogen. The protein was suspended in 5 mM Tris/HCl buffer (pH 7.4) and dialyzed against the same buffer at 40C for 36-48 hr. The macromolecular material was centrifuged at 150,000 x gav for 1 hr and the supernatant was applied to an affinity column ofmannose-Sepharose 6B (22). The SP-A bound to the affinity matrix in the presence of Ca2 + and was eluted with 2 mM EDTA. Further purification and removal of EDTA were accomplished by gel filtration using Bio-Gel A-5m (Bio-Rad). Electrophoretic analysis of the purified SP-A was performed as described (18). Complete details of this purification will be published elsewhere. A similar purification scheme In all preparations, more than 96% of the radioactivity was precipitated by treatment with 10% (wt/vol) trichloroacetic acid. At the highest specific activities utilized only 0.41% of monomeric SP-A molecules were modified by iodination, and this low level of modification yielded preparations that

Secretagogues Increase the Expression of Surfactant Protein A Receptors on Lung Type II Cells

“…The capacity of SP-A to function as the modulator of surfactant secretion and turnover appears to involve the binding of SP-A to type II cells, probably via specific cell surface receptors (Murata et al, 1993;Wright et al,, 1989;Kuroki et al, 1988). Previously we have reported that secretagogues enhanced the uptake of lipid and protein components of lung surfactant by isolated perfused lungs (Fisher et al, 1991) and by type II cells (Chinoy et al, 1993).…”

“…Several studies have shown that SP-A stimulates the incorporation of phospholipid by primary cultures of type II cells Tsuzuki et al, 1993;Bates et al, 1994). SP-A also functions to inhibit surfactant phospholipid secretion by type II cells Kuroki et al, 1988). Thus, SP-A may play a pivotal role in the regulation of surfactant turnover.…”

“…Thus, SP-A may play a pivotal role in the regulation of surfactant turnover. Type II cells express a high affinity receptor for SP-A (Kuroki et al, 1988, Wright et al, 1989) that recognizes the carboxyl-terminal portion of the SP-A molecule (Wright et al, 1989;Murata et al, 1993). Using an anti-idiotypic antibody approach, two groups have identified type II cell membrane proteins specific for SP-A with molecular masses of either 30-kDa (Strayer et al, 1993) or 55 kDa (Stevens et al, 1995).…”

Lactoferrin and surfactant protein A exhibit distinct binding specificity to F protein and differently modulate respiratory syncytial virus infection

“…Monoclonal antibody (mAb) against RSV protein F (B12) was produced as described previously [35,36]. Surfactant was isolated from bronchoalveolar lavage fluids of patients with alveolar proteinosis [37], and SP-A was purified from delipidated surfactant as described previously [4]. Human milk LF was purchased from Sigma (St. Louis, MO).…”

“…It belongs to the collectin subgroup of the C-type lectin superfamily. In addition to the interaction with the surfactant lipids and alveolar type II cells [3,4], SP-A plays an important role in the innate immune system of the lung. SP-A has been reported to bind to various spectra of microorganisms and enhance phagocytosis by alveolar macrophages [5,6].…”