Fibroblasts in healthy adult lung are quiescent, synthesizing little collagen. We studied lung biopsies from 30 patients with pulmonary fibrosis, using immunohistochemistry with monoclonal antibodies against the propeptides of type I collagen to localize fibroblasts actively synthesizing collagen. Adjacent sections were stained with antibodies to type III and IV collagen, fibrin, cytokeratin, plasma fibronectin, or EDIIIa-containing "cellular" fibronectin (cFN). In rapid pulmonary fibrosis, including the proliferative phase of diffuse alveolar damage, organizing pneumonia, and subacute idiopathic fibrosis, collagen-synthesizing cells were numerous in organizing exudate filling airspaces but were also seen in the interstitium of the alveolar walls, interlobular septa, and walls of blood vessels. The new matrix deposited in the airspaces also contained type III collagen and EDIIIa-containing fibronectin. In chronic pulmonary fibrosis, more than half of the biopsies showed foci of collagen synthesis and cFN deposition near the air-tissue interface. The foci were consistently localized outside remnants of basal lamina and therefore within airspaces. The results indicate that (1) fibrosis in chronic idiopathic pulmonary fibrosis results mainly from organization of exudate within airspaces, just as it does after acute lung injury, and (2) during this process, fibroblasts increase their synthesis of collagen and fibronectin coordinately. Foci of active matrix deposition provide evidence for the progressive nature of chronic pulmonary fibrosis.

Kd of 2 X 10-11 M. At higher concentrations, SP-D also caused Ca"+-dependent agglutination of Y1088 that was inhibited by a-glucosyl-containing saccharides, antisera to the carbohydrate-binding domain of SP-D, or Y1088 LPS. Lectin blots showed specific binding of 125I-SP-D to Y1088 LPS, as well as LPS from other several strains of enteric Gram-negative bacteria. Immunogold studies demonstrated strong and uniform surface labeling of the bacteria. Rat and human bronchoalveolar lavage (BAL) caused Ca++-dependent agglutination of E. coli that was dose dependent and inhibited by competing saccharides or anti-SP-D. SP-D was selectively and efficiently adsorbed from rat BAL by incubation with E. coli, and incubation of E. coli with radiolabeled rat type II cell medium revealed that SP-D is the major E. coli-binding protein secreted by freshly isolated cells in culture. We suggest that SP-D plays important roles in the lung's defense against Gram-negative bacteria. (J. Clin. Invest. 1992. 90:97-106.)

No abstract

Surfactant protein D (SP-D) is a collagenous, surfactant-associated, carbohydrate-binding protein that is synthesized by pulmonary epithelial cells. In the present studies, we examined the expression of SP-D and SP-D mRNA during late fetal (day 17, 19, 21) and early postnatal (day 5) rat lung development using immunochemical, cell-free translation, and Northern hybridization assays. SP-D mRNA and immunoreactive SP-D protein were first detected in guanidine extracts of whole rat lung at 21 days of gestation and reached even higher concentrations during the postnatal period. Likewise, immunoperoxidase studies of rat lung using affinity-purified antibodies to SP-D showed no staining at day 17 or 19, but demonstrated strong cytoplasmic staining of cuboidal epithelial cells lining immature airspaces at day 21 and strong cytoplasmic staining of type II and nonciliated bronchiolar cells in adult lung. SP-D also appeared in amniotic fluid by day 21 and was partially purified by affinity chromatography on maltosyl-agarose under conditions used for the isolation of rat lung SP-D. These studies indicate that the production of SP-D is increased shortly prior to birth, and that the increases in total lung SP-D and SP-D mRNA are temporally correlated with SP-D secretion and the appearance of SP-D in amniotic fluid.

Invest. 1995Invest. . 95:2699Invest. -2710

Abstract. Osteoclasts are the principal resorptive cells of bone, yet their capacity to degrade collagen, the major organic component of bone matrix, remains unexplored. Accordingly, we have studied the bone resorptive activity of highly enriched populations of isolated chicken osteoclasts, using as substrate devitalized rat bone which had been labeled in vivo with L-[5-3H]proline or 45Ca, and bone-like matrix produced and mineralized in vitro by osteoblast-like rat osteosarcoma cells.When co-cultured with a radiolabeled substrate, osteoclast-mediated mineral mobilization reached a maximal rate within 2 h, whereas organic matrix degradation appeared more slowly, reaching maximal rate by 12-24 h. Thereafter, the rates of organic and inorganic matrix resorption were essentially linear and parallel for at least 6 d when excess substrate was available.Osteoclast-mediated degradation of bone collagen was confirmed by amino acid analysis. 39% of the solubilized tritium was recovered as trans-4-hydroxyproline, 47% as proline. 10,000 osteoclasts solubilized 70% of the total radioactivity and 65% of the [3H]-trans-4-hydroxyproline from 100/~g of 25-50-/~m bone fragments within 5 d. Virtually all released tritium-labeled protein was of low molecular weight, 99% with Mr -10,000, and 65% with Mr _< 1,000. Moreover, when the 14% of resorbed [3H]proline-labeled peptides with Mr ~ 2,000 were examined for the presence of TC A and TC B, the characteristic initial products of mammalian collagenase activity, none was detected by SDS PAGE. In addition, osteoclast-conditioned medium had no collagenolytic activity, and exogenous TC A and TC B fragments were not degraded by osteoclasts. On the other hand, osteoclast lysates have collagenolytic enzyme activity in acidic but not in neutral buffer, with maximum activity at pH 4.0. These data indicate that osteoclasts have the capacity to resorb the organic phase of bone by a process localized to the osteoclast and its attachment site. This process appears to be independent of secretion of neutral collagenase and probably reflects acid protease activity.

CP4 is a collagenous glycoprotein (43 kDa, reduced) synthesized by rat type II pulmonary epithelial cells in primary culture (Persson et al., 1988). In order to better characterize this protein, CP4 was isolated from rat bronchoalveolar lavage and EDTA extracts of lung surfactant by adsorption to barium sulfate and elution with sodium citrate followed by reverse-phase HPLC. Amino acid analysis of purified CP4 demonstrated 4-hydroxyproline (Hyp), hydroxylysine (Hyl), and acid-labile components coeluting with Hyl glycosides. In addition, gas-phase amino-terminal microsequencing of two CP4 CNBr peptides demonstrated nonoverlapping collagenous sequences comprised of nine and six Gly-X-Y triplets, containing a total of four residues of Hyp and two of Hyl. There was less than 50% sequence homology of these peptides with the cDNA-derived sequence of the collagenous domain of rat SP-A. Two-dimensional IEF/SDS-PAGE resolved the protein into a charge train of basic isoforms (pI approximately 6-8), similar to those of newly synthesized CP4 and the class D surfactant proteins (Phelps & Taeusch, 1985). Gel filtration of nondenatured CP4 on 4% agarose showed a high apparent molecular mass complex comprised of disulfide-bonded trimers of the 43-kDa subunits. Antibodies to purified lavage CP4 showed specific binding to newly synthesized and surfactant-associated CP4. We propose that CP4 be designated "surfactant protein D" (SP-D) in accordance with an accepted nomenclature for surfactant-associated proteins.

The proteolytic activity of matrix metalloproteinases is involved in normal and disease-related remodeling processes. One member of this family, matrilysin, can degrade a wide spectrum of connective tissue proteins, suggesting that this enzyme is involved in numerous and diverse biologic processes. In fact, recent studies have shown that matrilysin is expressed in developing hair follicles and glands. Using in situ hybridization and immunohistochemistry, we examined the sites of matrilysin expression in normal and diseased adult skin. In normal mature skin, matrilysin mRNA and protein was strongly and consistently expressed in ductal cells and in some secretory cells of all eccrine and apocrine glands and was not found in any other cell type. A similar tissue distribution was also found in numerous benign inflammatory skin lesions, and prominent expression of matrilysin mRNA and protein was also found in glandular disorders such as axillary hidradenitis and sweat gland tumors. These findings indicate that matrilysin is a constitutive product of the epithelium of dermal glands and that its expression may not be related to a disease-specific or remodeling process. Because of its extensive expression in dermal glands, we assessed whether matrilysin might be produced by all exocrine glands. Indeed, we detected matrilysin mRNA and immunoreactive protein in the ductal and glandular epithelium of mammary and parotid glands, pancreas, liver, prostate, and the serous acini of peribronchial glands of the lung. Thus, our findings indicate that matrilysin is constitutively produced by exocrine epithelial cells throughout the body. Because of its broad catalytic activity, we speculate matrilysin may participate in the normal function of exocrine glands by preventing glandular obstruction.