Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues

A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room temperature. When bound to membrane, PAS IV was resistant to digestion by a number of proteinases, although after solubilization with non-ionic detergents, the protein was readily degraded. Amino acid analysis of the purified protein revealed a high percentage of amino acids with nonpolar residues. The location of PAS IV was determined in bovine tissues by using immunofluorescence techniques. In mammary tissue, PAS IV was located on both the apical surfaces of secretory epithelial cells and endothelial cells of capillaries. This glycoprotein was also detected in endothelial cells of heart, liver, spleen, pancreas, salivary gland, and small intestine. In addition to mammary epithelial cells, PAS IV was also located in certain other epithelial cells, most notably the bronchiolar epithelial cells of lung. The potential usefulness of this protein as a specific marker of capillary endothelial cells in certain tissues is discussed.     

Purification and characterization of a differentiation-specific sialoglycoprotein of lactating-guinea-pig mammary tissue.

A large acidic glycoprotein, PAS-I, was purified from the fat-globule membrane of guinea-pig milk. Threonine and serine accounted for over 30 mol% of the amino acids, and galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose and sialic acid were the principal sugars detected. On a molar basis, sialic acid accounted for over 60% of the total sugar. Removal of sialic acid by treatment with neuraminidase revealed the presence of binding sites for peanut (Arachis hypogaea) agglutinin, a lectin specific for the sugar sequence beta-D-Gal-(beta 1----3)-D-GalNac (the T antigen). The distribution of PAS-I-related epitopes, defined by five monoclonal antibodies, was determined in the mammary gland and in other guinea-pig tissues. PAS-I was maximally expressed on the apical surfaces of secretory cells in lactating mammary tissue and was either absent, or present in much lower amounts, in the glands of virgin or pregnant animals. PAS-I epitopes were not detected in liver, heart, spleen, pancreas, ovary, uterus, lung or intestine, either by immunofluorescence microscopy or by immunoblotting techniques. Several of the PAS-I-specific antibodies bound to mucins of high Mr in human fat-globule membrane, and similarities and differences between PAS-I and the human mucins are discussed. PAS-I and epitopes of this glycoprotein will be useful as indicators of differentiation in mammary cells and of markers of the apical surface of these cells during lactation.     

Monoclonal antibodies prepared against PAS-I butyrophilin and GP-55 from guinea-pig milk-fat-globule membrane bind specifically to the apical pole of secretory-epithelial cells in lactating mammary tissue

Monoclonal antibodies to the three major glycoproteins of guinea-pig milk-fat-globule membrane were isolated. The specificity of these antibodies was determined by solid-phase immunoassays and by immunoblotting and autoradiographic techniques after one- and two-dimensional gel electrophoresis. The antibodies bound to PAS-I, a sialoglycoprotein of Mr greater than or equal to 200 000 and the glycoproteins butyrophilin and GP-55, of Mr 63 000 and 55 000, respectively. Immunolocalization studies showed that all three proteins were highly concentrated in the apical pole of secretory-epithelial cells in mammary tissue during lactation. PAS-I, butyrophilin or GP-55, were not detected in either the basal cytoplasm of mammary epithelial cells or in myoepithelial cells, capillary endothelial cells or other cells found in the mammary gland. These proteins were either present in small amounts or were absent from mammary tissue taken in late pregnancy. The monoclonal antibodies characterized in this study will therefore be useful as probes for studies of the biogenesis of apical membrane proteins in mammary epithelial cells during lactation.     

PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV).

PAS IV is a 78-kDa (bovine) to 80-kDa (human) integral membrane glycoprotein of unknown function which is found in mammary epithelial cells. We now report the purification of human PAS IV and native bovine PAS IV from the milk fat globule membrane (MFGM), a preparation of apical plasmalemma from epithelial cells of lactating mammary tissue. N-Terminal sequence analyses of human and bovine PAS IV revealed homology to the N-terminal sequence of the 88-kDa human endothelial and platelet glycoprotein CD36. The similarity of MFGM PAS IV to platelet CD36 was further established by immunoblots of purified platelet CD36 and MFGM PAS IV with MFGM PAS IV specific antiserum. The removal of N-linked oligosaccharides from PAS IV and CD36 by treatment with endoglycosidase F reduced the apparent Mr of both proteins to approximately 57,000. These data suggest that PAS IV and CD36 are similar if not identical polypeptides that undergo cell type specific glycosylation.     

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes.

1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes.

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.     

Localization of pulmonary surfactant protein during mouse lung development

During lung development type II alveolar epithelial cells produce extracellular pulmonary surfactant. Polyclonal antibodies were produced against nonserum proteins associated with human surfactant. The present studies were designed (i) to determine if mouse surfactant proteins were antigenically cross-reactive with polyclonal antibodies directed against human surfactant proteins; and (ii) to determine surfactant protein localization during fetal, neonatal, and adult mouse lung development. Two-dimensional gel electrophoresis studies in conjunction with immunologic techniques provided evidence that mouse and human surfactant proteins shared antigenic determinants. The major monomeric form of mouse surfactant protein in a glycoprotein of approximately Mr 35,000 under reducing conditions. A less abundant form was identified as a Mr 45,000 polypeptide. Immunohistochemical localization showed that type II cells contain surfactant protein at Theiler stage 26. A gradient of immunostaining was localized within alveolar surfaces. The antigen was not detected in heart, blood vessels, or pulmonary interstitial cells. Surfactant protein was detected lining alveolar surfaces in mature adult lung. The distribution of this protein during fetal and neonatal lung morphogenesis suggests that this extracellular constituent of pulmonary surfactant may be extremely useful as a phenotypic marker with which to evaluate normal and abnormal lung development.     

Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk.

A major periodate--Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70 000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2, 17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.     

Isolation and chemical and immunochemical characterization of the peanut-lectin-binding glycoprotein from human milk-fat-globule membranes.

Membrane glycoprotein with high Mr (HMr-MGP) was purified from neuraminidase-treated Triton X-100-solubilized human milk-fat-globule membranes by peanut-agglutinin (PNA) affinity chromatography. The high carbohydrate content (75%), blood-group-A activity and typical monosaccharide composition (L-fucose, D-galactose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine in the proportions 0.26:1.00:1.85:1.30) indicate that the isolated HMr-MGP is a mucinous substance. Fractionation of the oligosaccharides from alkaline-borohydride-treated HMr-MGP on Bio-Gel P-2 suggest that the PNA-binding sites are located mainly on longer (tetra- to deca-saccharide) alkali-labile bound oligosaccharide chains. Polyclonal antibodies raised against the HMr-MGP showed an antigenic distribution in histological sections that was comparable with the distribution of peroxidase-labelled-PNA-binding sites in both normal and malignant breast tissues. The positive immunohistological staining of some other tissue components with this antibody indicates that HMr-MGP is not strictly breast-associated. The functional role of HMr-MGP is unknown, but, since its expression is dependent on the differentiation state of secretory epithelial cells, it serves as a differentiation antigen that can be used for better functional characterization of breast cancers.     

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.     

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. II. Biogenesis of milk-fat-globule membrane proteins

Guinea-pig mammary tissue was perfused in vitro, radiolabelled with [35S]methionine and intracellular protein precursors of the milk-fat-globule membrane (FGM) recovered by immunoabsorption techniques. Labelled xanthine oxidase was solely detected in post-microsomal supernatants and butyrophilin in carbonate-washed membranes. A major glycoprotein (Gp 55), was initially present in a membrane-bound form, but after longer perfusion times a fraction of this protein was recovered in the post-microsomal supernatant. These results are discussed with reference to formation of the apically-derived FGM.     

Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, (Na+ +K+)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in (3H-mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated Mr = 58000), there are several minor surface glycopeptides (Mr = 76000, 86000 and 92000-100000) which are apparent extrinsic membrane components, and two surface glycopeptides (Mr = 42000 and 130000) which are intrinsic membrane components.     

Identification and characterization of the principal proteins of the fat-globule membrane from guinea-pig milk

The milk-fat-globule membrane (MFGM) was isolated from guinea-pig milk and the membrane-associated proteins and glycoproteins characterized by electrophoretic techniques. Major components of the membrane included PAS-I, a sialoglycoprotein of Mr greater than or equal to 200000, the redox enzyme xanthine oxidase and the glycoprotein, butyrophilin. Membrane preparations also contained two other glycoproteins, GP-80 and GP-55, of Mr 80000 and 55000, respectively. Comparison of guinea-pig xanthine oxidase and butyrophilin with proteins from bovine MFGM by peptide mapping procedures, showed that the two proteins in both species were similar, but not identical. GP-55 may also be related to glycoproteins of Mr 45000 and 48000 in the bovine membrane. The integral and peripheral components of guinea-pig MFGM were identified by treating membrane preparations with sodium carbonate solutions at high pH and by partitioning the membrane proteins in solutions of Triton X-114. By these criteria xanthine oxidase and GP-55 appeared to be peripheral components and GP-80 an integral protein of the membrane. PAS-I and butyrophilin displayed hydrophilic properties in Triton X-114 solutions, but could not be removed from membrane preparations with sodium carbonate. Possible reasons for these ambiguous data are discussed. The observed similarity between several of the proteins of guinea-pig and bovine MFGM implies that these proteins may have specific functions related to milk secretion in mammary tissue, e.g. in the budding of milk-fat globules or the exocytosis of milk protein and lactose at the apical surface.     

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. II. Immunolocalization and microinjection studies

Using two monoclonal antibodies described in the preceding paper we determined by immunofluorescence microscopy the distribution of an integral membrane protein of the desmosomal domain, the major glycopolypeptide of Mr 165,000 (bovine muzzle epidermal desmosome band 3; desmoglein) in various normal tissues, tumors and cultured cell lines from several mammalian species. This protein was detected in dotted or streak-like arrays along cell boundary structures which were known to contain non-membrane-integrated desmosomal plaque proteins such as desmoplakins. This is true for epithelial, i.e. cytokeratin-expressing cell types, for the desmin-producing myocardiac and Purkinje fiber cells of the heart, and for certain vimentin-containing cells such as arachnoidal and meningiomal cells and dendritic follicular cells of lymph nodes. However, on the basis of both immunoblot and immunocytochemical reactions, the protein is absent from non-desmosomal adhering junctions, including those devoid of desmoplakin but containing another plaque protein, plakoglobin ("band 5 protein"). We have used these antibodies to localize their epitopes with respect to the cell membrane. By immunoelectron microscopy we found that both epitopes are located in the desmosomal plaques, and this was confirmed by microinjection of purified antibodies into living cultured cells which resulted in labelling of the plaques. From these findings, taken together with previous analyses and localizations of the carbohydrate moieties of this glycoprotein, we conclude that desmoglein is a transmembrane glycoprotein which projects into--and contributes to--the desmosomal plaque structure. This glycoprotein represents a general component of true desmosomes and it is coexpressed with obligatory desmosome-specific plaque proteins such as desmoplakin I. The potential value of this glycoprotein as a desmosomal and cell type marker in histology and tumor diagnosis is discussed.     

Identification of a novel serum protein secreted by lung carcinoma cells

The murine anti-human lung tumor monoclonal antibody L3 recognizes antigens found both in the medium of cultured carcinoma cells and in normal human serum. Sequential immunoprecipitation experiments indicate that the L3 antigen is also recognized by a previously described monoclonal antibody directed against a melanoma-associated antigen [Natali, P. G., Wilson, B. S., Imai, K., Bigotti, A., & Ferrone, S. (1982) Cancer Res. 42, 583-589]. This antibody precipitated a Mr 76000 glycoprotein from metabolically labeled extracts of the lung carcinoma cell line Calu-1 and a Mr 94 000 glycoprotein from labeled culture medium. Pulse-chase experiments suggested a precursor-product relationship between these molecules. Analysis of glycosidase sensitivities of the two forms indicated that maturation of carbohydrate side chains correlated with the apparent increase in molecular weights. L3 antigenic activity, measured in a competitive radiometric cell binding assay, was purified more than 90-fold from serum-free medium of Calu-1 cells and more than 3000-fold from normal human serum. The major immunoreactive components purified from culture medium and serum were identical with respect to apparent molecular weight, electrophoretic mobility, pI, glycosidase sensitivity, and V8 protease fingerprints. In addition, the sequence of the amino-terminal 16 N-terminal amino acid residues of the major immunoreactive species from both sources was identical. The properties of the L3 antigen did not correspond to those of any known protein, suggesting that this serum protein has not been previously characterized.     

Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.     

Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to greater than 200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Extracellular Amastigote-Like Forms of Leishmania panamensis and L. braziliensis. II. Stage- and Species-Specific Monoclonal Antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania . Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to >200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.