Characterization of subclones of Madin-Darby canine kidney renal epithelial cell line

Heterogeneity in Madin-Darby canine kidney (MDCK) epithelial cells has been reported, however, its details have not been well described. In the present study, we show that subclones obtained from a MDCK cell line could be divided into two morphologically and biochemically distinct cell types with different hormonal responsiveness. Clones of the first type, motile clones, which had extended and flattened cytoplasm, were devoid of carbonic anhydrase activity. Clones of the second type, nonmotile clones, formed colonies of cuboidal cells and showed carbonic anhydrase activity. Motile clones synthesized cAMP in response to arginine vasopressin, prostaglandin E1, and isoproterenol but not glucagon. In contrast, nonmotile clones responded to all of these hormones. These findings suggest MDCK cells have multiple cellular origins. The motile clones have characteristics similar to the principal cells of the collecting system, whereas the nonmotile clones may be derived from the thick ascending limb or the intercalated cell. Our studies also demonstrate a significant influence of culture condition on MDCK cellular behavior (carbonic anhydrase activity, Na+/K+-ATPase activity and vasopressin responsiveness). Therefore, physiologic and biochemical experiments with MDCK cells must be interpreted with reservations about cellular heterogeneity as well as differences induced by culture conditions.     

Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions.

The glycosphingolipids (GSLs) of two sublines of Madin-Darby canine kidney (MDCK) cells, an epithelial cell line, were characterized by t.l.c., antibody overlay and mass spectrometry. The major characteristic which distinguishes the two MDCK cell strains is their trans-epithelial electrical resistance which is typically of the order of 3000 ohm.cm2 for strain I and 100 ohm.cm2 for strain II cells. Strain I and II cells were equally rich in glycolipids, the cellular GSL/phospholipid ratio being 0.04. However, while the phospholipid patterns were identical, the GSLs showed striking differences, and each cell strain expressed appreciable amounts of GSLs that were not found in the other strain. Both cell types possessed neutral GSLs with one, two or three carbohydrate moieties. The monoglycosylceramide accounted for 50% of the total GSLs in each strain. However, while in strain I cells over 90% of this monoglycosylceramide was monoglucosylceramide, in strain II cells over 90% consisted of monogalactosylceramide. In addition, MDCK strain II cells selectively expressed GSLs belonging to the globo series (26% of its neutral GSLs), including globoside and Forssman antigen, a globoside derivative. MDCK strain I cells, on the other hand, expressed another series of GSLs with 4-7 carbohydrate moieties characterized by the common sequence Hex-HexNAc-Hex-Hex-Cer. The presence of two fucosylated GSLs in these series was established. Both MDCK strain I and II cells contained negatively charged GSLs, the major component of which was the ganglioside GM3. MDCK strain II cells in addition expressed sulfatide, the sulfated derivative of galactosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of cytotoxic thyroglobulin-specific T cell hybridomas

Clones of cytotoxic thyroid-specific T cell hybridomas were generated by fusion of thyroglobulin-primed, "in vitro"-boosted CBA lymph node cells with the AKR-derived lymphoma cell line BW 5147. One hundred and thirty one clones were obtained. Among them, 15 were able to induce the lysis of 51Cr-labeled syngeneic thyroid epithelial cells after 5 h of incubation at 37 degrees C. Two T cell clones, HTC1 and HTC2, were further studied. These clones, which exhibit cell surface characteristics of cytotoxic cells, were specific for only syngeneic thyroid cells (allogeneic thyroid cells or syngeneic epithelial cells were never lysed by these hybridomas). Moreover, by using Ag-pulsed syngeneic macrophages as targets, syngeneic cytotoxicity was shown to be specific for thyroglobulin and not for a nonrelated Ag. The lysis obtained with these autoreactive thyroid-specific T cell clones is restricted to class I major histocompatibility Ag. This property is assessed by both the blocking of syngeneic cytotoxicity toward thyroid epithelial cells or thyroglobulin-pulsed macrophages only by anti-class I mAb and by the detection of specific lysis of target cells exclusively when effector hybrid cells and target thyroid epithelial cells or thyroglobulin-pulsed macrophages shared at least class I major histocompatibility Ag.     

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix

The pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture. In this study, a new method for obtaining heterocellular cultures of AT1- and AT2-like alveolar epithelial cells maintained for 7 d on a rat tail collagen-fibronectin matrix supplemented with laminin-5 is described. These cultures contain cells that appear by their morphology to be either AT1 cells (larger flattened cells without lamellar bodies) or AT2 cells (smaller cuboidal cells with lamellar bodies). AT1-like cells stain for the type I cell marker aquaporin-5, whereas AT2-like cells stain for the type II cell markers surfactant protein C or prosurfactant protein C. AT1/AT2 cell ratios, cell morphology, and cell phenotype-specific staining patterns seen in 7-d-old heterocellular cultures are similar to those seen in alveoli in situ. This culture system, in which a mixed population of phenotypically distinct alveolar epithelial cells are maintained, may facilitate in vitro studies that are more representative of AT1-AT2 cell interactions that occur in vivo.     

Ultrastructure of the endolymphatic sac in the mouse.

The ultrastructure of the endolymphatic sac (ES) in the mouse was examined by light and electron microscopy. This organ was divided into three parts: proximal, intermediate and distal. In the proximal portion of the ES, the epithelium consisted of thin squamous cells. The epithelial cells had acquired basolateral processes, numerous small vesicles and well-developed Golgi apparatus. In the intermediate portion, the epithelium consisted of columnar or cuboidal cells. The epithelial cells could be classified into two types: type I and type II. The type I cells had abundant microvilli, pinocytotic vesicles, vacuoles, multivesicular bodies, lysosomes and mitochondria. The type II cells had fewer numbers of these organelles. A few free-floating cells could be observed in the lumen of this intermediate portion, most of which were macrophages. In the distal portion, the epithelium consisted of squamous or cuboidal cells. The epithelial cells had a few cytoplasmic organelles. In the ultrastructural study, each portion of the mouse ES was found to have a very distinct morphological feature. It was suggested that each of these three portions has a different function.     

Alloreactive bovine T lymphocyte clones: an analysis of function, phenotype, and specificity

A bovine alloreactive cell population was subjected to complement-dependent lysis with monoclonal antibody (mAb) IL-A11. The original population and the population depleted of cells bearing the determinant recognized by mAb IL-A11 were cloned. Parent cultures and 21 clones were examined for cytolytic function and for expression of determinants recognized by mAb IL-A11 and two additional mAb, IL-A12 and IL-A17. Clones could be classified according to maximal achievable levels of cytolysis by using Theileria parva-infected bovine lymphoblastoid target cells. In this way, three groups were identified--one capable of high level cytolysis, one of intermediate levels, and one group comprising apparently noncytolytic clones. The clones in the first group reacted with mAb IL-A17; those in the second and third groups, with mAb IL-A11 and IL-A12. It was shown that cytotoxicity effected by IL-A17+ clones could be inhibited by this mAb and also by a mAb directed to MHC class I determinants on target cells. Conversely, cytotoxicity effected by IL-A11+/IL-A12+ clones could be inhibited by mAb IL-A11 and by a mAb directed to MHC class II determinants on target cells. The levels of expression of class I and class II determinants on target cells correlated with the levels of killing by clones of the IL-A17+ phenotype and clones of the IL-A11+/IL-A12+ phenotype, respectively. The results indicate that cytotoxic bovine T lymphocyte clones specific for class I MHC antigens and both cytotoxic and noncytotoxic clones specific for class II MHC antigens can be obtained. Further, their specificity for class I or class II antigens can be determined by phenotyping with mAb.     

Virulence of Porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype

Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and DeltafimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor alpha5beta1-integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL-1beta and IL-6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.     

Reversion of the 8-azaguanine resistant phenotype of variant Chinese hamster cells treated with alkylating agents and 5-brono-2'-deoxyuridine.

From cultures of V79 Chinese hamster cells, 10 independent clones of 8-azaguanine resistant cells were isolated and subcultured. Cells from all ten clones were resistant to 1 mg/ml levels of 8-azaguanine (8-AzG), contained less than 3% of the wild type levels of the enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT), and were unable to grow in HAT medium. The ten clones were classified according to the conditions under which they reverted to the wild type phenotype. Clones in classes I and II reverted spontaneously with frequencies of 40-10(-5) and about 3-10(-5) respectively, and the reversion frequency was independent of the density of cells of all but one of the clones in the culture medium used. Class II clones evinced increased reversion frequencies with ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to a lesser extent with 5-bromo-2'-deoxyuridine (budR), suggesting that these clones contained point mutations in a locus which controls HGPRT activity. The processes of reversion and toxicity appeared to be associated. Class III clones did not revert spontaneously or with BUdR and MNNG, but did revert with EMS. The reversion frequency of class I clones was not increased after treatment with EMS, MNNG or BUdR.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.     

Expression of MHC antigens on murine trophoblast and their modulation by interferon

Primary cultures of defined populations of mouse trophoblast, isolated from mature placentas, were analyzed for their MHC antigen expression and for the modulatory effect of interferon (IFN) by antibody- and complement-mediated cytotoxicity and flow cytofluorometry. The cells were obtained from placentas by enzymatic digestion, followed by Percoll gradient fractionation, and are large, fetally derived epithelial cells, which we previously characterized and identified as trophoblast cells. After 2 days in culture, a significant proportion of the trophoblast cells were susceptible to antibody- and complement-mediated lysis by anti-paternal strain alloantisera (40%) and, to a lesser degree, by an anti-class I monoclonal antibody (20%). Flow cytofluorometric analysis indicated that 20 to 50% of the cultured trophoblast cells expressed low levels of paternal strain class I antigens as compared to L cell fibroblasts. After culture for 48 hr with IFN-alpha/beta or IFN-gamma, the percent of class I-positive cells was increased to 68 to 76%, as was the mean fluorescence intensity, which correlated with the increased percent of antibody- and complement-mediated specific lysis (73%). No expression of class II MHC antigen by the cultured trophoblast cells was detected, even after culture in the presence of IFN-gamma. The cultured trophoblast cells, when tested for alkaline phosphatase (AP) activity, were composed of strongly positive and weakly positive subpopulations. An inverse correlation between strength of AP activity and the expression of H-2 was observed by double staining. These results indicate that trophoblast cells cultured in vitro are able to express paternal strain class I but not class II MHC antigens, as has been reported in vivo, and that this expression can be modulated by IFN. Further study of these cells should provide important clues for the understanding of materno-fetal coexistence in the face of MHC antigen differences.     

Major histocompatibility complex-unrestricted cytolytic activity of human T cells. Analysis of precursor frequency and effector phenotype

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. These conditions were shown to expand a mean of 96% of cells cultured. All of the 198 clones generated by this method were T cells (CD2+, CD3+, CD4+ or CD2+, CD3+, CD8+) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. In addition, the activity was not inhibited by monoclonal antibodies directed against class I or class II nonpolymorphic MHC determinants. Killing, however, was inhibited by soluble monoclonal antibodies against the CD3 complex. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur or K562 was not mediated by a soluble factor secreted by the clones. Some of the clones retained their cytotoxic activity when grown in rIL-2 alone for 4 to 6 wk, whereas others exhibited markedly diminished cytotoxicity after maintenance in this manner. Clones that exhibited diminished or no cytotoxic activity after prolonged maintenance in rIL-2 could be induced to kill by stimulation with immobilized but not soluble monoclonal antibodies to CD3 in the absence of lectin. All of the clones examined expressed NKH1 and CD11b but none were CD16 positive. The degree of cytotoxicity of resting or activated clones could not be correlated with expression of these markers. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.     

On tight junction structure: Forssman glycolipid does not flow between MDCK cells in an intact epithelial monolayer

One model of tight junction structure suggests that lipids might flow from cell to cell within shared exoplasmic membrane leaflets. We tested this proposal by co-culturing two clones of MDCK epithelial cells, which differed in their content of Forssman glycolipid, and then staining by immunofluorescence with rabbit anti-Forssman Ig. In co-cultures grown on glass cover slips and on nitrocellulose filters, positive Forssman staining was restricted to sharply demarcated clusters of cells formed by the Forssman-positive clone. Integrity of tight junctions between the two clones was indicated on cover slips by the presence of individual domes (hemicysts) composed of both clones and on filters by the generation of transepithelial potential differences. These results suggest that glycolipids in the exoplasmic leaflet of cells in a tight epithelium do not flow to adjacent cells.     

Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.     

Cell differentiation of alveolar epithelium in the developing rat lung: ultrahistochemical studies of glycoconjugates on the epithelial cell surface

Glycoconjugates on the surface of pulmonary epithelial cells were ultrahistochemically examined in the fetal, neonatal and adult rat lung. Lectin and colloidal iron staining procedures were performed in combination with digestion using carbohydrate-degrading enzymes or methylation. The glycoconjugate composition of columnar cells at 16 days gestation was similar to that of cuboidal cells at 19 days gestation. Glycoconjugate differentiation on the cell surface occurred at 20 days gestation, and especially the loss of soybean agglutinin (SBA) binding sites could be detected on type II cells. The contents of Ricinus communis agglutinin-I (RCA-I) and Concanavalin A (Con A) binding sites on type II cells also began to decrease. On the contrary, the content of sulfated saccharides decreased on the surface of type I cells during development. Glycoconjugate differentiation on both type I and II cells was completed with the disappearance of hyaluronic acid and peanut agglutinin (PNA) binding sites; type I and II cells acquired a similar histochemical composition to that on adult type I and II cells at 5 days after birth. Both type I and II cells share a common early precursor cell, that is, the cuboidal epithelial cell at the canalicular stage.     

A functional analysis of hematopoietic growth factor production from class I and class II human alloreactive T cell clones

Class I and Class II human alloreactive T cell clones or their conditioned media were mixed with progenitor cell-enriched null cells to assess their ability to stimulate human hematopoietic progenitor cell (HPC) growth. Optimal release of erythroid, myeloid or megakaryocyte colony-stimulating factors occurred after 72 hours and required contact of cloned T cells with irradiated stimulator cells expressing the appropriate major histocompatibility complex (MHC) determinants recognized by the T cells. Individual clones were quite heterogeneous in their capacity to release hematopoietic growth factors. Clones that produced optimal levels of factors that stimulated granulocyte-macrophage colony growth did not always produce equivalent amounts of factors that stimulated erythroid colony growth and vice versa when tested against identical target cells. Class II clones released nearly twice as much interleukin 3 activity as Class I clones. Class II clones that lacked cell-mediated lympholytic (CML) activity against B or T lymphoblastoid targets were consistent stimulators of HPC growth. In contrast, Class I or Class II clones that contained CML activity either poorly stimulated or inhibited HPC growth. These CML-positive clones produced greater amounts of gamma interferon. Our findings may have important implications for HPC growth following allogeneic mismatched bone marrow transplantation.     

Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse

Primary cultures were initiated from marrow, spleen, and bone explants of an adult H-2K^b-tsA58 transgenic mouse (immortomouse). All cultures were initiated in immortalizing conditions, and an additional marrow culture was first incubated for 1 week in standard conditions and then switched to immortalizing conditions. Marrow cells immediately immortalized were designated the marrow immediate population (MIP); those immortalized after 1 week were termed the marrow delayed population (MDP). MIP and MDP cells both contained a mixture of fibroblastic or flattened cells, and the MIP cells contained an additional subpopulation of adipocytic (Oil Red-O positive) cells. Alkaline phosphatase expression was induced by dexamethasone (10⁻⁷ M) in MDP cells while MIP, spleen, and bone explant cells had only a low level of expression. MDP and MIP cells differentiated into bone when combined with porous calcium phosphate ceramics and implanted subcutaneously into nude mice while bone- and spleen-derived cells did not. Clones were isolated from the MDP and MIP cell populations and tested for differentiated phenotypes. Some MIP-derived clones exhibited adipocytic characteristics while MDP-derived subclones were negative. Histologic examination of porous ceramic implanted clones showed that all of the clones had osteogenic potential. Clones exposed to either dexamethasone, human recombinant bone morphogenetic protein-2, or horse serum plus hydrocortisone showed differences in expression of adipocytic or osteogenic markers. These immortalized cultures have retained both adipocytic and osteogenic potential even after 1 year of continuous culture, and provide a model system for clonal analysis of the developmental potential of marrow-derived mesenchymal precursor cells. © 1996 Wiley-Liss, Inc.     

Accessibility of glycolipid and oligosaccharide epitopes on rabbit villus and follicle-associated epithelium

The initial step in many mucosal infections is pathogen attachment to glycoconjugates on the apical surfaces of intestinal epithelial cells. We examined the ability of virus-sized (120-nm) and bacterium-sized (1-microm) particles to adhere to specific glycolipids and protein-linked oligosaccharides on the apical surfaces of rabbit Peyer's patch villus enterocytes, follicle-associated enterocytes, and M cells. Particles coated with the B subunit of cholera toxin, which binds the ubiquitous glycolipid GM1, were unable to adhere to enterocytes or M cells. This confirms that both the filamentous brush border glycocalyx on enterocytes and the thin glycoprotein coat on M cells can function as size-selective barriers. Oligosaccharides containing terminal beta(1,4)-linked galactose were accessible to soluble lectin Ricinus communis type I on all epithelial cells but were not accessible to lectin immobilized on beads. Oligosaccharides containing alpha(2, 3)-linked sialic acid were recognized on all epithelial cells by soluble Maackia amurensis lectin II (Mal II). Mal II coated 120-nm (but not 1-microm) particles adhered to follicle-associated enterocytes and M cells but not to villus enterocytes. The differences in receptor availability observed may explain in part the selective attachment of viruses and bacteria to specific cell types in the intestinal mucosa.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Monoclonal antibody identification of a type II alveolar epithelial cell antigen and expression of the antigen during lung development

A monoclonal antibody identifying an antigen expressed by rat type II alveolar epithelial cells, but not by type I epithelial cells or other mature lung cells, was produced by immunization of mice with cells of the rat L2 cell line. The antigen recognized by the antibody was present on the microvillous luminal surface of type II epithelial cells. In adult rat lung, only type II epithelial cells bound the antibody. During fetal development the antigen was expressed by cuboidal epithelial cells lining the respiratory ducts of the first divisions of the tracheal bud, but not by epithelial cells lining the esophagus or trachea. The antigen continued to be expressed by cuboidal epithelial cells lining the larger respiratory ducts until approximately 19 days gestational age. Thereafter, expression was increasingly limited to selected single cells or clusters of two to four cuboidal cells in the smallest ducts. By the 21st postnatal day, the antigen was expressed only by type II alveolar epithelial cells. Type II alveolar epithelial cells isolated from adult lung and the L2 cell line in culture expressed the antigen on the cell surface. A protein of approximately 146,000 Mr was isolated by immunoadsorption of the antigen from non-ionic detergent extracts of type II cells and L2 cells. Preliminary studies of the binding of the antibody to other rat tissues indicate that the antibody binds to renal proximal tubular epithelial cells of the kidney and the luminal surface of the small bowel epithelial cells.     

Concomitant loss of transforming growth factor (TGF)-beta receptor types I and II in TGF-beta-resistant cell mutants implicates both receptor types in signal transduction

A panel of 71 chemically mutagenized Mv1Lu mink lung epithelial cell clones were selected based on their resistance to the growth inhibitory action of transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2. Characterization of TGF-beta receptors in these mutants indicates that the TGF-beta-binding membrane proteoglycan, betaglycan, is apparently normal in all of them. However, 14 of the mutant clones are defective in TGF-beta receptor type I, and 22 clones are simultaneously defective in receptor types I and II. The clones with type I receptor defects fall into two distinct phenotypes, called R and LR. The R phenotype is characterized by the lack of detectable type I receptors, and has been previously described (Boyd, F. T., and Massagué, J. (1989) J. Biol. Chem. 264, 2272-2278). LR mutants are characterized by expression of low levels of type I receptor and are, like the R mutants, completely resistant to growth inhibition by TGF-beta 1 or -beta 2. Mutant clones that are simultaneously defective in receptor types I and II fall into three distinct phenotypes. These included DRa mutants which are characterized by lack of detectable receptor types I and II, DRb mutants which are characterized by low expression of both receptor types and an anomalously fast electrophoretic mobility of the type II receptor protein. All mutants that have a low level of type II receptor are also defective in type I receptor. In addition to the loss of growth inhibitory response, the receptor-defective mutants described here have lost all other responses to TGF-beta 1 and -beta 2 known to occur in parental Mv1Lu cells. The defects present in these mutant clones are not encountered in clones isolated from nonmutagenized parental Mv1Lu cells or in mutagenized cells that had not been exposed to selection with TGF-beta. The results implicate TGF-beta receptor types I and II in the mediation of a common set of cellular responses to TGF-beta. Furthermore, the high relative frequency of isolation of DR mutants raises the possibility that receptor types I and II interact as part of a common signaling TGF-beta receptor complex.