Immersion oil for high-resolution live-cell imaging at 37 degrees C: optical and physical characteristics.

The use of normal immersion oil, developed for 23 degrees C, at 37 degrees C greatly compromises both axial resolution and signal intensity. We developed and characterized an immersion oil for optimal performance in live-cell imaging at 37 degrees C. We quantify the improvements in resolution and intensity obtained when using the new oil instead of its standard 23 degrees C counterparts.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cells in vitro.

1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OMe) is an analogue of the naturally occurring 2-lysophosphatidylcholine belonging to the class of antitumor lipids. Previously, we demonstrated that ET-18-OMe modulates cell-cell adhesion of human breast cancer MCF-7 cells. In the present study, we tested the effect of ET-18-OMe on adhesion, invasion and localisation of episialin and E-cadherin in MCF-7/AZ cells expressing a functional E-cadherin/catenin complex. The MCF-7/6 human breast cancer cells were used as negative control since their E-cadherin/catenin complex is functional in cells grown on solid substrate but not in suspension. The function of E-cadherin, a calcium-dependent transmembrane cell-cell adhesion and signal-transducing molecule, is disturbed in invasive cancers by mutation, loss of mRNA stability, proteolytic degradation, tyrosine phosphorylation of associated proteins and large cell-associated proteoglycans or mucin-like molecules such as episialin. Episialin, also called MUC1, is an anti-adhesion molecule that by its large number of glycosylated tandem repeats can sterically hinder the adhesive properties of other glycoproteins. ET-18-OMe inhibited the E-cadherin functions of MCF-7/AZ cells as measured by inhibition of fast and slow aggregation and by the induction of collagen invasion. These effects were enhanced by MB2, an antibody against E-cadherin and blocked by monoclonal antibodies (MAbs) 214D4 or M8 against episialin. ET-18-OMe had no influence on tyrosine phosphorylation of beta-catenin and the E-cadherin/catenin complex remained intact. Transcription, translation, protein turnover and cell surface localisation of episialin were not altered. ET-18-OMe induced finger-like extensions with clustering of episialin together with E-cadherin and carcinoembryonic antigen but not with occludin. In cells in suspension, ET-18-OMe caused a shift in the flow-cytometric profile of episialin toward a lower intensity for MCF-7/AZ cells. In contrast with MCF-7/AZ cells, the adhesion-deficient and noninvasive MCF-7/6 cells showed neither morphotypic changes nor induction of aggregation nor invasion in collagen I upon treatment with ET-18-OMe. Co-localisation of episialin with E-cadherin was rarely observed. We conclude that in the human breast cancer cells MCF-7/AZ, E-cadherin and episialin are key molecular players in the regulation of promotion and suppression of cell-cell adhesion and invasion.

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes.

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface. In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.

Vascular-endothelial-cadherin modulates endothelial monolayer permeability.

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than 'jagged', VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.

The cellular target of histatin 5 on Candida albicans is the energized mitochondrion.

Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability of Candida albicans cells was studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion.

Localization in situ of costimulatory molecules and cytokines in B-cell non-Hodgkin's lymphoma.

Costimulatory molecules are essential in cognate interactions between T and B lymphocytes. To study the prerequisites of functional interactions between malignant B cells and intermingled T cells in B-cell non-Hodgkin's lymphomas (B-NHL), we examined the expression of CD40, CD80 and CD86 and their ligands CD40 ligand (CD40L, CD154), CD28 and CTLA4 (CD152) using immunohistochemistry and confocal laser scanning microscopy. Almost all mucosa-associated lymphoid tissue (MALT) NHL were positive for CD40 and CD80 and in nine out of 14 cases were positive for CD86. The majority of follicle centre cell lymphomas (FCCL) expressed CD40, but were heterogeneous in their expression of CD80 and CD86. Most diffuse large cell lymphomas (DLCL) were CD80+, but lacked expression of CD86. These patterns reflect the differences in phenotype of normal marginal-zone B cells (as counterparts of MALT NHL) and germinal centre cells (as counterparts of FCCL and DLCL). Counter-receptors on T cells were detectable in 13 of 14 MALT NHL, 12 of 16 FCCL but only occasionally in DLCL (three of 12 cases). A subgroup of FCCL was identified with T-cell expression of CD40L, CD28 and CTLA4 simultaneously with strong expression of CD40 and CD86 on the tumour B cells. These results indicate that MALT NHL and a subset of FCCL are most optimally equipped for functional interactions with T cells. This may be supported by the demonstration of cytokine production - mainly in T cells - in MALT NHL [interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-10] and FCCL (IL-2, IFN-gamma) and to a lesser extent in DLCL.

Automatic detection of stable and unstable chromosome aberrations visualized by three-color imaging after fluorescence in situ hybridization with a painting and a pancentromeric DNA probe.

Two-color fluorescence in situ hybridization (FISH) in combination with digital image analysis was used to develop an automatic system for the detection and classification of chromosome aberrations. Algorithms were developed for the automatic thresholding of the three digitized images: an FITC image representing specific painted chromosomes, a TRITC image representing the centromeres of all chromosomes, and a DAPI image representing all the counterstained chromosomes. A further algorithm was developed for the automatic classification of the different types of chromosome aberrations, such as translocations, dicentrics, and fragments. For this study, a dataset of 252 metaphases were digitized and analyzed automatically as well as manually. Of these metaphases, 81.3% could be correctly classified by the algorithm. The error rate was reduced to 9.3% by automatically excluding the detected clusters and artifacts. The average analysis time per metaphase was 34.5 s without any user intervention.

Ligand-independent role of the beta 4 integrin subunit in the formation of hemidesmosomes.

Recently, we have shown that a region within the beta4 cytoplasmic domain, encompassing the second fibronectin type III (FNIII) repeat and the first 27 amino acids of the connecting segment, is critical for the localization of alpha6 beta4 in hemidesmosomes. In addition, this region was shown to regulate the distribution of HD1/plectin in transfected cells. In order to investigate the function of the beta4 extracellular and cytoplasmic domains in the assembly and integrity of hemidesmosomes, we have constructed chimeric receptors consisting of the extracellular and transmembrane domains of the interleukin 2 receptor (IL2R), fused to different parts of the beta4 cytoplasmic domain. These chimeras are expressed as single subunits at the plasma membrane. The results show that the first and the second FNIII repeat, together with the first part of the connecting segment (in total a stretch of 241 amino acids spanning amino acids 1,115 to 1,356) are both essential and sufficient for the localization of beta4 in pre-existing hemidesmosomes. Moreover, expression of the IL2R/beta4 chimeric constructs in COS-7 and CHO cells, which do not express alpha6 beta4 or the bullous pemphigoid (BP) antigens but do express HD1/plectin, revealed that the stretch of 241 amino acids is sufficient for inducing the formation of type II hemidesmosomes. Expression of the IL2R/beta4 chimeras in a keratinocyte cell line derived from a patient lacking beta4 expression, showed that amino acids 1,115 to 1,356 can also induce the formation of type I hemidesmosomes. We further demonstrate that type I and II hemidesmosomes can also be formed upon adhesion of alpha6 beta4-expressing cells to fibronectin. These findings establish that the beta4 extracellular domain is not essential for the induction of hemidesmosome assembly. Moreover, they demonstrate that binding of alpha6 beta4 to ligand, and heterodimerization of alpha6 with beta4, are not required for hemidesmosome formation. This indicates that the assembly of hemidesmosomes can be regulated from within the cell.

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

Inhibition of invasion of epithelial cells by Tiam1-Rac signaling.

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.

Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200-10,000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.

A minimal region on the integrin beta4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells.

The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within beta4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with alpha6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 could not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.

In vivo binding of the salivary glycoprotein EP-GP (identical to GCDFP-15) to oral and non-oral bacteria detection and identification of EP-GP binding species.

Extra Parotid Glycoprotein (EP-GP) is a glycoprotein isolated from human saliva, having homologues in several other body fluids. The biological role of EP-GP and its homologues is unknown. Recently, EP-GP was shown to bind in vitro to the bacterium Streptococcus salivarius HB. In contrast, no binding to a number of other oral microorganisms could be demonstrated. In the present study we have determined whether binding of EP-GP to bacteria occurs in vivo in saliva and in other EP-GP containing body fluids. Therefore the presence of EP-GP on bacteria in vivo was determined by analyzing oral, skin and ear floras by confocal fluoresence microscopy using specific antibodies. About 12% of the in vivo oral flora had EP-GP present on their surface, while approximately 5% of the bacteria from ear canal or skin was positive for EP-GP. IgA was detected on approximately 65% of the salivary bacteria, whereas the high-molecular weight mucin (MG1) and cystatin C were not detectable on any oral bacterium. Using a replica-plate assay, a number of EP-GP binding strains in saliva were isolated and identified as Gemella haemolysans, Gemella morbillorium, Streptococcus acidominimus, Streptococcus oralis, Streptococcus salivarius and Streptococcus parasanguis. Bacteria from the ear canal and skin bacteria were identified as Staphylococcus hominis. It is concluded that EP-GP is selectively bound in vivo to several oral and non-oral bacterial species.

Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.

The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance.

A susceptibility gene for alveolar lung tumors in the mouse maps between Hsp70.3 and G7 within the H2 complex.

Lung tumor susceptibility (LTS) in the mouse is influenced by multiple loci within the H2 complex. We compared the LTS of two H2 congenic strains with intra H2 recombinations, B10.A(1R) and B10.A(2R), whose genetic difference has been reduced to a region of approximately 50 kilobases within the C4-H2D interval, between Hsp70.3 and G7. After transplacental induction with N-ethyl-N-nitrosourea the load of alveolar lung tumors in strain B10.A(2R) is significantly higher than in strain B10.A(1R) (P < 0.001). For papillary tumors no significant differences were observed. We conclude that the alveolar lung tumor load is influenced by an LTS gene located within the Hsp70.3-G7 interval.

Monoclonal antibody M3 used in tissue polypeptide-specific antigen assay for the quantification of tissue polypeptide antigen recognizes keratin 18.

Recently, a new 'specific tissue polypeptide antigen (TPA)' test was introduced and designated tissue polypeptide-specific antigen (TPS); it is based on the monoclonal antibody (MAb) anti-TPS, M3. We have tested the specificity of this antibody by immunocyto- and immunohistochemistry, gel electrophoresis and immunoblotting. MAb M3 bound to intermediate filaments of epithelial cells and revealed a staining pattern identical to cytokeratin (CK) 18-specific MAb (DE-K18) on tissue sections of various human tissues. On immunoblots of proteins extracted from various epithelial cell lines, M3 reacted with a 45-kD protein corresponding to CK18, and on immunoblots of proteins isolated from MCF-7 culture fluid M3 stained three bands, 45, 33 and 29 kD. The same bands were stained with CK18-specific MAb, indicating that they represent CK18 and its degradation products. TPA, used as a tumor marker in clinical diagnoses and follow-up, was shown to be a degradation product of CK 8, 18 and 19. In contrast to TPA, MAb M3 did not stain CK8 and CK19 present on immunoblots.

Derivation of tumorigenic and non-tumorigenic mouse alveolar type-II cell lines from fetal type-II cells after a combined in vivo/in vitro carcinogen treatment.

Alveolar type-II cells were isolated from the lungs of fetuses (day 18 of gestation) of the A/WySnAf (A/Sn) mouse strain, which were treated in utero at day 15 with the directly-acting carcinogen N-ethyl-N-nitrosourea (ENU). The isolated type-II cells were again treated with ENU during their initial growth in vitro. After a prolonged culture period, 5 cell lines were obtained, which were identified as type-II cell lines. Differences between cell lines were found with respect to contact-inhibited growth, cell doubling time and ability to grow in a serum-free medium. Two out of the 5 cell lines produced highly invasive type-II cell carcinomas after s.c. injection of 5 x 10(6) cells into nude mice. Thus, both tumorigenic and non-tumorigenic mouse alveolar type-II cell lines were derived after this combined in vivo and in vitro carcinogen treatment of fetal mouse alveolar type-II cells. This offers the possibility of studying in vitro the factors thought to influence lung tumorigenesis in vivo. In addition, our findings strongly suggest that alveolar type-II cells are the progenitor cells of malignant mouse lung tumors.

MHC and non-MHC genes in lung tumor susceptibility in the mouse: implications for the study of the different lung tumor types and their cell of origin.

Lung tumorigenesis in the mouse is controlled by multiple genes, which until now largely escaped detection because of the limitations of the available genetic tools. Therefore, we have developed the Recombinant Congenic Strains (RCS), which can be used to separate and map individual tumor susceptibility genes. The two strains, B10.O20/Dem and O20/A (used to produce the RCS series O20.c.B10.O20/Dem) differ in number, type, and degree of malignancy of lung tumors. Thus the genetic control of the different aspects of lung tumorigenesis can now be analyzed using RCS. The tests on the role of the Major Histocompatibility Complex (MHC; H-2 in mice) in lung tumorigenesis show that the H-2 influence is different for alveolar tumors and papillary tumors. In addition, only the development of papillary tumors is influenced by glucocorticoid administration concomitant with the transplacental carcinogen treatment. The MHC influence on lung tumor development is probably related to H-2 effects on the regulation of lung differentiation. In the H-2 congenic strains used papillary tumors developed early, whereas alveolar tumors appeared later, if at all. This differential impact of genetic and hormonal factors on the development of alveolar and papillary tumors suggests that they arise either from different cell types or from a common cell type at different stages of differentiation.

Fetal mouse alveolar type II cells in culture express several type II cell characteristics found in vivo, together with major histocompatibility antigens.

Alveolar type II cells were isolated from fetal mouse lung by differential adherence and obtained in monolayer culture. Cultures display a high degree of purity as shown by histochemical and immunocytochemical staining procedures. Seventy-five percent of cells stained positive with specific anti-lavage serum mouse (SALS-M), an antiserum specific for (pre)alveolar type II cells of the mouse, and osmiophilic bodies were present in 82% of cells. These and other characteristics of type II cells in culture correspond to those of alveolar type II cells in fetal mouse lung. The pattern of reactivity of these cells with various anti-cytokeratin antibodies is described, and we show that, in contrast to rat type II cells, they do not exhibit alkaline phosphatase activity. Identity of the type II cell cultures was shown by their specific phospholipid composition and surfactant protein A (SP-A) content. The fetal alveolar type II cells in culture were found to synthesize and express class I but not class II major histocompatibility complex (MHC) antigens. The possibility to culture fetal alveolar type II cells of the mouse and the availability of genetically well-defined inbred and transgenic mouse strains opens ways to study the genetics of type II cell differentiation and function. Also, the in vitro availability of alveolar type II cells, the progenitor cells of mouse lung tumors, will enable us to study in vitro several of the processes involved in lung tumorigenesis in the mouse.