Immunologic conservation of the fiber cell beaded filament

Lenses were obtained from the eyes of four different classes of Chordates, including Mammalia (rat, mouse, cow, human), Aves (chicken), Amphibia (tiger salamander), and Osteichthyes (steelhead), as well as from one Mollusca (squid). Buffer soluble, urea soluble and urea insoluble fractions were prepared from each, and probed by western blot analysis for the presence of the lens fiber cell 115 and 49 kD beaded filament proteins. Application of both polyclonal and monoclonal antibodies revealed that an immunologic homologue to the bovine fiber cell 115 kD protein is present in all examples of Chordates tested, and that this homologue possessed properties very similar to those of its bovine counterpart. Both monoclonal and polyclonal antibodies revealed an immunologically cross-reactive homologue in squid as well, but suggested that the squid protein had a native molecular weight of closer to 70-80 kD. A monoclonal antibody to the bovine 49 kD beaded filament protein was successful at identifying an immunologic homologue to this protein in mouse, chicken, and tiger salamander. Ultrastructural analysis of rat, human, and fish lenses showed that a beaded filament was present in these lenses, which was indistinguishable from that seen in the bovine lens. In the squid a filamentous, beaded structure was observed, but it differed from that seen in the bovine lens. We conclude from the data presented that the beaded filament, and its constituent proteins, are well-conserved. This data should facilitate the identification of lens cytoskeletal proteins and structure in a wide range of animal models, and establish that probes for these proteins may be of broad applicability.     

Immunocytochemical identification of Müller's glia as a component of human epiretinal membranes

Sections of seven human epiretinal membranes of diverse pathologic origin were labeled with antibodies to cellular retinaldehyde binding protein (CRALBP) and two intermediate filament proteins: glial fibrillary acidic protein (GFAP) and vimentin. All of the membranes studied contained heterogeneous cell populations that exhibited diverse morphologic characteristics. Double labeling with both anti-GFAP and anti-CRALBP positively identified one of the cellular components in these membranes as Müller's glia. In addition, other epiretinal cells exhibited immunolabeling patterns consistent with those found in fibrous astrocytes and retinal pigment epithelial (RPE) cells normally. The results demonstrate that a double-labeling method using CRALBP antibodies, in combination with antibodies to other appropriate antigens, can be used to distinguish between the different epiretinal cell types.     

The Mr 115 kd fiber cell-specific protein is a component of the lens cytoskeleton

Electron microscope level immunocytochemistry was used to localize a lens fiber cell-specific protein with an Mr of 115 kd. Affinity-purified polyclonal antibodies were utilized on sections of detergent-extracted, acrylic-embedded lens cortical fiber cells. Monoclonal antibodies were utilized for pre-embeddment labelling of a subcellular fraction of lens fiber cells generated by homogenization, and high-speed centrifugation. The results indicate that the Mr 115 kd antigen is a component of the lens fiber cell cytoskeleton, specifically the beaded filament (BF), a cytoskeletal element thought to be unique to the differentiated lens fiber cell.     

Intermediate filaments in the human retina and retinoblastoma. An immunohistochemical study of vimentin, glial fibrillary acidic protein, and neurofilaments

Fifty-five retinoblastoma specimens were studied by a sensitive immunoperoxidase method to determine the intermediate filament types present in human retina and retinoblastoma. Polyclonal antiserum against vimentin and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and to the 200 kD neurofilament triplet protein were used. In the human retina, Müller's cells coexpressed vimentin and GFAP in most instances, probably as a reactive phenomenon. Surprisingly, the horizontal cells did not stain with any of the antibodies used, and may thus lack intermediate filaments. Also, the meshwork of neural fibers in the inner plexiform layer was unusually sparse. Retinoblastoma cells were found to be devoid of all intermediate filament types studied. The tumors contained, however, vimentin and GFAP in the stromal cells. All neurofilament-positive cells in retinoblastoma apparently derived from infiltrated retina. One retinoblastoma eye was also studied by indirect immunofluorescence microscopy of frozen sections with identical results.     

Infantile subconjunctival and anterior orbital fibrous histiocytoma. Ultrastructural and immunohistochemical studies

A fleshy, polypoidal and partially lobulated lesion that protruded between the eyelids from the medial caruncular region and that infiltrated the contiguous anterior orbital tissues developed over 1 month in a 9-month-old infant. The microscopic features of the tumor included a plump spindle cell population, more polygonal cells, early xanthoma cell transformation, infiltrating lymphocytes and eosinophils, and multinucleated giant cells, the last not exhibiting classic Touton characteristics. The histopathologic differential diagnosis ranged among fibrous histiocytoma, juvenile xanthogranuloma, and eosinophilic granuloma (histiocytosis-X). Results of electron microscopy disclosed abundant rough-surfaced endoplasmic reticulum, a paucity of lysosomes, and no Langerhans' (Birbeck) granules. Immunohistochemistry corroborated the fibrohistiocytic nature of the tumor, because histochemical stains for the enzymes alpha-1-antichymotrypsin and lysozyme, and monoclonal or polyclonal antibodies against common leukocytic antigen and S-100 protein, were negative--whereas they would have been expected to be positive in various combinations in the different histiocytic proliferations. Vimentin was identified in the tumor cells; this is an intermediate cytoplasmic filament almost always present in mesenchymal proliferations. The distinctions between fibrous histiocytomas of stromal cell origin and true histiocytic proliferations of bone marrow cell provenance are explored.     

Resisting the effects of aging: a function for the fiber cell beaded filament

PURPOSE: The beaded filament is a cytoskeletal structure that has been found only in the lens fiber cell. It includes phakosin and filensin, two divergent members of the intermediate filament family of proteins that are also unique to the fiber cell. The authors sought to determine what function the beaded filament fulfills in the lens. METHODS: Light microscopy and electron microscopy were used to characterize structural changes that occurred in previously generated phakosin and filensin knockout mice. Immunocytochemistry and electron microscopy were used to define the distribution of phakosin, filensin, and beaded filaments. RESULTS: In phakosin and filensin knockout mice, initial lens development and the early phases of fiber cell differentiation proceed in a manner largely indistinguishable from that of wild type. Fiber cells elongate, undergo organelle elimination, and, in the organelle-free zone, develop the unique paddlelike extensions that characterize cells in this region. Subsequent to those stages, however, fiber cells undergo loss of the differentiated fiber cell phenotype and loss of the long-range stacking that characterizes fiber cells and that has been considered essential for clarity. CONCLUSIONS: The beaded filament is not required for the generation of the differentiated fiber cell phenotype but is required to maintain that differentiated state and the long range order that characterizes the lens at the tissue level.     

A comparative immunohistochemical study of human corneotrabecular tissue

Using in situ immunohistochemical techniques and a broad panel of antibodies directed against intermediate filament proteins, vascular endothelial markers, neuroectodermal/neuroendocrine markers, and monoclonal antibodies raised against human corneal endothelial cells (HCECs), a comparative phenotypical analysis was performed on HCECs, keratocytes, trabecular cells, and cells lining the canal of Schlemm. The coexpression of cytokeratins and neurofilaments by HCECs argues in favor of a neuroectodermal origin, which is further supported by the fact that they stain positive for neuron-specific enolase (NSE) and that they express neural cell adhesion molecules (N-CAM) at their surface. The expression of NSE and N-CAM also applies to the trabecular cells. The cells lining the canal of Schlemm were found to share many immunophenotypical features with vascular endothelial cells (i.e., factor-VIII-related antigen and BMA-120), rather than with HCECs. This was further supported by the reactivity of two monoclonal antibodies (i.e., 9.3 E and 5.52 H) that were raised against HCECs, and which labelled vascular endothelium and cells lining Schlemm's canal. Offprint requests to: B. Foets     

Limiting the proliferation and reactivity of retinal Müller cells during experimental retinal detachment: the value of oxygen supplementation.

PURPOSE: To assess the role of hypoxia in inducing the proliferation, hypertrophy, and dysfunction of Muller cells in detached retina and the effectiveness of supplemental oxygen in limiting these reactions. METHODS: Retinal detachments were produced in the right eye of each of 13 cats; the cats survived surgery for 3 days, during which six were kept in normoxia (room air, 21%) and seven in hyperoxia (70% oxygen). Retinas were labeled for proliferation with an antibody (MIB-1) to a cell cycle protein (Ki-67), for evidence of hypertrophy employing antibodies to the intermediate filament protein glial fibrillary acidic protein (GFAP) and to beta-tubulin and for disturbance of glutamate neurochemistry employing antibodies to glutamate to a glutamate receptor (GluR-2) and to glutamine synthetase. RESULTS: Results from the two animals kept in normoxia after retinal detachment confirmed previous reports that detachment caused the proliferation of Muller cells, the hypertrophy of Muller cell processes, and the disruption of glutamate recycling by Muller cells. Oxygen supplementation during detachment reduced Muller cell proliferation and hypertrophy and reduced the abnormalities in the distributions of glutamate, GluR-2, and glutamine synthetase. CONCLUSIONS: Oxygen supplementation reduced the reaction of retinal Muller cells to retinal detachment, limiting their proliferation and helping to maintain their normal structure and function. In the clinical setting, oxygen supplementation between diagnosis and reattachment surgery may reduce the incidence and severity of glial-based complications, such as proliferative vitreoretinopathy.     

Lens intermediate filaments

The ocular lens assembles two separate intermediate filament systems sequentially with differentiation. Canonical 8-11 nm IFs composed of Vimentin are assembled in lens epithelial cells and younger fiber cells, while the fiber cell-specific beaded filaments are switched on as fiber cell elongation initiates. Some of the key features of both filament systems are reviewed.     

Microfilaments, microtubules, and intermediate filaments in cultured corneal fibroblasts

Corneal stromal fibroblasts play an important role in wound healing. Proteins from all three cytoskeletal classes (microfilament, microtubule, and intermediate filament) are involved in the control of various cellular events, such as motility, cell adhesion, shape changes, intracellular transport, and mitosis. By epifluorescent light microscopy, we studied the intracellular distributions of actin (microfilament), tubulin (microtubule), and vimentin (intermediate filament), as well as vinculin (a junctional protein connecting microfilaments to the cell membrane), in cultured corneal fibroblasts. Mutual positional relationships between actin and the other cytoskeletal proteins were investigated by double-labeling. Particular attention was paid to the leading edge of spreading or migrating fibroblasts and to their cell-to-cell contacts.     

Development- and differentiation-dependent reorganization of intermediate filaments in fiber cells

PURPOSE. To define the remodeling of lens fiber cell intermediate filaments (IF) that occurs with both development and differentiation. METHODS. Prenatal and postnatal mice were probed for the IF proteins phakosin, filensin, and vimentin, using light microscope immunocytochemical methodology. RESULTS. The pattern of vimentin accumulation in elongating fiber cells changed with development. Early in development vimentin first emerged predominantly as focal accumulations in the basal region of both epithelial and primary fiber cells. A light diffuse cytoplasmic staining was also noted. Later in embryonic development, and through maturity, vimentin in fiber cells was predominantly associated with the plasma membrane with no anterior-posterior polarity. Phakosin and filensin were first detected in the very latest stages of primary fiber elongation and continued to accumulate well after cells had completed elongation. Initially, these proteins accumulated in the anterior half of the fiber cells and were cytoplasmic in distribution. After P13, the pattern of initial distribution in differentiating fiber cells changed to a predominantly plasma membrane localization. Neither beaded filament protein showed focal basal accumulations. In mature lenses, all three proteins ultimately disappeared from the nuclear fiber cells. CONCLUSIONS. Beaded filament protein accumulation lags significantly behind both primary and secondary fiber cell elongation, suggesting a functional role subsequent to elongation. The subcellular distribution of vimentin and the beaded filament proteins showed marked differences within the cell, with differentiation, and with development. The differences in time of initial synthesis and in distribution of these IF proteins may bear on hypotheses about the role of IFs in fiber cell elongation and in structural-functional polarity of the fiber cell.     

Identification and immunolocalization of actin cytoskeletal components in light- and dark-adapted octopus retinas.

Photoreceptors in the octopus retina are of the rhabdomeric type, with rhabdomeres arising from the plasma membrane on opposite sides of the cylindrical outer segment. Each rhabdomere microvillus has an actin filament core, but other actin-binding proteins have not been identified. We used immunoblotting techniques to identify actin-binding proteins in octopus retinal extracts and immunofluorescence microscopy to localize the same proteins in fixed tissue. Antibodies directed against alpha-actinin and vinculin recognized single protein bands on immunoblots of octopus retinal extract with molecular weights comparable to the same proteins in other tissues. Anti-filamin identified two closely spaced bands similar in molecular weight to filamin in other species. Antibodies to the larger of the Drosophila ninaC gene products, p174, identified two bands lower in molecular weight than p174. Anti-villin localized a band that was significantly less in molecular weight than villin found in other cells. Epifluorescence and confocal microscopy were used to map the location of the same actin-binding proteins in dark- and light-adapted octopus photoreceptors and other retinal cells. Antibodies to most of the actin-binding proteins showed heavy staining of the photoreceptor proximal/supportive cell region accompanied by rhabdom membrane and rhabdom tip staining, although subtle differences were detected with individual antibodies. In dark-adapted retinas anti-alpha-actinin stained the photoreceptor proximal/supportive cell region where an extensive junctional complex joins these two cell types, but in the light, immunoreactivity extended above the junctional complex into the rhabdom bases. Most antibodies densely stained the rhabdom tips but anti-villin exhibited a striated pattern of localization at the tips. We believe that the actin-binding proteins identified in the octopus retina may play a significant role in the formation of new rhabdomere microvilli in the dark. We speculate that these proteins and actin remain associated with an avillar membrane that connects opposing sets of rhabdomeres in light-adapted retinas. Association of these cytoskeletal proteins with the avillar membrane would constitute a pool of proteins that could be recruited for rapid microvillus formation from the previously avillar region.     

Actin filament localization and distribution in the young adult mouse cornea: a correlative immunofluorescent and cytochemical study

The localization and distribution of actin filaments was determined in the corneal epithelium and endothelium of the young adult Swiss Webster mouse by correlative indirect immunofluorescence using rabbit anti-skeletal muscle actin antiserum and by in situ labelling with heavy meromyosin subfragment-1 (HMM-S1). A diffuse fluorescent staining was observed in the cytoplasm of epithelial and endothelial cells and in stromal keratocytes. In addition, a highly fluorescent punctate cytoplasmic staining was seen only in the corneal epithelium. Ultrastructurally, HMM-S1 decorated actin filament bundles were generally distributed within the cytoplasm of glycerinated superficial, wing and basal epithelial cells and within pedicle-like processes on the posterior surface of superficial and wing cells. Actin filament bundles also were seen in close association with intermediate filament bundles in the epithelium. HMM-S1 labelled filaments were observed in the endothelial cytoplasm anteriorly near Descemet's membrane, posteriorly near the anterior chamber and adjacent to lateral cell borders. Actin filaments in epithelial and endothelial cells appeared to insert into the plasma membrane.     

Lens membrane fraction associated intermediate filaments of different aged rats

PURPOSE: To describe the intermediate filament proteins vimentin, filensin and phakinin associated with different fractions isolated from neonatal, 10 day old and 20 day old rat lenses. METHODS: Fractions were isolated by differential and density gradient centrifugation of lens homogenates from neonatal, 10 day old and 20 day old rats. Aliquots of the 8 M urea soluble proteins of each fraction were separated by SDS PAGE, transferred to PVDF membranes, the membranes were probed with antibodies to vimentin, filensin or phakinin, and analyzed by computer. RESULTS: Over the 20 day growth period, the water soluble fraction increased and the most abundant membrane fraction was characterized by a significant increase in its urea insoluble protein and a significant decrease in its urea soluble protein. There were no significant quantitative changes in any of the other fractions. The concentration of each intermediate filament protein was greatest in the cytoskeletal fraction and over the 20 day period, the amount of vimentin associated with this fraction dramatically decreased, and the amounts of filensin and phakinin dramatically increased. Among the membrane fractions, the greatest concentration of each intermediate filament protein was found in the non sedimenting membrane fraction (NSMF) which was the least abundant fraction recovered. Filensin and phakinin associated with the other three major membrane fractions increased over the 20 day growth period, but the level of vimentin did not significantly change. CONCLUSIONS: The NSMF may represent a domain of the lens plasma membrane particularly important in interaction between plasma membrane and cytoskeleton and as the membrane-cytoskeleton protein architecture of rat lens changes over the first 20 days of life, the changes are readily detected in the different membrane fractions.     

In vitro filament-like formation upon interaction between lens alpha-crystallin and betaL-crystallin promoted by stress

PURPOSE: To determine whether alpha-crystallin is capable of forming filament-like structures with other members of the crystallin family. METHODS: Water-soluble crystallins were isolated from calf lenses and fractionated into alpha-, betaH-, betaL-, and gamma-crystallins according to standard procedures. Chaperone-like activity of alpha-crystallin was determined in control and UV-A-irradiated lenses by the heat-induced aggregation assay of betaL-crystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament formation was examined by transmission immunoelectron microscopy using specific antibodies directed against the crystallins. Involvement of intermediate filament constituents was excluded by the results of Western blot analysis, which were all negative. Moreover, the in vitro amyloid fibril interaction test using thioflavin T (ThT) was also performed. RESULTS: At the supramolecular level heating at 60 degrees C has no effect on the morphologic appearance of alpha-crystallin as observed by transmission electron microscopy. Moreover alpha-crystallin obtained from UV-A-irradiated lenses shows a virtually identical shape. However, heating in the presence of betaL-crystallin results in the formation of filament-like alphabeta-hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against alpha- or betaL-crystallin. Parallel experiments with alpha-crystallin derived from UV-A-irradiated lenses showed even more pronounced filamentous structures, compared with the controls. Nonetheless, we were able to show that the UV-light treatment affected the chaperone-like capacity of alpha-crystallin, as revealed by a diminished ability to inhibit in vitro denaturation of betaL-crystallin. To exclude the presence of cytoskeletal contamination in the crystallin preparations, vimentin antibodies were also tested. These latter experiments were negative. The filamentous nature of the hybrids was further confirmed by the results obtained with the ThT assay earlier applied for the detection of amyloid fibrils. CONCLUSIONS: Crystallin hybrids have previously been detected in the water-soluble lens crystallin fraction. Our findings indicate that such endogenous hybrids, formerly called "rods," may result from stress-induced interaction between alpha-crystallin and other lens constituents such as betaL-crystallin. Because the hybrid formation is enhanced when alpha-crystallin from UV-A-irradiated lenses is used as one of the two components of the hybrid, one can only speculate that this formation may be one of the factors leading to UV-A cataract.     

Expression of the 55-kD/64-kD corneal keratins in ocular surface epithelium.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells. Immunofluorescence microscopic analysis of frozen sections of guinea pig and bovine tissue revealed that the 55-kD antiserum labeled corneal and limbal epithelium. In addition, the antiserum stained a subpopulation of peripheral limbal cells that were distributed in both basal and suprabasal layers of the epithelium. The monoclonal antibody AE5 was used to investigate the distribution of the 64-kD polypeptide in guinea pig and bovine tissue. Immunoblotting analysis revealed that AE5 antibodies recognized a 64-kD polypeptide in guinea pig cornea, but recognized a 66-kD polypeptide in bovine cornea. Immunofluorescence microscopic analysis of guinea pig tissue revealed that AE5 antibodies labeled suprabasal layers of corneal epithelium, in suprabasal layers of limbal epithelium, and in groups of cells in the peripheral limbal epithelium. We discuss the possibility that the ocular epithelial cells recognized by either the 55-kD or the 64-kD antibodies in the peripheral limbus may play a role in the reepithelialization of the cornea after wounding.     

Polygonal arrays of actin filaments in human lens epithelial cells. An aging study

In order to determine the importance of lens actin filament configuration to lens accommodation, the pattern of actin filaments in the epithelium was studied in human lenses from different decades of life spanning the accommodative and non-accommodative years. Polygonal arrays of microfilaments were demonstrated in whole mounts of epithelium from normal and cataractous lenses using rhodamine phalloidin, an actin-specific, fluorescent-labeled probe. Tangential section transmission electron microscopy (TEM) studies confirmed that these arrays consist of central vertices and interconnecting filament rays, which line the apical end of each epithelial cell and appear to attach to the lateral membrane. These polygonal arrays were present in human lenses ranging from 25-94 yr of age. Measurements of intervertex distance showed remarkable constancy throughout the ages studied. In view of these findings, it is proposed that a possible function of these polygonal arrays is to stabilize the lens epithelium during lens flattening.     

S100 immunophenotypes of uveal melanomas.

To determine whether ocular melanomas are immunophenotypically identical to cutaneous melanomas, 34 primary and metastatic choroidal melanomas representing all major histotypes defined by the Callender's classification, plus one melanoma of the iris and one conjunctival melanoma, were subjected to a panel of immunostains designed to distinguish anaplastic biopsies of cutaneous melanomas from carcinomas and lymphomas. All ocular melanomas were found to express the intermediate filament vimentin but not keratin, and all but 2 were melanotic by immunostaining. Thirty-three of 34 (97%) choroidal melanomas were strongly stained with a rabbit polyclonal antibody (P-S100) developed against the S100 protein family. In contrast, none of 14 spindle cell type primary lesions was stained with a monoclonal antibody (MAB-079) specific for both S100 alpha and S100 beta, the best-characterized S100 polypeptides. Furthermore, only 2 of 5 epithelioid and 3 of 10 mixed-cell-type melanomas were weakly reactive. Overall, 14.7% (5 of 29) were stained. In comparison, MAB079 stained 85% of all cutaneous melanomas. Five metastases of choroidal melanomas (spindle B, epithelioid, and mixed cell types) from different organ sites also were stained by P-S100 but not by MAB079. These findings were corroborated by immunostaining with another monoclonal antibody (MAB4D4) specific for S100 beta. Differential staining by the polyclonal but not the monoclonal antibodies suggests the possible presence of a variant S100 polypeptide(s) in choroidal melanomas. Since S100 alpha, S100 beta, and related proteins appear to be physiologically important, additional studies of these S100 proteins may shed light on the etiology or pathology of choroidal melanomas.     

Kinetics of cyclic AMP-dependent accumulation of novel intermediate filament proteins in cultured chick lens cells.

PURPOSE: To refine the parameters affecting the accumulation of cytoskeletal markers of lens fiber terminal differentiation. METHODS: Primary cultures of chick lens annular pad cells were treated with a lipid soluble cyclic AMP analog under various culture conditions. The accumulation of beaded filament proteins, unique markers of lens fiber terminal differentiation, was quantified with an ELISA assay. The incorporation of beaded filament proteins into macromolecular structures was followed with immunofluorescence microscopy. RESULTS: In a time- and dose-dependent manner, beaded filament protein levels were increased in cyclic nucleotide treated cells. The addition of serum to treated cells caused a further dose-dependent increase in beaded filament protein levels. The continuous presence of cyclic nucleotides for maximal beaded filament protein accumulation was also established. At the light microscopic level, cyclic nucleotide treatment produced much more extensive multilayering of cells and lentoid formation. Macromolecular structures containing beaded filament proteins also increased in both abundance and complexity after cyclic nucleotide treatment and were restricted to the multilayers/lentoids. CONCLUSIONS: These results indicate that multiple mechanisms (including cyclic AMP, serum factors, and the degree of cell-cell interactions) affect the accumulation of beaded filament proteins during the normal differentiation of lens fibers.     

Intermediate filament, laminin and integrin expression in lacrimal gland acinar cells: comparison of an immortalized cell line to primary cells, and their response to retinoic acid.

PURPOSE: The goal of this study was to characterize intermediate filament, integrin and laminin expression by rabbit lacrimal gland acinar cells in culture, to determine whether retinoic acid (RA) alters expression of these proteins and to compare primary cells to an immortalized rabbit lacrimal gland acinar cell line using flow cytometric analysis. METHODS: Primary cells, maintained in serum free medium, were exposed to 10(-6) M retinoic acid for 24 hours. Immortalized cells were grown in defined medium with Nu-Serum and exposed to retinoic acid. Cells were labeled with monoclonal antibodies to cytokeratins (AE1, AE2, AE3, AE5, CK10/13, CK18), integrins (alpha(3), alpha(6), alpha(V), beta(1), beta(2), beta(3) and beta(4)), laminin, or vimentin and with FITC-conjugated secondary antibodies. Cells were analyzed for antigen expression by flow cytometry and immunocytochemistry. RESULTS: Primary and immortalized cells expressed type I and type II epithelial cytokeratins (AE1 and AE3), cytokeratin 18, and cytokeratin 3 (AE5) Both cell types were negative to AE2 and CK10/13. Primary and immortalized cells expressed vimentin in culture, with immortalized cells expressing this protein at higher levels. Lacrimal acinar cells appear to synthesize laminin which was detected intracellularly in both cells types. Integrins alpha(6) (CD49f) and alpha(V) (CD51) were expressed by primary and immortalized cells. Expression of integrin alpha(6) was 10-fold higher in immortalized cells compared to primary cells. Retinoic acid increased integrin alpha( V) expression by primary and immortalized cells 1.3-fold and 3-fold, respectively, and caused a slight increase in integrin alpha(6) expression by primary cells. Both cell types also expressed integrins beta( 1), beta(2) and beta(3), but beta(4) was detected only in immortalized cells. Lacrimal acinar cells do not express integrin alpha(3). CONCLUSIONS: Expression of cytokeratins, laminin and integrins by primary and immortalized cells was similar, suggesting that the immortalized cell line is a good model for the study of lacrimal structure and function. Since retinoic acid up-regulated only integrin alpha(V), but not cytokeratins, these cells appear to be highly differentiated. Flow cytometry is a useful method for analysis of protein expression by lacrimal acinar cells.