Identification of the alpha6 integrin as a candidate receptor for papillomaviruses.

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [35S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha6beta4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha6 or beta4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha6beta4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha6 integrin subunit and that integrin complexes containing alpha6 integrin complexed with either beta1 or beta4 integrins may act as a receptor for PV binding and entry into epithelial cells.     

Immunocytochemical identification of cell types in human mammary gland: variations in cellular markers are dependent on glandular topography and differentiation

Antiserum to epithelial membrane antigen and three monoclonal antibodies (MAb) to milk-fat globule membranes immunocytochemically stain only epithelial cells, whereas a fourth reacts also with myoepithelial cells in inter- and intralobular ducts of human breast. Staining with peanut lectin shows a gradual increase for epithelial cells, from little or no staining in ducts through variable staining in ductules to intense staining in secretory alveoli. Antisera and MAb to vimentin, smooth-muscle actin, MAb to the common acute lymphoblastic leukemia antigen and to a glycoprotein of 135 KD stain myoepithelial cells in main ducts, but this staining is reduced in inter- and intralobular ducts and ductules. MAb to epithelial-specific keratin 18 stain a minor population of ductal epithelial cells, the major population of epithelial cells in interlobular (ILD) and extralobular terminal ducts (ETD), and epithelial cells in a minority of ductules. In lactating glands most epithelial cells in ductules are stained, but the alveolar and myoepithelial cells are unstained. Keratin MAb PKK2 and LP34 strongly stain myoepithelial cells, but only a minor population of epithelial cells in main ducts. However, these MAb stain principally the epithelial cells in ILD, ETD, and a minority of ductules. In lactating glands most epithelial cells are stained in ductules, but the myoepithelial and not the alveolar cells are stained intensely in secretory lobules. It is suggested that the unusual staining pattern of cells found principally in the ILD, ETD, and some ductules may represent regions of growth and/or subpopulation(s) of cells intermediate between epithelial and myoepithelial cells.     

A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors.

A new member of the integrin superfamily of adhesion receptors was isolated from human epithelial cells. Analogously to other integrins, this molecule is a heterodimer comprised of structurally unrelated subunits, both glycosylated. Unequivocal amino-acid sequence homologies were observed between these subunits and integrin alpha and beta chain sequences, indicating that this epithelial heterodimer is a novel integrin. No obvious serologic cross-reactivities were detected with other integrins. The beta chain of the epithelial integrin displayed a mol. wt significantly higher than other integrin beta chains, possibly due to a large sialic acid content. Integrin heterodimers are grouped into three families, based on which of three beta chains (beta 1, beta 2 and beta 3) they contain. Therefore, the epithelial integrin may represent the prototype of a fourth integrin family, because it contains a structurally distinct beta chain. The designation alpha E beta 4 is proposed for this novel human integrin.     

A Novel Recognition Site on Laminin for the α3β1 Integrin

Synthetic peptide GD-2 is a sequence of amino acids derived from the carboxy-terminal long arm of the A chain of laminin. Previous studies have shown that peptide GD-2 promotes the adhesion of human squamous cell carcinoma (SCC) cells as well as a variety of other cell lines. In this study, we attempted to identify the receptor that SCC cells use to adhere to peptide GD-2. Monoclonal antibodies (mAbs) against a human SCC cell line were generated. One of these mAbs, ASC-1, bound to the surface of SCC cells as determined by flow cytometry. This mAb inhibited SCC cell adhesion to peptide GD-2 and laminin, but not fibronectin or type IV collagen, suggesting that mAb ASC-1 binds to the SCC receptor for the peptide GD-2 sequence of laminin. MAb ASC-1 immunoprecipitated a complex composed of two components of 135 and 116 kDa. Immunoadsorption of ASC-1-binding material from the SCC cell extract by incubation with mAb ASC-1 resulted in the removal of the α3β1 integrin from the extract. Immunohistochemical staining of tissue from a normal human tongue and from a patient with SCC of the tongue revealed that mAb ASC-1 stained the surface of epithelial cells that were in contact with the basement membrane, as well as those cells located two to three layers above the basement membrane. This mAb also stained blood vessels in the squamous tissue. This staining pattern was identical to that observed when the same tissues were stained by a mAb against the α3 integrin subunit. In summary, by use of a new mAb, ASC-1, that recognizes the α3β1 integrin, we have determined that the α3β1 integrin mediates SCC cell adhesion to the peptide GD-2 sequence within laminin.     

Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes

Epiligrin is a new glycoprotein in most epithelial basement membranes (BMs) and is a ligand for cell adhesion via integrin alpha 3 beta 1. In the extracellular matrix of human foreskin keratinocytes (HFKs), epiligrin contains three disulfide-bonded, glycoprotein subunits, E170, E145, and E135, based on molecular size in kilodaltons. Epiligrin, immunopurified with MAb P1E1, induced cell adhesion and localization of integrin alpha 3 beta 1 in focal adhesions (FAs). Cell adhesion to epiligrin was inhibited with an anti-alpha 3 beta 1 MAb. Epiligrin also colocalized with integrin alpha 6 beta 4 in hemidesmosome-like stable anchoring contacts (SACs). alpha 3 beta 1-FAs encircled alpha 6 beta 4-SACs in a complex adhesion structure. alpha 3 beta 1 and epiligrin localized in BM junctions of epithelial cells primarily in organs of endodermal/ectodermal origin. In epidermis, epiligrin was detected in the lamina lucida of BMs. alpha 3 beta 1 localized in plasma membranes of basal cells in contact with epiligrin and also in lateral/apical membranes. Epiligrin is the ligand of an adhesion super complex composed of alpha 3 beta 1-FAs and alpha 6 beta 4-SACs (hemidesmosomes).     

Immunohistochemical localization of activated EGF receptor in human eccrine and apocrine sweat glands.

Epidermal growth factor (EGF) is secreted into sweat from secretory cells of human sweat glands. The function of EGF in sweat is poorly understood. The biological function of EGF is exerted by the binding of EGF to the receptor (EGFR) and its activation. Therefore, we immunohistochemically localized the activated form of EGFR in human eccrine and apocrine sweat glands to assess the functional importance of the EGF-EGFR system in human sweat glands. Frozen sections of human skin were stained with a monoclonal antibody (MAb) specific for tyrosine-phosphorylated (activated) EGFR and with an MAb that stains both activated and non-activated EGFR. In the secretory portion of eccrine sweat glands, nuclei of the secretory cells were stained with the anti-activated EGFR MAb. In coiled and straight portions of eccrine sweat ducts, nuclei of luminal and peripheral cells were stained with the antibody specific for activated EGFR. Luminal cell membranes and luminal cytoplasm of inner ductal cells possessed non-activated EGFR. In the secretory portion of apocrine sweat glands, activated EGFRs were present in cytoplasm and nuclei of secretory cells. These data suggest that EGF, already known to be present in the cytoplasm of secretory cells in eccrine and apocrine sweat glands, activates EGFR in the nuclei of secretory cells themselves in an intracrine manner. Because ductal cells do not express EGF, EGF in the sweat secreted from the secretory cells should activate EGFR in the ductal cells in a paracrine manner. (J Histochem Cytochem 49:597-601, 2001)     

Preferential localization of CD8+ alpha E beta 7+ T cells around acinar epithelial cells with apoptosis in patients with Sj?gren's syndrome.

The T lymphocytes that infiltrate the exocrine glands in Sj?gren's syndrome (SS) play a key role in damaging glandular epithelial cells, but the mechanisms of this damage by T lymphocytes are not fully understood. To determine the cellular basis of this phenomenon, we focused our attention on the T lymphocytes around acinar epithelial cells in SS. We showed that CD8+ but not CD4+ T lymphocytes were located around the acinar epithelial cells and that a majority of these CD8+ T lymphocytes possess an unique integrin, alpha E beta 7 (CD103). The acinar epithelial cell adherent with alpha E beta 7 (CD103)+ CD8+ T lymphocytes was apoptotic. Both the perforin/granzyme B and Fas/Fas ligand pathways were implicated in the process of programmed cell death in lacrimal glands. These results suggested that alpha E beta 7 integrin, by interacting with E-cadherin, mediates the adhesion between CD8+ T lymphocytes and acinar epithelial cells in SS and participates in inducing epithelial cell apoptosis, leading to secretory dysfunction of exocrine glands, a hallmark of SS.     

Localization of laminin alpha4-chain in developing and adult human tissues.

Recent studies suggest important functions for laminin-8 (Ln-8; alpha4beta1gamma1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln alpha4-chain. Immunoreactivity for the Ln alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln alpha4- and Ln alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln alpha4-chain was found as M(r) 180,000 protein.     

Identification of antibodies against HAI-1 and integrin alpha6beta4 as immunohistochemical markers of human villous cytotrophoblast.

Syncytiotrophoblast and invasive extravillous trophoblast arise from a common stem cell, namely villous cytotrophoblast, but have very different characteristics. The study of the differentiation process relies on the availability of suitable markers for these different cell types of developing placenta. In this work, we have produced monoclonal antibodies that are specific to human villous cytotrophoblast. Monoclonal antibody (MAb) MG2 was specific to villous cytotrophoblast across gestation, and recognizes hepatocyte growth factor activator inhibitor type 1. MAb MD10 stained villous cytotrophoblast across gestation and also some endothelial cells, particularly in the second or third trimester. MAb MD10 recognizes human integrin alpha6beta4. As a test for specificity, the novel MAbs were also used for staining of frozen tissue from human colon carcinoma. The results show that the two antibodies can be used as tools to study human villous cytotrophoblasts and also human tumors. The MG2 antibody seems most specific and promising for the study of various aspects of human villous cytotrophoblast.     

Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes

The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

Alpha(v)beta(6) integrin-A marker for the malignant potential of epithelial ovarian cancer.

The mechanism(s) responsible for the progression of non-metastatic or borderline ovarian cancer to invasive Grade I/III ovarian cancer is still unknown. An epithelium-restricted integrin, alpha(v)beta(6), is present in malignant epithelia but not in normal epithelia. We studied the relative expression and distribution of alpha(v)beta(6) integrin in early and late-stage invasive (Grade I and Grade III) and non-invasive (benign and borderline) ovarian tumors of serous, mucinous, endometrioid, and clear-cell carcinoma subtypes, to assess its potential as a marker for epithelial ovarian cancer progression. Sixty-six specimens, including eight normal, 13 benign, 14 borderline, 13 Grade I, and 18 Grade III tumors were evaluated by immunohistochemistry (IHC) using a monoclonal antibody (MAb) against alpha(v)beta(6) integrin. Normal ovarian surface epithelium was negative for alpha(v)beta(6) integrin expression. All 45 carcinomas studied were positive, and the staining intensity significantly correlated with the grade of the tumor. The Grade III carcinomas of all types showed strong staining intensity. Only mucinous benign tissues were positive, and no reactivity was observed in benign serous neoplasms. On the basis of these observations, we hypothesize that the expression of alpha(v)beta(6) integrin is associated with epithelial ovarian cancer and that a gradual increase in the expression of the molecule may be a correlative index of the progression of this disease.     

Alpha 2u-globulin in modified sebaceous glands with pheromonal functions: localization of the protein and its mRNA in preputial, meibomian, and perianal glands

alpha 2u-Globulin, the principal urinary protein of the male rat, has extensive sequence homology with many lipid binding proteins. The highest concentration of alpha 2u-globulin is found in the preputial gland, a holocrine secretory organ with pheromonal function. Meibomian and perianal glands are two other modified sebaceous glands with holocrine secretory cycles and pleiomorphic peroxisomes capable of synthesizing pheromonal lipids. Immunocytochemical examination shows the presence of alpha 2u-globulin in the acinar cells of all three of these modified sebaceous glands. Whereas in the preputial gland all of the acinar cells exhibit immunoreactivity, in the meibomian and perianal glands only selective cells contain alpha 2u-globulin. In the case of the preputial gland, in addition to the acinar cells some stratified epithelial cells also were immunoreactive. In the perianal and meibomian glands, keratinocytes lining nearby hair shafts and select cells of accessory oil glands stained for alpha 2u-globulin. In situ hybridization with a cloned cRNA probe confirmed the immunocytochemical data. Presence of the alpha 2u-globulin mRNA in these glands was also established by Northern blot analysis. Immunoelectron microscopic examination of preputial alpha 2u-globulin showed the presence of this protein in secretory granules of various maturational stages. Immunolabeled alpha 2u was also found in attached vesicles containing protein and lipid inclusions. The lytic cells were not only loaded with alpha 2u-globulin but also contained sharp-edged, irregularly shaped electron-dense granules which stained heavily for this protein. Specific localization of alpha 2u-globulin and its mRNA in three pheromone-producing sebaceous glands and its structural homology with known lipid binding proteins indicate a pheromone carrier role of alpha 2u-globulin.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

Production of monoclonal antibodies against a novel glycoprotein synthesized and secreted by dog thyroid C-cells.

A monoclonal antibody (MAb) that reacted only with thyroid C-cells was raised against cell suspensions from dog thyroid glands, to examine a glycoprotein secreted by C-cells. After chronically-induced hypercalcemia and administration of an anti-thyroid drug, reaction products for the antibody markedly decreased in C-cells, coinciding with alterations in calcitonin immunoreactivity. The antigen recognized by the MAb appears to be a secretory protein. The MAb reacted with C-cells from a wide variety of mammalian species, including rats, mice, hamsters, cattle, cats, rabbits, and monkeys. Furthermore, tumor cells of human medullary thyroid carcinoma, which is derived from C-cells, were immunoreactive to the MAb. Exceptionally, C-cells from guinea pigs and pigs were not stained with the MAb. No crossreactivity was observed in any of the dog tissues examined. Immunoblot analysis demonstrated that the MAb recognized a single prominent band at a molecular weight of approximately 79,000. The 79 KD band reacted with various digoxigenin-labeled lectins, including GNA, DSA, SNA, and MAA; it is a glycoprotein containing mannose, N-acetylglucosamine, and sialic acid. Dog thyroid C-cells were also densely stained with these lectins. The results indicate that thyroid C-cells synthesize and secrete a specific glycoprotein in addition to peptide hormones.     

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Human marrow stromal precursors are alpha 1 integrin subunit-positive

In this work we studied the expression of adhesion molecules on primate human and non-human marrow stromal cells (primary cultures and lines) and on human CD34(+) hematopoietic normal and leukemic precursors. Differential expression of alpha1 integrin subunit was observed, since this molecule was intensely expressed by marrow stroma but not detected on CD34(+) cells. We used this difference to select, in fresh bone marrow samples, alpha 1-positive cells. We found that all stromal precursors giving rise to colony-forming units-fibroblasts (CFU-F) were present in the alpha 1-positive fraction. No colonies were detected in the alpha 1-negative fraction even after 2 weeks of culture. Phenotypic studies of stromal cells derived from alpha1-positive cells and grown in long-term marrow culture indicated that these cells were similar to stromal cells from primary cultures. We also observed early upregulation of alpha 4 and alpha 2 integrin subunits in cultures derived from alpha1-positive cells with maximal expression by day 10 (26 and 51%, respectively) preceding a gradual decline to low to nil values at day 30 (4.5 and 12%). These data indicate that alpha 1 integrin subunit is a marker for both mature stromal cells and stromal precursors, while alpha 2 and alpha 4 integrin subunits are expressed primarily by immature cells.     

Immunohistological localization of alpha 1-microglobulin in normal rat tissues

The tissue distribution of rat alpha 1-microglobulin (alpha 1-m) was studied by indirect immunofluorescence in various rat tissues using a polyvalent rabbit antiserum to the purified antigen and a monoclonal antibody (H23) to the human homologue, in parallel with a polyclonal anti-rat IgA antiserum. It was found that all tissues stained by anti-IgA were also alpha 1-m positive; these tissues included tissues of the stomach, duodenum, ileum, colon, pancreas, trachea, esophagus and jejunum. However, the observation that IgA plasma cells as well as secretory cells, while positively stained by anti-IgA, are alpha 1-m negative suggests that the association between IgA and alpha 1-m occurs at a postsecretory stage, after the IgA molecules have been transported across the epithelial cells. Additionally, hepatocytes were intensely stained by anti-alpha 1-m antibodies, indicating that the liver, as already suggested by metabolic studies on isolated guinea-pig liver explants, may be responsible for the synthesis of this protein. Among lymphoid tissues, an intense and homogeneous staining was observed in the thymus and the white pulp of the spleen. Sections of lymph nodes, however, showed differential staining; apart from a few isolated dendritic cells in the mantle region of the lymphoid follicles, the germinal centers and medullary cords showed no staining with anti-alpha 1-m antibodies. The paracortical cells, macrophages in the subcapsular sinus, and interfollicular lymphocytes showed intense cytoplasmic staining with anti-alpha 1-m antibodies. In other tissues, macrophages, monocytes, tissue histiocytes, and dendritic cells were alpha 1-m positive. Although they confirm the presence of alpha 1-m in the lymphoid tissues, as already reported in man, these results show that the protein is also present in hepatocytes and in exocrine fluids containing IgA. Since alpha 1-m, like secretory component, can bind to IgA to form stable complexes, these two heavily glycosylated proteins may have similar biologic properties.     

Occurrence of oligosialic acids on integrin alpha 5 subunit and their involvement in cell adhesion to fibronectin.

Integrin alpha(5)beta(1), a major fibronectin receptor, functions in a wide variety of biological phenomena. We have found that alpha 2-8-linked oligosialic acids with 5 < or = degree of polymerization (DP) < or = 7 occur on integrin alpha(5) subunit of the human melanoma cell line G361. The integrin alpha(5) subunit immunoprecipitated with anti-integrin alpha(5) antibody reacted with the monoclonal antibody 12E3, which recognizes oligo/polysialic acid with DP > or = 5 but not with the polyclonal antibody H.46 recognizing oligo/polysialic acid with DP > or = 8. The occurrence of oligosialic acids was further demonstrated by fluorometric C(7)/C(9) analysis on the immunopurified integrin alpha(5) subunit. Oligosialic acids were also found in the alpha(5) subunit of several other human cells such as foreskin fibroblast and chronic erythroleukemia K562 cells. These results suggest the ubiquitous modification with unique oligosialic acids occurs on the alpha(5) subunit of integrin alpha(5)beta(1). The adhesion of human melanoma G361 cells to fibronectin was mainly mediated by integrin alpha(5)beta(1). Treatment of cells with sialidase from Arthrobacter ureafaciens cleaving alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids inhibited adhesion to fibronectin. On the other hand, N-acetylneuraminidase II, which cleaves alpha 2-3 and alpha 2-6 but not alpha 2-8 linkages, showed no inhibitory activity. After the loss of oligosialic acids, integrin alpha(5)beta(1) failed to bind to fibronectin-conjugated Sepharose, indicating that the oligosialic acid on the alpha(5) subunit of integrin alpha(5)beta(1) plays important roles in cell adhesion to fibronectin.     

Laminin-2 stimulates the proliferation of epithelial cells in a conjunctival epithelial cell line

Laminin-2 (LN-2, alpha2beta1gamma1) is a basement membrane-associated laminin isoform usually considered in the context of muscle and nerve tissues. To test the hypothesis that LN-2 can additionally modulate epithelial cell biology, an analysis of the role of LN-2 in cell adhesion, activation of signalling intermediates and proliferation was undertaken. A virally transformed human conjunctival epithelial cell line (HC0597) was utilized in this study. Adhesion assays using function-inhibiting antibodies demonstrated that alpha3beta1 integrin is essential for the rapid attachment of conjunctival epithelial cells to LN-2. Bromodeoxyuridine (BrdU) incorporation analyses revealed that, compared with LN-1 or LN-10, LN-2 significantly promotes epithelial proliferation. Phosphorylation of the signalling intermediates Erk1/2 and Akt-1 was observed within 15 min of cell adhesion to LN-2. Inhibiting alpha3beta1 integrin function decreased total cellular phosphotyrosine levels, specifically inhibited phosphorylation of Erk1/2 and Akt-1, and dampened the proliferation response of epithelial cells adherent to LN-2. Inhibition of Erk or Akt activation inhibited cell proliferation in a dose-dependent manner. However, the inhibition of Erk resulted in a stronger suppression of proliferation compared with Akt inhibition. From these results, it is concluded that human conjunctival epithelial cells adhere to immobilized LN-2 using alpha3beta1 integrin. alpha3beta1 integrin/LN-2 signalling, transduced primarily through an Erk pathway, enhances epithelial cell proliferation. These results demonstrate that LN-2 can impact on epithelial cell biology in addition to nerve and muscle, and provide information regarding the role of this isoform in ocular surface epithelial cells.