Anti-laminin Serum Research Articles

Recognition and lysis by natural killers of tumor cells with participation of laminin

According to the data obtained in the present work, the receptor complex of mouse natural killers (NK) includes laminin, antibody to which blocks EK-activity (NKA regardless of the presence of complement. Preincubation of mouse splenocytes with anti-laminin serum led to a decrease in their NKA towards tumor cells-targets (CT), the NKA activity decreasing 2 times with respect to cultivated cells of rat hepatoma HTC, while 10 times - to cultivated cells of human erythroblastosis K562. Pretreatment of aplenocytes with noraml nonimmune serum did not lead to a change of NKA. Quite different was the pattern after the tumor cell preincubation with anti-laminin serum: pretreatment of CT K562 led to a twofold decrease in sensitivity of these cells to NK-lysis, whereas the pretreatment of CT K562, on the contrary, made them twice sensitive to NK-lysis. Electrophoretic separation of protein of CT plasma membranes with subsequent immunoblotting with anti-laminin immune serum revealed the presence oflaminin on HTC cell plasma membrane, which was identified as laminin 8/9 by the mass-spectrometry method, while no laminin was detected on K562 cells. Preincubation of splenocytes with laminin did nor affect NKA with respect to CT K562 and HTC. Pretreatment of CT K562 and HTC with laminin decreased the NKA to zero. The obtained data allow suggesting a doubtless participation of laminin and its receptors in CT cytolysis by NK.

Tissue-culture surfaces with mixtures of aminated and fluorinated functional groups. Part 2. Growth and function of transgenic rat insulinoma cells (βG I/17)

Interactions of transplantable cells with synthetic polymers can influence the function of biohybrid artificial organs. This study explored growth and secretion of human insulin by βG I/17 cells cultured on surfaces bearing diamine groups (N2), trifluoropropyl groups (F3) and mixtures of the two. Cells cultured on high-F3 and high-N2 surfaces spread well, grew rapidly and produced >1.8 mol lactate per mol glucose consumed, closely resembling cells grown on the permissive control, glass. On one mixed surface, with a molar ratio of 33 N2 groups : 67 F3 groups, cells had a lower lactate/glucose ratio, adopted a rounded form, grew slowly and were quick to form emergent aggregates, similar to cultures on the inhibitory control, untreated polystyrene. Cultures on surfaces with higher F3 content secreted the most insulin and, in the case of the highest-F3 surface, showed improved responsiveness to secretagogues. Hormone secretion was roughly 50% greater when cells were grown on F3 surfaces conditioned by earlier cultures of βG I/17. Incubation of conditioned surfaces with high concentrations of a polyclonal anti-laminin serum prior to re-plating partially abolished this improvement in secretory function. Polymers bearing trifluoropropyl groups appear to be attractive candidates for use in the artificial endocrine pancreas. Surface coatings that include laminin might promote function of transgenic insulinoma cells in vitro and in vivo.

Immunochemical analysis of laminin during postnatal development of the rat submandibular gland

Anti-laminin serum was used to investigate distribution patterns and chain composition of laminin during postnatal development of rat submandibular gland. Immunohistochemical analysis showed that in glands of newborn rats laminin was not uniformly present around growing acini. Staining was frequently weak or absent in clefts formed between adjacent cells. This irregular staining pattern decreased progressively over the subsequent periods, and in 30-day-old animals immunoreactivity was observed only at the periphery of glands. Immunoblot analysis showed that laminin was composed of bands corresponding to the alpha 1, beta 1 and gamma 1 polypeptides. The correlation between the pattern of laminin expression and gland maturation suggests a role of laminin in the functional maturation of acinar cells.

Immunochemical characterization and distribution of laminin in the rat tongue

Anti-laminin serum was used to investigate the distribution and composition of laminin in the rat tongue. Immunohistochemical analysis showed that laminin is a useful marker to study the microscopic anatomy of the tongue. Laminin is expressed in most tissues that form this organ, such as smooth and striated muscle, epithelium, nerves, and endothelium, allowing the observation of fine details of neuromuscular junctions, taste papillae and microvasculature. Western blot analysis revealed that laminin extracted from the tongue differs from EHS tumor laminin. Tongue laminin contains B chains, while A chain seems to be present only in small amounts.

Isolation of sympathonectin; a substrate-bound protein which induces preferential growth of sympathetic fibers in vitro

A protein named ‘sympathonectin’, was purified from chick heart cell-conditioned medium (HCM), on the basis of its ability to direct the neurite outgrowth of cultured sympathetic neurons. The most purified fraction showed a doublet band on SDS-PAGE with an apparent molecular weight of 370 kDa. The biological activity of sympathonectin was over 100 times higher than that of laminin. Immunoblotting with anti-sympathonectin of the 100,000 × g pellet (particulate) fraction of HCM identified a distinct 370 kDa band; this molecule did not react to the anti-laminin serum. The immunohistochemical analysis showed that the antibody against sympathonectin stained heart tissue but not skeletal muscle tissue, whereas anti-laminin serum stained both tissues. These results suggest that sympathonectin may play a role during sympathetic innervation of target organs.

Immunohistochemical Localization of Laminin in the Peridontal Ruffini Endings of Rat Incisors: A Possible Function of Terminal Schwann Cells.

Ruffini endings in the periodontal ligament of rodents are ensheathed by a special type of terminal Schwann cell with a particularly developed rough endoplasmic reticulum and Golgi apparatus, and further enveloped by a characteristic multi-layered structure. In order to reveal the functional significance of the structures, localization of a laminin molecule in the periodontal Ruffini endings of rats was immunohistochemically investigated at the levels of light and electron microscopy. Immunostaining using an anti-laminin serum clearly demonstrated the profiles of the Ruffini endings as well as those of the blood vessels. Ultrastructurally, reaction products for laminin were deposited in the entire thickness of the multi-layered structure, supporting the idea that this structure is derived from the basal lamina. The basal lamina, immunoreacting with laminin antiserum, was penetrated by periodontal collagen fibers, possibly serving as an adhesive device between the Ruffini endings and surrounding collagen fibers. The laminin immunoreactive materials were also recognized in the vesicles and caveolae of the terminal Schwann cells which tended to gather at the interstitial surface of the cells. The terminal Schwann cells are therefore believed to be directly involved in the formation of the multilayered basal lamina through the active production of its materials.

Immunocytochemical identification of myoepithelial cells in normal and neoplastic rat mammary glands with the lectins Griffonia simplicifolia-1 and pokeweed mitogen.

Peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) histochemically stain only the myoepithelial cells and not the epithelial or fibroblastic cells of rat mammary glands preserved in methacarn or glutaraldehyde and embedded in paraffin. This pattern of staining occurs in other rat exocrine glands except the pancreas, but is the reverse of that seen in most lining epithelium. The histochemical binding of GS-1 and PWM to myoepithelial cells is inhibited specifically by D-galactose and by polymers of N-acetylglucosamine, respectively. GS-1 and its subcomponent, GS-1-B4, also bind to extracellular structures similar to those stained by anti-laminin serum. At the ultrastructural level, both conjugated GS-1 and PWM bind to the plasma membrane of the myoepithelial cells, as well as to the adjacent basement membrane. Non-metastasizing rat mammary tumors produced by dimethylbenz[a]anthracene, by derivative epithelial stem-cell lines, and by a transplantable tumor all contain more elongated myoepithelium-like cells as well as cuboidal epithelium-like cells; both cell types are neoplastic. The more elongated myoepithelium-like cells are stained by GS-1 and PWM, whereas the cuboidal epithelium-like cells are unstained. Moderately and strongly metastatic rat mammary tumors produced by epithelial cell lines and by transplantable tumors, respectively, contain no such neoplastic cells that bind either lectin. We suggest that the carbohydrate receptors for GS-1 and PWM are consistent markers for the presence of the myoepithelial cell in normal and tumorous rat mammary glands.

Distribution of laminin in the murine pituitary

The distribution and cellular localization of the glycoprotein laminin were investigated by light and electron microscopic immunocytochemistry in the adult murine pituitary gland. Immunoblots confirmed that laminin was the only protein in the pituitary gland of the adult male mouse to react with antilaminin serum. Laminin immunoreactivity was demonstrated at the light microscopic level simultaneously with that of beta-follicle stimulating hormone (beta-FSH) and beta-luteinizing hormone (beta-LH). In addition to its distribution is basal laminae, laminin immunoreactivity was coincidently expressed in gonadotrophs with the immunoreactivities of beta-FSH and beta-LH. Electron microscopic immunocytochemistry was employed on aldehyde-fixed sections embedded in L.R. White. Sites of binding of primary antisera to laminin were identified with affinity-purified secondary antisera directly coupled to 20 nm particles of colloidal gold. Three antisera recognizing laminin were compared and found to result in an identical pattern of immunoreactivity. Laminin was found extracellularly only in formed basal laminae in all three lobes of the pituitary and was not found in extracellular matrices of connective tissue. Laminin immunoreactivity was also found intracellularly in gonadotrophs but in none of the other endocrine or non-endocrine cells of the anterior lobe. Within gonadotrophs, only secretory granules were labeled. The majority, but not all, secretory granules were labeled in each of the gonadotrophs examined, and the proportion of granules labeled with laminin could not be increased by doubling the concentration of anti-laminin serum. Laminin immunoreactivity segregated with the subset of secretory granules containing beta-FSH. In contrast, laminin immunoreactivity was absent in the smaller subset of secretory granules that contain serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)

Method for epitope selection of antisera for immunohistology.

Polyclonal anti-laminin serum was affinity-purified on paraformaldehyde-fixed laminin on a nitrocellulose filter. The purified antibodies were tested for their specificity in immunohistological stainings on frozen sections of paraformaldehyde-fixed tissue. As compared to the initial polyclonal serum, the purified antibodies increased the specificity of antigen detection, since all background caused by nonspecific reactions was eliminated. This technique promises to be very useful for immunohistological analysis using light and electron microscopy.

Role of laminin in maintenance of type II pneumocyte morphology and function.

Loss of differentiated function by type II pneumocytes plated on plastic surfaces was demonstrated by decreased lamellar body content, increased cellular protein, and rapid cellular flattening, changes that were retarded modestly by plating cells on laminin-coated surfaces. Laminin surfaces also inhibited [3H]thymidine (THM) incorporation into cellular DNA by 40% compared with plastic at 40 h, but did not alter an additional mitogenic effect of rat serum over fetal calf serum. In contrast, cells plated on the laminin-rich basement membrane-like gel formed from an extract of EHS mouse sarcoma, matrix gel (MG), maintained a high content of intracellular lipids in lamellar inclusions and retained a rounded morphology for at least 3 days. MG markedly inhibited THM incorporation and morphological changes when cells were cultured on this surface or when MG was formed over cells initially plated on plastic for various intervals. The importance of the laminin component of MG was demonstrated when these surfaces were pretreated with a highly specific antilaminin serum. Type II cells commenced flattening on the treated MG surface, and THM incorporation increased with the same time course as did control cells on plastic. The data suggest that short-term culture and study of differentiated type II pneumocytes may require a laminin-rich substratum. THM incorporation into type II cell DNA provides an important early and sensitive index of cell-basement membrane interaction and subsequent maintenance of function.

Cell-free translation products of basement membrane RNA from the EHS tumor

The biosynthetic products of the Engelbreth-Holm-Swarm (EHS) tumor and the cell-free translation products of EHS tumor cell RNA were characterized. Six distinct gene products (three laminin polypeptides, entactin/nidogen, and two collagen IV chains) comprising the basement membrane matrix were identified by a combination of proteolytic digestion and immunologic techniques. Analysis of the cell-free translation products using EHS tumor RNA precipitated by anti-laminin serum confirms earlier evidence that there are at least two B chains encoded by different genes. The anti-laminin serum also recognized entactin/nidogen, which was further identified by specific immunoprecipitation with anti-entactin serum. Radiolabeled laminin A chains, synthesized by the EHS tumor in organ culture, were also identified by the anti-laminin serum but were not detected among the cell-free translation products of EHS tumor RNA. Pulse-chase studies of EHS tumor in organ culture as well as in vitro translation of EHS tumor RNA suggest that the precursor forms of α1(IV) and α2(IV) collagen chains are nearly identical in size, with apparent molecular weights of 170 000. The mRNAs encoding these two polypeptides migrate differently on sucrose gradients. It is likely that glycosylation and hydroxylation of collagen IV account for the major differences in molecular weight of mature α1(IV) and α2(IV) chains in the EHS tumor matrix.

Enhanced levels of attachment of fibronectin-primed Treponema pallidum to extracellular matrix

Freshly extracted Treponema pallidum organisms treated with exogenous human fibronectin (Fn) (Fn-primed treponemes) showed a 6- to 15-fold increased level of attachment to Fn-coated cover slips and to extracellular matrix (ECM) when compared with unprimed treponemes. Treponemes primed with collagen or laminin showed no similar enhanced binding to immobilized Fn or ECM. Preexposure of immobilized Fn and ECM to anti-Fn serum but not to anti-collagen or anti-laminin serum prevented treponemal adherence. Also, the presence of proteoglycanlike molecules such as dextran sulfate or heparan sulfate inhibited Fn-primed treponemal attachment to Fn or ECM. In contrast Fn-primed treponemes did not exhibit elevated levels of attachment to eucaryotic cell monolayers. To understand the increased tropism of Fn-primed T. pallidum organisms for Fn and ECM-like surfaces, we radiolabeled freshly extracted treponemes with [35S]methionine and examined them for the presence of surface immunoreactive Fn. Magnetic protAspheres and glass beads coated with monospecific anti-Fn serum bound only 20 to 30% of radiolabeled treponemes. Nonadherent treponemes failed to bind to gelatin-agarose, further confirming the absence of surface Fn or Fn-like material. Fn-free organisms, however, did attach to Fn-coated cover slips and to cell monolayers like treponemes of the original population. Incubation of Fn-free treponemes with human Fn resulted in almost total binding of organisms to anti-Fn antibody on glass beads and also produced increased attachment to Fn-coated cover slips and ECM. These results suggest that enhanced interactions between T. pallidum and the host are dependent on the presence of Fn on syphilis spirochetes and the specific location and orientation of Fn in vivo.

Dedifferentiation of Rat Mammary Myoepithelial-Like Cell Lines After Passage In Vivo or Cloning In Vitro<xref ref-type="fn" rid="FN2">2</xref>

Myoepithelial-like cell lines from normal mammary glands of neonatal Ludwig Wistar rats, rat mammary (Rama) 401 and Rama 704E, were injected into fat pads of syngeneic animals or were single-cell cloned in vitro. Rama 401 produced tumors that were predominantly composed of elongated cells, while the subclones of both cell lines yielded multilayered structures of elongated cells when grown on floating 0.3% collagen gels in vitro. Immunocytochemical analysis of histologic sections for markers of myoepithelial cells revealed that anti-actin-myosin and human keratin sera failed to stain the Rama 401 tumor cells or subclones of both cell lines on collagen gels, but both were stained with antilaminin serum. Immunofluorescent analysis of cultures of Rama 401 tumors showed that the resulting elongated cells failed to stain with antikeratin serum, but abundant staining was observed with antilaminin and antivimentin sera, as in the tumors. Ultrastructural analysis of the Rama 401 tumor cells identified intermediate junctions and extracellular basement membrane-like material in the vicinity of plasma membrane-associated pinocytotic vesicles, but neither true desmosomes nor myofilamental bundles were observed. Thus growth of rat mammary myoepithelial-like cells as tumors in syngeneic animals or as subclones in vitro can lead to selective loss of myofilaments and prekeratin-containing intermediate filaments. Similar relatively undifferentiated elongated cells may be responsible for some of the cellular heterogeneity observed in certain carcinogen-induced rat mammary tumors.

Laminin production by murine melanoma cells: possible involvement in cell motility.

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [35S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [35S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the Mr = 950 kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the Mr = 400 kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (Mr = 200 kD) and as a disulfide-linked B dimer (Mr = 400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells.

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.

Isolation and Characterization of Clonal Cell Lines From a Transplantable Metastasizing Rat Mammary Tumor, TR2CL<xref ref-type="fn" rid="FN2">2</xref>

The metastasizing rat mammary cell strain from the Ludwig Institute for Cancer Research (London Branch) which was originally developed from a benign rat mammary tumor induced by N-methyl-N-nitrosourea (CAS: 684-93-5), yielded single-cell-cloned lines of isometric epithelial cells [rat mammary (Rama) 600-Rama 621] and one line of elongated cells (Rama 622); the former had a higher estrogen receptor content than the latter. All the representative epithelial cell lines tested (Rama 600, 603, and 617) failed to convert to elongated, myoepithelial-like cells or droplet cell/doming, alveolar-like cells in vitro. All representative cell lines tested induced tumors in syngeneic F344/N rats and CBA nu/nu mice, but only the epithelial lines metastasized to lungs and local lymph nodes in rats and to lungs in nude mice. The involved lungs and lymph nodes contained mainly intravascular thrombi and deposits in the subcapsular sinus, respectively. Tumors and metastases from the representative epithelial cell lines contained acinar and glandular structures together with an elongated cellular component. The Rama 622 tumors contained mainly spindle cells. Antisera to rat milk fat globule membranes and human keratins stained some of the epithelial and elongated cells in the Rama 600 tumors; less staining was observed in the Rama 622 tumors. None of the tumor cells stained with antiserum to myosin. Anti-laminin serum delineated a fragmented basement membrane in glandular elements and stained weakly the cytoplasm of the more elongated tumor cells. Ultrastructural analysis confirmed the identity of epithelial cells in the Rama 600 tumors, but no well-differentiated myoepithelial cells were seen in either type of tumor. Since nonmetastasizing epithelial cells isolated directly from carcinogen-induced benign rat mammary tumors can differentiate to myoepithelial-like cells in vitro or when growing as tumors in animals, it is suggested that the development of the malignant phenotype is associated with a loss of this differentiating ability.

An antigen cross-reacting with anti-laminin sera is found in the submembranous cortical region of various cells in culture

Laminin is a complex extracellular matrix molecule consisting of one A-subunit (M r400KD) and 3 B-subunits (M r220KD) and is found in the basement membrane. Even though it is now apparent that different cell types are synthesizing laminin-like molecules, the role of these molecules in different systems is not well understood. We have characterized laminin and raised specific antiserum in rabbits. The distribution of laminin was studied by indirect immunofluorescence in different cells such as PFHR-9, WI-38, MRC-5, CHO, 3T3, WI38VA132RA, RAW264-7 and Ki3T3. All normal and transformed cells display a high amount of intracellular submembranous network-like component cross-reacting with antilaminin serum (anti-Lm) and not with anti-fibronectin (anti-Fn) serum as seen by immunofluorescence in permeabilized cells. Preabsorption of anti-Lm with increasing amounts of laminin progressively decreased the staining of the submembranous network. Anti-Lm sera from four other laboratories also showed similar staining pattern. The structural and non-secretory nature of this submembranous staining was confirmed by (a) inhibiting protein synthesis in 0.5% serum and 4 μg/ml puromycin and (b) by immunoelectron microscopy of permeabilized cells. Immunoprecipitation of 3H-leucine labelled cellular proteins with anti-laminin sera showed proteins of M r 220-210 KD in SDS-PAGE fluorography. These studies suggest that an antigen(s) crossreacting with anti-Lm sera is localized in the membrane associated cytoskeletal region where spectrin/fodrin family of proteins have been localized.

Loss of Myoepithelial Cell Characteristics in Metastasizing Rat Mammary Tumors Relative to Their Nonmetastasizing Counterparts<xref ref-type="fn" rid="FN2">2</xref>

A series of WF rat mammary tumors comprising one transplantable nonmetastasizing line (MT-W9), two predominantly lymphatic (SMT-2A and SMT-077) and one lymphatic and hematogenous (MT-450) metastasizing transplantable lines, and 7,12-dimethylbenz[a]anthracene (DMBA)-induced nonmetastasizing primary tumors was examined for the presence of epithelial and myoepithelial cell characteristics with the use of immunocytochemical techniques. Tumor cells staining for myosin were only occasionally observed in a basal orientation in glandular structures in sections of DMBA-induced and MT-W9 tumors; anti-laminin serum stained the peripheries of the glandular structures in the DMBA-induced and MT-W9 tumors but failed to stain the SMT-2A and SMT-077 tumor cells. In the nonmetastasizing tumors immunologically detectable keratin occurred mainly in the outer cellular layer of glandlike structures, whereas milk fat globule membrane immunoreactivity occurred primarily in the luminal cells. Both these types of immunoreactivity were observed in MT-450 tumor cells, but the pattern of keratin staining was random. No such immunoreactivity occurred in SMT-2A or SMT-077 tumors. No tumor cell-associated staining for fibronectin was seen in any of the tumors examined, although host stromal components stained intensely. The nonmetastasizing tumors contained cuboidal epithelial cells with lumen formation, surface microvilli, and intercellular junctional complexes, together with a relatively undifferentiated elongated cell component. Other elongated cells showed hemidesmosomes, pinocytotic vesicles, tonofilaments, and small bundles of cytoplasmic filaments, suggesting gradations toward a myoepithelial phenotype. The MT-450 tumors were ultrastructurally similar to the nonmetastasizing tumors, but no features of myoepithelial cells were seen, although some cuboidal epithelial cells exhibited prominent tonofilaments. The SMT-2A and SMT-077 tumors consisted of nests of cuboidal-like cells with highly pleomorphic nuclei and much intercellular collagen. The results indicate a progressive loss of cellular differentiation characteristics, particularly those of the myoepithelial cell with increasing malignancy in this system.