Laminin-coated Plates Research Articles

Expression and Function of β1 Integrins on Adherent and Nonadherent Ehrlich Ascites Tumor Cells

Differences in integrin-mediated cell-matrix adhesion between two types of Ehrlich ascites tumor (EAT) cells, adherent and nonadherent EAT cells, have been studied. The adherent EAT (a-EAT) cells adhere to and spread on laminin- or fibronectin-coated plates, whereas the nonadherent EAT (na-EAT) cells do not. The adhesion of a-EAT cells to laminin or fibronectin requires the presence of both Ca 2+ and Mg 2+. Anti-human fibronectin receptor (α 5β 1 integrin) antiserum blocks a-EAT cells from adhering to both laminin- and fibronectin-coated plates. An RGD-containing peptide (GRGDSP) inhibits the adhesion of a-EAT cells to fibronectin-coated but not to laminin-coated plates. Taken together, these data suggest that a-EAT cells interact with laminin and fibronectin via different β 1 integrins. As determined by immunofluorescence flow cytometry assay, both a-EAT and na-EAT cells express similar amounts of surface antigen(s) recognized by the same anti-α 5β 1 integrin polyclonal antibody that blocks a-EAT cells from adhering to laminin and fibronectin. The α 5 and β 1 subunits expressed on both types of EAT cells appear to have the same molecular weight when analyzed by immunoblotting. The same amount of 125I-labeled membrane protein was isolated from both types of EAT cells on immobilized laminin and fibronectin affinity columns. These data indicate that na-EAT cells express similar amounts of cell surface integrins, and these integrins, when isolated, react with laminin and fibronectin in the same manner as the integrins of a-EAT cells, even though na-EAT cells are unable to adhere to laminin and fibronectin. Previous work has shown that both types of EAT cells express Neu5Ac α2,3 Gal-beta; 1, 4 GleNAe residues on their cell surface and that they both react with Maackia amurensis lectin which specifically recognizes this carbohydrate structure. Here we show that both types of EAT cells attach to M. amurensis lectin-coated surfaces, but only a-EAT cells spread; this suggests that the differences between these two types of cells in responding to extra cellular matrix molecules may reside in some differences within the cell.

Alternative splicing of the neurofibromatosis 1 gene correlates with growth patterns and neuroendocrine properties of human small-cell lung-carcinoma cells.

Two distinct transcripts, type I and type II, of the neurofibromatosis I (NFI) gene are generated by alternative splicing in the region corresponding to the gene's GTPase-activating protein-related domain (GRD). Relative expression levels of these 2 transcripts were previously correlated to neural differentiation. Since small-cell lung carcinoma (SCLC) often exhibits neuroendocrine properties, we analyzed the type-I to type-II mRNA ratio in 15 SCLC cell lines, using reverse transcriptase and polymerase chain reaction methods. The type-I mRNA was predominant in 10 cell lines; 8 of them grew as floating aggregates in culture and had high L-dopa decarboxylase (DDC) activity. The other 5 lines predominantly expressed type-II mRNA, adhered to the culture substrate, and expressed low or undetectable levels of neural cell-adhesion molecule (NCAM) antigen and DDC activity. N2+, one of the subclones of NCI-N417 cells, exhibited a higher type-I to type-II ratio after the cells had adhered to a laminin-coated plate and had emitted neurite-like processes. These findings provide evidence that alternative splicing patterns of NFI mRNA correlate with the mechanisms that regulate the growth patterns and neuroendocrine properties of SCLC cells in vitro.

Role of laminin in the morphogenetic cascade during coculture of Sertoli cells with peritubular cells.

Observations summarized in this article demonstrate an essential role of laminin during the restructuring processes that occur during coculture of Sertoli cells with testicular peritubular cells. The data presented indicate that laminin becomes detectable on the free surfaces of Sertoli cells only after reaggregation of Sertoli cells begins, coincident with the initiation of repolarization at a specific stage of the morphogenetic cascade. We infer that laminin deposited at this time serves as a cohesion molecule that permits peritubular cells to come into close contact with Sertoli cells and subsequently to spread along the free surfaces of Sertoli cells. These conclusions and inferences are based on the following experiments. Cycloheximide-treated peritubular cells in culture in MEM containing cycloheximide readily attach to laminin-coated polystyrene surfaces. By contrast, added peritubular cells do not attach onto monolayers of Sertoli cells in monoculture or onto Sertoli cells plated on top of peritubular cells and maintained in coculture for periods of up to 48 h. In cocultures maintained for 6 days, however, labeled peritubular cells readily adhere to the free surfaces of reaggregated Sertoli cells. Laminin, but not fibronectin, appears on the free surfaces of the reaggregated Sertoli cells at this time, coinciding with the period of initial mound formation. The addition of antilaminin IgG, but not antifibronectin IgG, blocks the attachment of cycloheximide-treated peritubular cells to laminin-coated plates and also blocks the subsequent migration of peritubular cells required to form a monolayer. Similarly, anti-laminin IgG inhibits the attachment and spreading of labeled peritubular cells seeded on the free surfaces of reaggregated Sertoli cells in mounds generated during the morphogenetic cascade. We interpret the combined data to indicate that the appearance of laminin on the free surfaces of Sertoli cells is required to permit peritubular cells to adhere and subsequently to migrate on Sertoli cell surfaces, resulting in the formation of a tubule-like structure.

Follicle-stimulating hormone (FSH) increases FSH receptor messenger ribonucleic acid while decreasing FSH binding in cultured porcine granulosa cells.

The goal of the present studies was to compare direct effects of porcine FSH (pFSH) on [125I]pFSH-binding sites with effects of pFSH on FSH receptor mRNA in cultured porcine granulosa cells. Cells from immature follicles were cultured on laminin-coated plates in serum-free medium for up to 6 days in the absence or presence of pFSH (1-100 ng/ml) or cholera toxin (0.04-400 ng/ml), which activates adenylyl cyclase independently of the FSH receptor. RRA indicated that [125I] pFSH binding to cells cultured without stimulator increased more than 10-fold with time in culture. Addition of pFSH to cultures resulted in a dose-dependent decrease in binding, assessed after removal of bound pFSH. Equilibrium saturation binding analysis indicated that pFSH (10 ng/ml) caused a 39% decrease in binding sites in cells cultured for 6 days. At the same time, pFSH increased progesterone production 9.5-fold. Cholera toxin (4 ng/ml) increased [125I]pFSH binding 110% and progesterone production 8.9-fold. Northern hybridization analysis of cultured granulosa cell mRNA using a porcine FSH receptor cDNA revealed three transcripts for the FSH receptor [2.2, 3.5, and 4.2 kilobases (kb)], with the major transcript being 4.2 kb in length. Addition of either pFSH (10 ng/ml) or cholera toxin (10 ng/ml) to cultures of granulosa cells increased the intensity of pFSH receptor transcripts compared with control values, with the 4.2-kb message remaining predominant. Hybridization with a porcine LH receptor cDNA revealed different transcripts (2.4, 4.0, 4.7, 7.0, and 11.0 kb), with the major transcript being 4.7 kb in length. Addition of either pFSH or cholera toxin to the cultures increased the intensity of all LH receptor transcripts; however, cholera toxin was more effective than pFSH. pFSH and cholera toxin increased the intensity of each species to different extents, although the 4.7-kb transcript remained predominant. These results indicate that exposure to FSH in culture results in down-regulation of the FSH receptor. Down-regulation is accompanied by increased FSH receptor mRNA levels, suggesting that FSH enhances FSH receptor synthesis.

Differential Effects of IL12 and IL2 on Expression and Function of Cellular Adhesion Molecules on Purified Human Natural Killer Cells

Accessory functions of cellular adhesion molecules (CAM) in activation, adhesion, migration, and cytotoxicity of natural killer (NK) cells are partly dependent on activation by cytokines. We studied effects of interleukin 12 (IL12) on expression and function of adhesion molecules on human NK cells and compared them to the effects mediated by IL2. Target binding of NK cells was significantly increased by IL12, leading to an increased level of conjugate formation with K562 target cells as well as enhanced binding to tumor monolayers. IL12 also induced significant levels of cytotoxicity against fresh tumor cell targets in purified human NK cells. IL12 significantly enhanced adhesion and subsequent migration of NK cells through 3-μm-pore-size polycarbonate filters. However, IL2 was a more potent activator of these functions, which have been shown to be partly mediated by CD2, CD58, β 2 integrins, and ICAM-1. As assessed by flow cytometry, IL12 also induced significant up-regulation in the proportion or mean fluorescence intensity of NK cells positive for the following activation markers and adhesion molecules: CD25, HLA-DR, CD69, CD71, CD56, CD2, and CD54. Among the β 2 integrins, IL12 selectively increased expression of CD11a on NK cells, although to a significantly lower level than that induced by IL2. IL12 had different in vitro effects than IL2 on expression and function of the β 1 integrins. Whereas IL2 induced marked up-regulation in expression of the β 1 integrins, CD49b, -c, -d, and -e, IL12 had no demonstrable effect over a wide range of concentrations. In addition, while IL2-activated NK cells showed significantly increased integrin-dependent adhesion to fibronectin- or laminin-coated plates, IL12-activated cells were less adherent to fibronectin and were unchanged in their adherence to laminin. Our data demonstrate that IL12 is involved in interactions of NK cells with fresh or cultured tumor cell targets, biologic substrates, or extracellular matrix molecules. Although the magnitude of its in vitro effects on adhesion-dependent functions of NK cells was significantly smaller than that of IL2, lower doses of IL12 were required to up-regulate functions of CAM, and this may be an attractive feature of IL12 as a potential therapeutic cytokine.

Glycyl-l-Glutamine Regulates the Expression of Asymmetric Acetylcholinesterase Molecular Forms in Cultured Cardiac Post-natal Myocytes

Asymmetric acetylcholinesterase (AChE) forms were associated with pre-natal but not post-natal ventricular myocytes, when myocytes were cultured in a defined medium on laminin-coated plates for 72 h. In contrast, globular AChE molecular forms were associated with both pre-natal and post-natal myocytes. Glycyl-l-glutamine (10-4 or 10-6M), but glycyl-d-glutamine or glycyl-l-glutamate, induced the expression of asymmetric AChE molecular forms by the cultured post-natal myocytes. Neither of the three dipeptides altered the specific activity of cell-associated AChE in the cultured post-natal ventricular myocytes. Tetrameric globular (G4) AChE was the main AChE form secreted by cultured pre-natal and post-natal cardiac myocytes. The secretion rate of AChE from post-natal myocytes was not affected by the addition of glycyl-l-glutamine. These results that glycyl-l-glutamine has trophic effect on at least one of the components of the post-synaptic cholinergic system in developing rat heart.

Response of human NK cells to IL-6 alterations of the cell surface phenotype, adhesion to fibronectin and laminin, and tumor necrosis factor-alpha/beta secretion.

In vitro effects of human recombinant IL-6 (1-1000 U/ml) on highly enriched human NK CD3-CD56+ cells (94% +/- 2; mean +/- SEM; n = 8), obtained from PBL were studied. IL-6 induced low levels of NK cell proliferation (7- to 30-fold during 6-day incubation), which was IL-2-independent, because IL-6 did not induce detectable IL-2 production by NK cells. Two-color flow cytometry analysis demonstrated that incubation of NK cells with IL-6 at the optimal concentration of 250 U/ml for 6 days significantly increased the proportion of NK cells expressing the following activation Ag: CD25 (26% +/- 17, mean +/- SEM vs 4% +/- 1 in control, n = 5), CD54 (44% +/- 17 vs 9% +/- 3), HLA-DR (29% +/- 13 vs 12% +/- 4), CD69 (45% +/- 7 vs 12% +/- 3), and CD71 (34% +/- 17 vs 6% +/- 2). The mean fluorescence intensity of these activation Ag was increased as well. IL-6 induced expression of CD49b (alpha-chain of VLA-2, 20% +/- 11 vs 2% +/- 1) and CD49c (alpha-chain of VLA-3, 43% +/- 17 vs 5% +/- 3), which are not expressed on resting NK cells. IL-6 also enhanced the fluorescence intensity of beta 1 integrins, CD49d, CD49e, and CD49f, expressed on NK cells. IL-6-stimulated NK cells showed significantly increased integrin-mediated adhesion to fibronectin- or laminin-coated plates (26 +/- 3 mean % cells adhering +/- SEM vs 15 +/- 4 in control for FN and 19 +/- 1 vs 11 +/- 1 for LM, p < 0.05 for both) as determined in a 3 h binding assay. As assessed by inhibition of adhesion using mAb to the VLA-2, -3, -4, -5, and -6, NK cell adhesion to fibronectin was mediated by VLA-4 and 5, and their adhesion to laminin by VLA-3 and -6. NK cells incubated in the presence of IL-6 were found to produce a factor cytostatic to WEHI-164 clone 13 target cells. This effect was partly, although significantly, blocked by neutralizing antibodies to TNF-alpha or TNF-beta. Our data demonstrate that IL-6 can directly activate human NK cells, but is a less potent NK cell activator, for all activation and functional parameters studied, than IL-2.

Effects of Decidual Cell Supernatants and Lymphokines on Murine Trophoblast Growth in Vitro1

Blastocysts were flushed from CD1 mice and were cultured in plastic or laminin-coated plates as an in vitro model of implantation. The purified cytokines epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) markedly stimulated 3H-thymidine incorporation; but recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF), which might be produced by alloantigen-stimulated T cells at the feto-maternal interface, had no growth-stimulating effect. Indeed, higher nonphysiological concentrations of GM-CSF manifested a toxic inhibition. Surprisingly, a purified nonrecombinant murine GM-CSF preparation induced proliferation of both blastocyst and ectoplacental cone trophoblast whereas recombinant murine (and human) GM-CSF had no effect, indicating that the growth stimulation may have been due to a contaminant. Decidual supernatants prepared on Days 5.5-6.5 of pregnancy from mice with high abortion rates (DBA/2-mated CBA/J) had no toxic or stimulating effect on blastocyst trophoblast outgrowth compared to similarly prepared supernatants from low-abortion-rate DBA/2-mated C3H/HeJ mice. These data suggest that it is not GM-CSF that is crucial for the trophoblast proliferation that determines the success of pregnancy.

A comparative study of the neutrophil stimulatory activity in vitro and pro-inflammatory properties in vivo of 72 amino acid and 77 amino acid IL-8.

IL-8, a potent neutrophil-activating protein, can be produced by many cell types including monocytes, lymphocytes, fibroblasts, neutrophils, and endothelial cells. Depending on the cell source, the N-terminal amino acid sequence of IL-8 displays heterogeneity that has been shown to confer differences in its neutrophil stimulatory activity in vitro. Despite these observations the relative potency of different IL-8 molecules in vivo is unknown. To address this question we have investigated the biologic activity of the two predominant forms of IL-8, the 72 and the 77 amino acid proteins, in vitro and in vivo. In vitro, human rIL-8(72) and human rIL-8(77) dose dependently induced adherence of rabbit peritoneal neutrophils and human neutrophils to laminin-coated plates and elevated cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2 loaded neutrophils. In these in vitro assays human rIL-8(72) was more potent than human rIL-8(77) while inducing comparable responses to human rC5a. With respect to enhancing [Ca2+]i, neutrophils desensitized to human rIL-8(72) failed to respond to human rIL-8(77). However, neutrophils fully desensitized to human rIL-8(77) could exhibit a partial response to human rIL-8(72). Further, human rIL-8(72) was approximately 10-fold more effective than human rIL-8(77) in displacing human [125I]rIL-8(72) from rabbit peritoneal neutrophils in a receptor-binding assay. In vivo, intradermally administered human rIL-8(72) and human rIL-8(77) induced 111In-neutrophil accumulation and edema formation in rabbit skin. In contrast to the in vitro studies, the two forms of IL-8 gave identical responses in vivo although they were less potent than human rC5a. Our results demonstrate that, in vitro, human rIL-8(72) is more potent than human rIL-8(77) in stimulating neutrophils. It may be that IL-8)72) has a greater affinity and/or efficacy for the neutrophil IL-8 cell-surface receptors. One possibility for the observation that both forms of IL-8 are equipotent in inducing inflammatory responses in vivo is that the extended form is proteolytically cleaved to the more biologically active IL-8(72).

Nonencephalitogenic CD4-CD8- V alpha 2V beta 8.2+ anti-myelin basic protein rat T lymphocytes inhibit disease induction.

Recently there has been a number of reports suggesting that CD4-CD8- T cells participate in the processes of inflammatory reaction. In an attempt to delineate the distinctive functions of double negative (DN) T lymphocytes in an autoimmune-induced disease, we isolated and cloned such T cells, along with control CD4+ cells, from Lewis rats immunized with guinea-pig myelin basic protein in CFA. Both clones proliferated in response to the guinea-pig myelin basic protein and its synthetic encephalitogenic peptide, and expressed the same TCR V genes homologous to the mouse V alpha 2 and V beta 8.2 families that appear to be the defining entity of experimental autoimmune encephalomyeltis (EAE). Moreover, the TCR D and J region gene products of the DN cell were found to be similar to another encephalitogenic rat T cell clone. The two T clones did not differ markedly in their ability to produce TNF and IL-2 and to adhere to vascular wall-derived extracellular matrix- and laminin-coated plates. Surprising, therefore, was the finding that, although the CD4+ T lymphocytes were capable of inducing EAE, the DN cells did not elicit disease but rather inhibited subsequent EAE induction. Thus, TCR V alpha 2V beta 8.2 and its junctional region gene products are not the only prerequisite segment for a T cell to become encephalitogenic. We suggest that the important determinants of the T cell ability to induce disease are features of the T cell, other than or in addition to, the T cell receptor.