Macrophages express cell surface laminin

Laminin, a non-collagenous extracellular connective tissue glycoprotein, was detected on the surface of mouse peritoneal macrophages. As determined by indirect immunofluorescence, as many as 60% of peritoneal macrophages elicited with thioglycollate expressed cell surface laminin. Only 14% of resident cells displayed detectable laminin. The expression of laminin increased with time post-injection. Concomitant with laminin expression, macrophages also displayed a receptor for the IB4 isolectin from Griffonia simplicifolia. This lectin, which binds methyl-alpha-D-galactopyranoside, may also react with the carbohydrate moeity of laminin. A small population of macrophages displayed both laminin and surface fibronectin. Unlike the difference in laminin expression between resident and thioglycollate-stimulated cells, there was no difference in cell surface fibronectin between these cell populations. Since laminin has been found to mediate cell attachment in other systems, expression of this molecule on the surface of stimulated macrophages may be important in cell-cell and cell-matrix adhesive properties of these cells.     

Regulation of cell attachment and cell number by fibronectin and laminin

We have examined the effect of laminin and fibronectin on the attachment and growth on type IV collagen of a line of mouse epithelial cells and a strain of adult human fibroblasts. Laminin stimulated attachment of the epidermal cells and fibronectin stimulated fibroblast attachment. At high concentrations (100 micrograms/ml), the attachment proteins altered the growth of cells in culture. The epidermal cells grew better in media containing fibronectin-free serum supplemented with laminin. Fibroblasts, on the other hand, grew best in media containing serum supplemented with fibronectin. These data suggest that laminin promotes epithelial cell growth whereas fibronectin promotes fibroblast growth. This observation was confirmed when these cells were cocultured in the presence of the attachment proteins or of their respective antibodies. The mouse epidermal cells grew best when laminin was added to cocultures of fibroblasts and epithelial cells. Fibroblasts grew best in the presence of antibody to laminin and poorly in the presence of antibody to fibronectin. Thus, fibronectin and laminin may participate in the regulation of cell populations in vivo and may be involved in epithelial-mesenchymal interactions.     

Laminin and fibronectin in normal and malignant neuroectodermal cells

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Sphingosine inhibits attachment of murine Lewis lung carcinoma cells to laminin and type IV collagen

The effect of sphingosine (SPH) on the adhesive properties of Lewis lung carcinoma (3LL) cells was investigated using plastic precoated with the extracellular matrix proteins, laminin, fibronectin, or type IV collagen. Treatment of 3LL cells with SPH (0.5-10 microM) resulted in a dose-dependent decrease in the ability to bind to laminin and type IV collagen but had little or no effect on attachment to fibronectin. Phorbol 12-myristate 13-acetate (PMA) selectively enhanced attachment of 3LL cells to laminin and collagen. The inhibitory effect of SPH on attachment to both proteins was competitively antagonized by PMA. These results suggest that SPH acts as a negative effector for cell attachment to laminin and collagen, and that the cell attachment process to both proteins might be regulated in part by protein kinase C.     

Substrate adhesion of rat hepatocytes: a comparison of laminin and fibronectin as attachment proteins

In previous studies rat hepatocytes have been shown to adhere to substrates composed of collagen or fibronectin. In the present communication, the basement membrane protein laminin is reported to mediated the attachment and spreading of hepatocytes. The cell attachment-mediating activity of laminin was compared with that of fibronectin. The activity of fibronectin was heat sensitive, whereas laminin retained its activity after boiling. On the other hand, reduction and alkylation or periodate oxidation of the proteins affected only the cell attachment activity of laminin. Preincubation of cells with soluble fibronectin inhibited initial cell attachment to fibronectin but not to laminin substrates, and, reversely, soluble laminin selectively inhibited cell attachment to laminin. These results suggest that attachment of cells to substrates of the two proteins involves different cellular receptors recognizing distinct and nonidentical structures in the proteins.     

Type XVII collagen (BP180) can function as a cell−matrix adhesion molecule via binding to laminin 332

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knock-down of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix.     

Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT)

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

Laminin inhibits the adhesion of a murine tumor of macrophage origin

The adhesive behavior of the M5076 reticulum cell sarcoma, a highly metastatic murine tumor of macrophage origin, was investigated in vitro both in the presence and in the absence of purified exogeneous laminin. Although laminin enhanced the adhesion of other murine cell lines to collagen-coated and plastic surfaces, it reduced the attachment to both substrates of M5076 cells, peritoneal macrophages and the macrophage cell line WEHI-3. Thus, the inhibition appeared to be related to the macrophage nature of the M5076 tumor. The effects of laminin were reversible and did not cause loss of viability or functional capacity of the M5076 cells. Laminin appeared to exert this inhibitory effect by binding to the substrates rather than binding to the cells. These studies indicate that laminin, a glycoprotein from the basement membrane, may either stimulate or inhibit cell attachment, depending on the type of cell.     

Differential properties of attachment of human fibroblasts to various extracellular matrix proteins

Human diploid fibroblasts (TIG-3) were shown to attach and spread onto substrata coated with collagen, fibronectin, laminin and vitronectin. The cell attachment to these proteins required divalent cations. Mg2+ stimulated the cell attachment to all the proteins, while Ca2+ alone was not effective for the attachment to collagen and laminin. A mild trypsin treatment had prevented cells from attaching to the laminin, while it had no effect on the attachment to the other proteins. The fibronectin fragment, which retained cell binding activity, inhibited the cells from attaching and spreading onto fibronectin, but it did not cause any inhibition on the other proteins. The synthetic peptide GRGDSP inhibited the cells from attaching and spreading onto fibronectin and vitronectin, while it did not cause any inhibition on collagen and laminin. In attempts to isolate distinct receptors for these proteins, we were able to purify proteins very similar to the fibronectin and vitronectin receptors of human placenta. Based on the differential properties of the attachment of TIG-3 cells to these proteins and biochemical data, we indicate that human diploid fibroblasts have distinctive binding sites (receptors) for collagen, fibronectin, laminin and vitronectin.     

Mononuclear phagocyte adherence in the presence of laminin : A possible marker of cellular differentiation

The effect of laminin on the vitro adhesive behavior of mononuclear phagocytes was investigated. Laminin significantly inhibited the adhesion of guinea pig, mouse, and rat alveolar or peritoneal macrophages and of human peripheral blood monocytes. Adhesion of these cells was unaffected by similar concentrations of fibronectin. Experiments performed with monocytes maintained in culture showed that the degree of laminin-mediated inhibition of adherence was dependent on the state of differentiation of the cells: the less mature the monocytes, the greater the degree of inhibition. Laminin also reduced the attachment capacity of polymorphonuclear leukocytes isolated from human peripheral blood. These results suggest a possible role for laminin in the regulation of the passage of cells across the basement membrane during inflammation.     

Biological activities of laminin

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.     

Cranial and trunk neural crest cells use different mechanisms for attachment to extracellular matrices.

We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the beta 1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to alpha 1 intergrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and Bronner-Fraser, M. (1991) Development 113, 1069-1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, alpha 1 integrin and beta 1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties.     

The role of murine cell surface galactosyltransferase in trophoblast: laminin interactions in vitro

Implantation of the mouse embryo involves the invasion of the secondary trophoblast giant cells of the ectoplacental cone (EPC) into the uterine decidua. The mechanisms of this event are poorly understood. The putative substrate molecules found in the decidua which could support trophoblast invasion include laminin, fibronectin, and collagen type IV. EPCs dissected from Day 7.5 embryos were cultured on all three matrices. Galactosyltransferase (GalTase) was localized by immunolabeling on trophoblast cell surfaces when grown on laminin but not the other matrices. Perturbations of the enzyme:substrate complex with alpha-lactalbumin, uridine diphosphogalactose, anti-GalTase, and pregalactosylation of the matrix did not affect rates of EPC attachment. However, decreased rates of migration or altered morphologies of spreading cells were observed. Laminin, and not fibronectin or collagen type IV, could be galactosylated with both exogenous GalTase or EPC outgrowths. Digests of galactosylated laminin produced a glycoconjugate substrate with a molecular weight of less than 10K. The results suggest that invasive secondary trophoblast cells possess a GalTase-mediated migration system that is functional on laminin.     

层粘连蛋白促进实体型瘤细胞粘着、铺展及~3H-TdR参人

 恶性肿瘤细胞或粘着于基膜以及在一定的基质上增殖对于肿瘤的浸润转移至关重要。层粘连蛋白(1aminin,LN)是基膜所特有的非胶原糖蛋白。我们研究了LN在肿瘤细胞粘着、铺展及增殖中的作用。通过测定粘着细胞所释放的LDH活性定量分析细胞粘着率。LN可显著增加体外培养的小鼠S180-V肉瘤及B16-MBK黑色素瘤细胞在玻璃及Ⅳ型胶原基质上粘着及铺展。纤维粘连蛋白(fibronectin,FN)亦有类似作用。而非糖蛋白(牛血清清蛋白)或其他糖蛋白(鸡卵清清蛋白或免疫球蛋白)均无类似作用。此外,LN或FN抗体可分别抑制细胞粘着于LN或FN。这些结果表明LN或FN促细胞粘着作用是专一性的。扫描电镜观察表明,粘着在LN敷盖的玻璃表面的瘤细胞呈多突扁平形、膜皱襞及微绒毛十分稀少,而粘着在裸露的玻璃表面者为球形,膜皱襞及微绒毛甚多。再者,~3H-TdR向粘着于LN基质上的瘤细胞群的参入量显著高于粘着在裸露玻璃面者。     

Response of sensory neurites and growth cones to patterned substrata of laminin and fibronectin in vitro

During neurite elongation in the developing peripheral nervous system, the distribution of laminin and fibronectin may provide preferred substrates for neurite elongation. In this study, the response of sensory neurites and growth cones to patterns of laminin or fibronectin applied to a background substrate of Type IV collagen was studied to determine any possible substrate preference. Neurites exhibited elongation restricted to a laminin pattern, but not a fibronectin pattern, indicating that sensory neurites prefer to elongate on laminin compared to Type IV collagen. When polylysine is included in the background substrate, neurite preference for laminin is decreased. Laminin also enhances neurite elongation and defasciculation and stabilizes growth cone protrusions. These results suggest an adhesive as well as a cytoskeletal involvement in the response to laminin, but direct adhesion estimates indicate that laminin decreases overall adhesion, arguing against an adhesive involvement. Regardless of the mechanism involved, the observed neurite preference for laminin is consistent with the hypothesis that spatial and temporal laminin distributions provide preferred pathways for peripheral neurite elongation.     

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10(-9) and 10(-7) M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Embryonic heart mesenchymal cell migration in laminin

Laminin, a large glycoprotein and major component of basement membranes, influences cell adhesion, migration, morphology, and differentiation. A peptide sequence, YIGSR, from the B1 chain of laminin has been found to correspond to an active site for cell adhesion. We report here that cardiac mesenchymal cells migrate vigorously within three-dimensional gels of laminin and that the YIGSR peptide will completely abolish this migratory activity. In contrast, migration of the mesenchymal cells into three-dimensional gels composed of collagen or collagen + laminin is not effected by YIGSR or other peptides (GRGDS, GRGDTP) reported to mediate cellular adhesion.