Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane.

We studied the adhesion mechanism of pancreatic carcinoma using in vitro adhesion and migration assays of stable cell lines and tumors grown from these cell lines in nude mice. We also compared the results with the expression profiles of laminins and their receptors in pancreatic carcinomas to evaluate the relevance of these mechanisms in vivo. All of the cell lines preferably adhered to laminin-5, irrespective of their capability to synthesize laminin-5. Cell migration was studied in the presence of hepatocyte growth factor, as it increased the speed of migration manyfold. Herbimycin A treatment and antibodies against the beta 1 and alpha 3 integrin subunits and laminin alpha 3 chain almost entirely blocked cell migration of the BxPC-3 cell line, whereas migration was nearly unaffected by RGD peptide and only moderately inhibited by antibody against the alpha 6 integrin subunit. Indirect immunofluorescence microscopy of wounded BxPC-3 cells suggested a rapid endocytosis of alpha 3 integrin subunit in the cells at the margin of the wound and a rapid, polarized rearrangement of the alpha 6 beta 4 integrin. Especially HGF-treated cultures showed a prominent cytoplasmic reaction for laminin-5 at the margin of the wound. Xenografted cells formed tumors that produced and deposited the same laminin chains as the in vitro cultures. Frozen sections of human pancreatic carcinomas showed reactivity for laminin chains suggestive for expression of laminin-1 and laminin-5. Both xenografted tumors and human pancreatic carcinomas also showed stromal reactivity for laminin-5. Electron microscopy of the human tumors suggested that this was due to an abundant reduplication the basement-membrane-like material around the nests of malignant cells. Our results suggest that pancreatic carcinomas synthesize and deposit laminin-5 in the basement membrane in an abnormal manner. Invading cells adhere to this newly produced basement membrane and migrate on it by using the alpha 3 beta 1 integrin receptor recognizing laminin-5.     

Alpha 6 beta 4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype.

We studied the expression and distribution of different laminin chains, the alpha 6 beta 4 integrin and type VII collagen, i.e., components of the epithelial adhesion complex, in gastric carcinomas and in suggested preneoplastic stages of this malignancy. Intestinal-type gastric carcinomas showed strong reactivity for laminin alpha 1, alpha 3, beta 1, and beta 3 chains, the components of laminin-1 and -5, at the interface between malignant cells and tumor stroma. The reactivities were continuous throughout the carcinomas, even in structures invading through the smooth muscle layers of the gastric wall. The expression of different laminin chains was accompanied by strong polarized reactivity for the alpha 6 beta 4 integrin, which is a receptor for both laminin-1 and laminin-5. Collagen type VII was only occasionally present at sites showing reactivity for laminin-5 and was totally absent from the cell islands invading through the gastric wall. Intestinalized gastric epithelium showed a similar expression pattern of laminins and the alpha 6 beta 4 integrin as the gastric carcinomas. Our results suggest that gastric carcinomas use the alpha 6 beta 4 integrin and newly deposited laminin-1 and -5, accompanied by the disappearance of type VII collagen, as their mechanism of adhesion during the invasion through surrounding tissues. Unlike in previous studies, the reactivity for the laminin-5 protein was not restricted to the invading cells but surrounded the malignant glandular structures throughout the tumor. Our results also show that both intestinal-type gastric carcinoma, and intestinal metaplasia mimic the gastric surface epithelium in the expression pattern of laminins and the beta 4 integrin subunit. This supports previous studies proposing a pathogenetic sequence from intestinal metaplasia to gastric carcinoma.     

Differential expression of laminin chains and their integrin receptors in human gastric mucosa.

The proliferating cells of the gastric mucosa are found among the pit and mucous neck cells. These cells migrate upward to renew the surface epithelium and downward to restitute the glandular cells. As the epithelial basement membranes (BMs) function as substrate for cell adhesion and migration as well as signals for their differentiation, we studied, by indirect immunofluorescence microscopy, the distribution of different laminin chains and their integrin receptors in adult human stomach. The immunoreactivity for laminin alpha 2 chain localized to the BMs of glands and the lower parts of the gastric pits whereas the laminin alpha 3 chain (laminin-5/kalinin) immunoreactivity was strictly confined to BMs underneath the surface epithelium and the upper parts of the pits. Proliferating mucosal epithelial cells, identified by Ki-67 antibodies, were confined to the areas containing both alpha 2 and alpha 3 laminin chains. The alpha 1, beta 1, and gamma 1 laminin chains were found in all BMs of the mucosa whereas the beta 2 chain was prominent in mucosal blood vessels and also detectable in some glands. Among the laminin integrin receptors, the alpha 3 and beta 4 subunits were seen to be expressed in cells along the BMs with the alpha 3 laminin chain. The alpha 6 integrin, on the other hand, was seen in all gastric epithelia. The present results demonstrate that in the adult human stomach laminin alpha 2 and alpha 3 chains show zonal distribution in BM underlying gastric mucosal epithelium whereas other laminin chains show a more general distribution.     

Laminin isoforms of lymph nodes and predominant role of alpha5-laminin(s) in adhesion and migration of blood lymphocytes

During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains and that the same chains, in addition to alpha2, are found in RFs. In functional studies with laminin isoforms covering all Lm alpha chains, alpha5-laminin (Lm-511) was the most adhesion- and migration-promoting isoform for human blood lymphocytes, followed by alpha3- (Lm-332) and alpha4- (Lm-411) laminins, and the lymphocytes used the alpha6beta1 integrin as the primary receptor for the alpha5-laminin. Moreover, Lm-511 strongly costimulated T cell proliferation, and blood lymphocytes were able to secrete alpha4- and alpha5-laminins following stimulation. The LN cell number in laminin alpha4-deficient mice compared with wild-type did not differ significantly. This study demonstrates a predominant role for alpha5-laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.     

Laminin receptors in the integrin family.

Integrins are membrane receptors, consisting of an alpha and a beta subunit, which are involved in cell adhesion. Their extracellular domain is able to bind to ligands such as laminin which occurs in basement membranes of various kinds of cells. Most of these integrins, with their intracellular domains, interact with the actin-containing cytoskeleton, via linking proteins such as vinculin and talin, while one of them interacts with the keratin filaments, via an as yet unknown linking molecule(s). Among more than eighteen integrins which have been identified to date, integrins alpha 3 beta 1 and alpha 6 beta 1 have been characterized as laminin receptors. They recognize the laminin long arm E8 fragment obtained after elastase digestion of the molecule. The binding requires the presence of divalent cations which bind to specific sites on the integrin alpha subunit. The affinities of the alpha 3 beta 1 and alpha 6 beta 1 integrins for murine and human laminin are different, which is probably depended on the existence of different isoforms of laminin. When cells have adhered to laminin, the alpha 6 beta 1 integrin localizes in focal contacts in which actin microfilaments are anchored to the plasma membrane. Whether another integrin, the alpha 6 beta 4 complex, of epidermal cells is also a laminin receptor has not yet been confirmed. The alpha 6 beta 4 integrin localizes in hemidesmosomes which are attachment structures to the substratum where intermediate (keratin) filaments are anchored.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin.

The surface of a normal ovary is covered by a monolayer of epithelial cells that rest on a basement membrane. The glycoprotein laminin is the major noncollagenous protein present in the basement membrane. The integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4 serve as cell surface receptors for laminin. During the progression of serous ovarian carcinoma, tumor cells are frequently exfoliated from the surface of the ovary, thereby losing contact with the basement membrane. This study was designed to determine whether alterations in integrin expression may be associated with the malignant phenotype of the primary ovarian tumor and exfoliated ovarian carcinoma cells in the ascites fluid. By immunohistochemical staining, the entire surface of epithelial cells of normal ovaries stained positively for beta 1, alpha 2, and alpha 3 integrins, whereas only the basal surface of the epithelial cells, where they are in contact with laminin, stained positively for alpha 6 and beta 4. The entire surface of epithelial cells of solid tumors from patients with serous ovarian carcinoma stained positively for beta 1, alpha 2, and alpha 3 integrins. In most cases, no intact basement membrane surrounded the tumor nests, and staining for alpha 6 and beta 4 was irregular. When present, the basement membrane stained positively for laminin, and the basal surface of the epithelial cells stained positively for alpha 6 and beta 4. Ovarian carcinoma ascites cells exhibited a distinct phenotype, with a significant decrease in expression of the alpha 6 and beta 4 integrin subunits. As alpha 6 and beta 4 integrin subunits are present at the basal surface of many epithelial cells and serve as receptors for laminin, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of alpha 6 and beta 4 integrin subunits.     

Laminin alpha1 chain in human renal cell carcinomas and integrin-mediated adhesion of renal cell carcinoma cells to human laminin isoforms

In human tissues, the laminin (Ln) alpha1 chain shows a restricted and developmentally regulated distribution in basement membranes (BMs) of a subset of epithelial tissues, including those of renal proximal convoluted tubules. The present study investigated the distribution of the Ln alpha1 chain in renal cell carcinomas (RCCs) and oncocytomas as well as in xenografted tumours induced in nude mice with four characterized RCC cell lines. These cell lines were also used in cell adhesion studies with purified laminins. By immunohistochemistry it was found that the Ln alpha1 chain is widely present in the BMs of RCCs, all of the specimens presenting immunoreactivity. High-grade RCCs tended to contain more BM-confined and stromal immunoreactivity than low-grade tumours, none of the grade 3 (G3) carcinomas being negative and all of the metastatic specimens showing partial or overall BM immunoreactivity. Double immunolabelling experiments showed that in RCC BMs but not in vessel walls, the Ln alpha1 chain was co-distributed with Ln alpha5, beta1, and beta2 chains, implying the presence of Ln-1/Ln-3 and Ln-10/Ln-11. In papillary RCCs, the Ln alpha1 chain co-localized with Ln-5. The oncocytomas lacked immunoreactivity for the Ln alpha1 chain. Xenografted tumours induced in nude mice showed BM-like deposition of the Ln alpha1 chain. In cell adhesion studies, mouse and human Ln-1 were equally effective in promoting cell adhesion of all RCC cell lines. For each cell line, Ln-10 and Ln-10/11 were equally effective adhesive substrates, all cell lines adhering more avidly to these laminins than to mouse or human Ln-1. As judged by inhibition assays employing specific integrin antibodies, adhesion of normal human renal proximal tubular epithelial (RPTE) cells and RCC cells from a G1 tumour to human Ln-1 was mediated mainly by alpha(6)beta(1) integrin, while only the G1 RCC cells adhered to mouse Ln-1 by using alpha(6)beta(1) integrin. For adhesion to Ln-10, RPTE cells and RCC cells from a G1 tumour used an unidentified beta(1) integrin. Cells from G3 tumours mainly used an alpha(3)beta(1) integrin complex for adhesion to mouse Ln-1 and to human Ln-1 and Ln-10. For all cells, adhesion to the Ln-10/11 mixture was mediated by an unidentified integrin complex or by other adhesion molecules. These results show that laminin trimers containing the alpha1 chain are, in contrast to oncocytomas, abundant in the BMs of RCCs. This is in keeping with their suggested origin from renal proximal tubular epithelium known for its capacity to produce the Ln alpha1 chain. The results also show that RCC cells utilize complex, mainly integrin alpha(3)beta(1)- and integrin alpha(6)beta(1)-mediated, mechanisms for adhesion to laminins. The adhesion to Ln-1 changes from integrin alpha(6)beta(1) to integrin alpha(3)beta(1) upon increasing malignancy and, especially for Ln-10 and Ln-10/11, other adhesion molecules of non-integrin type may contribute to the adhesion.     

Beta4 integrin and laminin 5 are aberrantly expressed in polycystic kidney disease: role in increased cell adhesion and migration

Extracellular matrix alterations have been suggested to be part of the early events occurring in Autosomal Dominant Polycystic Kidney Disease (ADPKD), a disease characterized by formation of renal cysts and progressive renal failure. Here we report that cDNA array analysis identified beta(4) integrin aberrant expression in ADPKD cells. Furthermore, laminin 5 (Ln-5), the main alpha(6)beta(4) integrin ligand, was also found to be abnormally expressed in ADPKD. Studies performed with ADPKD cyst-lining epithelial cells (CC) by comparison with normal tubular cells indicate that integrin alpha(6)beta(4)-Ln-5 interactions are involved in cellular events of potential importance for cystogenesis: 1) laminin 5 is a preferential adhesion substrate for CC, mainly through alpha(6)beta(4) interaction, 2) CC increased haptotactic and chemotactic motility depends on the presence of Ln-5 and requires integrin alpha(3)beta(1) cooperation, and 3) CC haptotactic or chemotactic migration is specifically increased by mAb-mediated beta(4) integrin ligation, through an alpha(3)beta(1) integrin-dependent and independent pathway, respectively. These results highlight the role of Ln-5 and alpha(6)beta(4) integrin in adhesive and motility properties of cyst-lining epithelial cells, and further suggest that integrins and extracellular matrix modifications may be of general relevance to kidney epithelial cell cyst formation.     

Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse.

Cells interact with the extracellular matrix through receptors, most commonly of the integrin family. We (Cachaco et al. [2003] Development 130:1659-1671) and others (Schwander et al. [2003] Dev. Cell 4:673-685) have demonstrated a role for beta1 integrins in mouse primary myogenesis. However, it is unclear what alpha subunits pair with beta1 during this process in vivo. Here, we determined alpha subunit expression patterns at embryonic day (E) 11.5-E14.5. Differentiated myotomal myocytes express all alpha subunits studied. As the muscle masses form both in trunk (E12.5) and limbs (E11.5-E12.5), laminin receptors alpha6beta1 and alpha7beta1 are undetectable, and an assembled laminin matrix is absent. Instead alpha1beta1, alpha4beta1, alpha5beta1, and an alpha v-containing integrin are expressed and unassembled laminin and fibronectin are abundant around myogenic cells. At E13.5-E14.5, alpha6beta1 and alpha7beta1 are expressed, and a laminin matrix forms around individual myotubes. Thus, myogenic cells change their integrin expression pattern during the course of primary myogenesis in the mouse, suggesting different roles for fibronectin- and laminin-containing matrices in this process.     

Expression of integrin family of cell adhesion receptors in rheumatoid synovium. Alpha 6 integrin subunit in normal and hyperplastic synovial lining cell layer.

Integrins are heterodimeric cell adhesion receptors. The beta 1 integrin subunit can be in a complex with multiple a subunits and form receptors for collagen, laminin, fibronectin, and vitronectin. We have characterized the distribution of eight integrin subunits in rheumatoid synovium, with special interest in the lining cell layer. The beta 1 integrin subunit was found in abundance in synovial stroma and in lining cells. The only alpha subunit seen constantly in lining cells was alpha 6. In complex with alpha beta subunit, alpha 6 forms a laminin receptor usually seen in epithelial or endothelial cells or in macrophages. The fact that laminin was found in the extracellular matrix around synovial cells suggests the importance of alpha 6 integrin in the adhesion of synovial lining cells. Furthermore, alpha 6 expression was noticeably weaker in strongly proliferative lining cell layers, indicating that the inflammatory process may regulate integrin expression. A potential connection between altered expression of cell adhesion receptors and the pathological behavior of rheumatoid lining cells is suggested.     

Adhesion of menstrual endometrium to extracellular matrix: the possible role of integrin alpha(6)beta(1) and laminin interaction

Previous in-vitro studies have shown that the endometrium preferentially adheres to the extracellular matrix (ECM) of the amnion and peritoneum. This interaction probably involves adhesion molecules, e.g. integrins. We evaluated the expression of integrins in naturally shed menstrual endometrium and the adhesion pattern of this tissue to different components of the ECM. To identify integrins and matrix components involved, blocking studies were performed. Most of the 15 menstrual tissue samples showed positive staining for each of the integrins investigated, except alpha(4)beta(1). Compared with binding to collagen IV, which was set at 100%, adhesion to collagen I was 93% (not significant), to fibronectin 87% (P < 0.05), and to laminin 74% (P < 0.05). Scanning electron micoscopy showed that endometrium adhered to laminin but hardly spread, whereas spreading was observed when layered on the other coatings. Compared with the control (which was set at 100%), incubation with 4B4, a monoclonal antibody against the integrin beta(1) subunit, showed a significant reduction of adhesion (to approximately 50%; P < 0.05) when layered on laminin and a smaller reduction (to 82-86%; P < 0.05) when layered on the other three coatings. Incubation with antibody GOH3 against integrin alpha(6)beta(1) resulted in a similar reduction in adhesion to laminin. Incubation with an RGD peptide significantly reduced adhesion (to 84%; P < 0.05) when plated on fibronectin. In conclusion, antegradely shed menstrual endometrium expresses various integrins. It shows preferential attachment to collagen IV and collagen I, when compared with fibronectin and laminin. Blockage of the integrin beta(1) subunit resulted in greatest disruption to adhesion when layered on laminin, implying that the interaction was mediated by the alpha(6)beta(1) integrin. Since this adhesion was not completely blocked, other mechanisms are likely to be involved.     

Integrin distributions in renal cell carcinomas of various grades of malignancy.

We studied 41 renal cell carcinomas, classified according to histologic grades G1 through G3, by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (MAb) against various integrin subunits, and the basement membrane (BM) components laminin and collagen type IV. Selected cases also were immunostained using the avidin-biotin-complex method. The alpha 3 and beta 1 integrin subunits were detected in tumor cells of all the carcinomas. All G1 carcinomas, like normal tubular epithelial cells, expressed the alpha 6 subunit, whereas it was lacking in 20% and 40% of G2 and G3 carcinomas, respectively. Furthermore, when alpha 6 was expressed, a lack of basally polarized organization of the subunit, coupled with disorganization of the BM components, correlated with histologic grade. Another feature that appeared to characterize the more anaplastic tumors was their high level (80%) of the alpha v subunit expression as compared with its absence in the G1 carcinomas. Stromal myofibroblasts, identified by double-labeling with anti-myosin, were often characterized by the expression of the alpha 1, alpha 3, alpha 5 and beta 1 subunits. These results indicate that changes in integrin expression in renal cell carcinomas may be correlated with their degree of histologic malignancy.     

Integrin alpha2beta1 on rat myeloma cells modulates interaction of alpha4beta1 integrin with vascular cell adhesion molecule-1 but not fibronectin

It is well established that alpha2beta1 integrin functions as a receptor for collagen and laminin; whereas alpha4beta1 integrin binds fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In the present study, we showed that rat myeloma YB2/0 cells constitutively expressed alpha4beta1 but not alpha2beta1 integrin. Transfection of cDNA of mouse a2 integrin subunit resulted in the expression of heterologous alpha2beta1 integrin on YB2/0 cells (YBmalpha2). The expression of alpha2beta1 conferred YBmalpha2 cells the ability to interact with collagen and laminin. In comparison with mock transfected YB2/0 cells (YBpF), YBmalpha2 cells exhibited increases in the binding and migration on VCAM-1; in contrast, both YBpF and YBmalpha2 were similar in their interactions with fibronectin or fibronectin fragment FN-40 that contains the binding site for alpha4beta1 integrin. The interaction of alpha4beta1 with VCAM-1 was further stimulated upon ligation with alpha2beta1-specific mAb. The use of specific inhibitory mAb demonstrated the role of alpha4beta1 in mediating the observed interactions with fibronectin and VCAM-1. Therefore, results show that expression of alpha2beta1 differentially regulated alpha4alpha1 integrin function by stimulating its interactions with VCAM-1 but not fibronectin. The in vivo significance of alpha2beta1 integrin expression was demonstrated by intravital videomicroscopy showing that ligation of alpha2beta1 enhanced alpha4beta1-mediated extravasation of YBmalpha2 cells in the liver.     

Defective laminin 5 processing in cylindroma cells

Cylindromas are benign skin tumors occurring as multiple nodules characteristically well circumscribed by an excess of basement membrane-like material. To determine the molecular defects leading to extracellular matrix accumulation, the ultrastructural, immunological, and biochemical properties of cylindroma tissue and isolated cells were analyzed. In cylindromas, hemidesmosomes are reduced in number, heterogeneous and immature compared to the normal dermal-epidermal junction. Expression of the alpha6beta4 integrin in tumor cells is weaker than in basal keratinocytes of the epidermis. Moreover, although in the epidermis alpha2beta1-integrin expression is restricted to the basal cell layer, it is found in all neoplastic cells within the nodules. Laminin 5 is present throughout the whole thickness of the basement membrane-like zone whereas laminin 10 is restricted to the interface adjacent to the tumor cells. Furthermore, laminin 5 is not properly processed and most of the alpha3A and gamma2 laminin chains remain as 165-kd and 155-kd polypeptides, respectively. Mature laminin 5 is thought to be necessary for correct hemidesmosome and basement membrane formation and its abnormal processing, as well as the low expression of alpha6beta4 integrins, could explain the lack of mature hemidesmosomes. Together, the results show that multiple molecular defects, including alteration of laminin 5 and its integrin receptors, contribute to structural aberrations of the basement membrane and associated structures in cylindromas.     

Functional sites in the laminin alpha chains

Laminins, heterotrimers composed of alpha, beta, and gamma chains, are multifunctional glycoproteins present in basement membranes. Laminins, the most important component of basement membranes during basement membrane assembly in early development, are involved in various biological activities such as cell adhesion, migration, growth, differentiation, tumor metastasis, and angiogenesis. Fully 15 laminin isoforms have been identified and are tissue- and/or developmental stage-specifically expressed. Integrins, dystroglycan, syndecans, and the other several cell surface molecules are cellular receptors for laminins. The globular domains located in the N- and C-terminus of the laminin alpha chains are critical for interactions with the cellular receptors. There are highly conserved functional sites and chain-specific functional sites among the laminin alpha chains. Additionally, laminins are processed by specific endogenous proteases and the processing regulates laminin functions. Binding of the functional sequences in laminins to the cellular receptors triggers intracellular signaling, followed by inducing various cell activities including cell spreading and migration. Laminins possess multifunctional sequences and are key molecules that determine cell fate.     

Laminin expression by human periodontal ligament fibroblasts

Our previous study demonstrated that a laminin-like molecule produced by periodontal ligament fibroblasts (PLFs) induces gingival epithelial cell chemotaxis. The aim of this study was to identify the laminin isoforms that are expressed by PLFs. Proteins in PLF-conditioned medium from serum-free cultures were separated by gel filtration followed by gelatin-affinity chromatography to remove fibronectin. Protein expression of laminin isoforms was determined using Western blotting, and mRNA expression was examined by RT-PCR. Partially purified laminin evoked gingival epithelial cell chemotaxis, and this activity was blocked by anti-integrin alpha3, alpha6, and beta1 antibodies. Although RT-PCR analysis showed PLFs expressed laminin alpha1 to alpha5, beta1 to beta3, gamma1, and gamma2 chain mRNAs, the predominant laminin chains detected by Western blotting were alpha4, alpha2, beta1, beta2, and gamma1. These results suggest that PLFs secrete mainly laminin-8/9 (alpha4beta1gamma1/alpha4beta2gamma1) and laminin-2/4 (alpha2beta1gamma1/alpha2beta2gamma1). PLF-derived laminins may be involved in the pathogenesis and progression of periodontitis by inducing apical migration of epithelial cells in certain circumstances.     

Distribution of laminin 5, integrin receptors, and branching morphogenesis during human fetal lung development.

The role of the epithelial adhesion ligand laminin 5 (LN5) in lung development has been poorly investigated. To determine its potential involvement in lung organogenesis, we used immunofluorescence microscopy to investigate the distribution of LN5 and its integrin (Int) receptors alpha2beta1, alpha3beta1, alpha6beta1, and alpha6beta4 during human fetal airway branching morphogenesis and respiratory epithelium differentiation. At the pseudoglandular and canalicular stages of airway development, LN5 and its constituent chains were localized in the basement membrane (BM) of the proximal respiratory tubules and in the cytoplasm of the epithelial cells forming the growing epithelial buds, which expressed Int alpha2beta1, alpha3beta1, and, transiently, alpha6beta1. At the alveolar and adult stages, LN5 and its constituent chains were localized both in the BM of evolving and differentiated bronchioles and in the alveolar parenchyma. The bronchiolar epithelium markedly expressed Int alpha2beta1 and alpha3beta1, whereas the alveolar parenchyma strongly expressed Int alpha2beta1, alpha3beta1, and alpha6beta1. Throughout fetal development and in the adult, LN5 and its constituent chains were detected both in the tracheal BM, regardless of the degree of epithelial differentiation, and in the cytoplasm of the cells at the invading front of the growing glandular ducts. Ultrastructural studies showed that nucleation of the hemidesmosomes (HDs) correlated with the differentiation of the tracheal epithelium. These results suggest that LN5 may play multiple roles during branching morphogenesis, by modulating proliferation and/or migration of the epithelial cells in the respiratory buds and by establishing branch points, through interaction initially with Int alpha6beta1 and later with Int alpha2beta1 and alpha3beta1. We also propose that LN5 may regulate the differentiation of the tracheal epithelium by means of Int-beta4, which governs HD nucleation.     

Developmentally regulated interactions of human thymocytes with different laminin isoforms

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.     

Oxidized laminin‐1 induces increased monocyte attachment and expression of ICAM‐1 in endothelial cells

Atherosclerosis has been associated with increased oxidative stress and monocyte recruitment by endothelial cells. Sub‐endothelial basement membrane proteins, such as laminins that play a central role in cell adhesion, are exposed to reactive oxygen species. In the present study monocyte attachment on human umbilical cord vein endothelial cells (HUVEC) that were preattached to oxidized or native laminin, was investigated. Intracellular cell adhesion molecule‐1 (ICAM‐1) expression by HUVEC was estimated by an enzyme‐linked immunosorbent assay. HUVEC attachment to oxidized or native laminin‐1 was examined using the Hemacolor kit. Anti‐alphaL, anti‐alphaM, anti‐alpha2 and anti‐beta2 integrin subunit antibodies were used in order to further investigate the above phenomena. HUVEC that were preattached to oxidized laminin expressed higher levels of ICAM‐1 and monocytes attached at a higher degree to these cells as compared to HUVEC that were preattached to native laminin. Incubation of monocytes with monoclonal antibodies against the alphaM and beta2 integrin subunits equalized the above mentioned differences. Moreover, HUVEC attached to oxidized laminin at a higher degree as compared to native laminin. This difference was equalized after incubation with the antibody against the alpha2 integrin subunit. These results indicate a modified interaction between HUVEC and the basement membranes in cases where laminin is oxidatively modified. This modified interaction results in increased ICAM‐1 expression by endothelial cells and consequently increased monocyte recruitment capacity.     

Leucocyte recruitment during enteric nematode infection

Resolution of infection with the intestinal nematode Trichinella spiralis depends on the host mounting a T helper 2 (Th2) response. It is known that both mast cells and T cells play a crucial role. We have previously shown that efficient migration of mast cells to the gut during infection depends on their expression of the integrin beta 7. beta 7 forms a heterodimer complex with either alpha E or alpha 4 integrin chains, alpha E beta 7 binding to E-cadherin expressed by epithelial cells and alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule (MAdCAM-1) on the endothelium. We were interested to know whether dysfunctional mast cell localization to the gut in the absence of beta 7 was due to the failure of alpha 4 beta 7 to bind to MAdCAM-1 or the failure of alpha E beta 7 to bind to E-cadherin. We used blocking monoclonal antibodies against alpha E (M290) or alpha 4 (PS2) or beta 7 (HB293) during T. spiralis infection of C57BL/6 mice and found that all antibody treatments reduced mastocytosis. In contrast, none of the antibody treatments prevented the migration of CD3(+) T cells into the intestine. These results indicate that during inflammation (a) there is integrin redundancy for lymphocytes but not for mast cells and (b) both alpha E beta 7 and alpha 4 beta 7 are crucial either for the entry of mast cells into the gut or for their maturation once they have entered.