Polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma: distinct architectural composition revealed by collagen IV, laminin and their integrin ligands (alpha2beta1 and alpha3beta1)

Aims: Polymorphous low‐grade adenocarcinoma (PLGA) and adenoid cystic carcinoma (ACC) are malignant salivary gland tumours bearing many similar histological patterns. This study was undertaken to show how the presence and distribution of collagen IV and laminin, and their ligands (integrin α2β1 and α3β1 components), can reveal histoarchitectural differences which distinguish these two entities. Methods and results: Five cases of ACC and five cases of PLGA from the archives of the Oral Pathology Department of the School of Dentistry of the São Paulo University were submitted to immunostaining with the antibodies to collagen IV, laminin, and integrins α2β1 and α3β1 using the streptavidin–biotin–peroxidase technique. Positive and negative controls were included. PLGA showed a thin line of collagen IV and laminin surrounding structures composed of a single cell layer. Integrins were expressed as a widespread and granular pattern. A thick line of collagen and laminin was observed around the neoplastic structures of ACC. Both integrins were expressed in intercellular spaces and around luminal spaces of tubular structures. Conclusions: Collagen IV and laminin, and their integrin ligands, are useful in demonstrating that neoplastic ductal units of PLGA are composed of a single cell layer, being distinct from ACC which contains structures composed of two layers of neoplastic cells.     

Second generation monoclonal antibodies to the human integrin alpha 6 beta 4

Second generation monoclonal antibodies to the alpha 6 beta 4 subunits of human integrins have been prepared. MAbs 450-9D, 10D, and 11A1 react at different sites on the beta 4 molecule and MAbs 450-30A1 and 33D react at the same site on the alpha 6 subunit. Double determinant (two-site) radioimmunoassays using combinations of these MAbs have been developed. Two assays for beta 4 distinguish between the whole beta 4 molecule and the beta 4 molecule truncated from the C-terminus (form c) while another assay measures the presence of alpha 6 subunits. Data from the two-site assays support the following conclusions: (1) Colon tumors and normal colon mucosa express large amounts of alpha 6 beta 4 although only form c of the beta 4 was detected; (2) There is no evidence for alpha 6 beta 1 expression in colon; however, some of this complex may be present in certain lung tumors. The extracellular domains of alpha 6 and beta 4 can associate with each other even if the cytoplasmic domain of the beta 4 subunit is not present. MAbs to specific domains of the beta 4 molecule may be useful in analyses of forms a and c in normal and malignant tissue. The fact that only the largest beta 4 molecule "a" retains the phosphorylation site may have functional significance.     

Expression of integrin alpha5 and integrin beta4 and their extracellular ligands fibronectin and laminin in human decidua during early pregnancy and its sex steroid-mediated regulation

The reorganization of the human endometrium is termed decidualization, which includes endometrial cell proliferation, differentiation, integrin switching and extracellular matrix (ECM) remodeling during early pregnancy. The present study aimed to investigate distribution patterns, staining intensity and sex steroid-mediated regulation of integrin alpha5 (CD49e), integrin beta4 (CD49f) expression and their ligands fibronectin and laminin during decidualization. Human tissue samples were evaluated in two groups, those collected in early days and those collected in advanced days of the first trimester. Correlating immunostaining was found between laminin and integrin beta4, and between fibronectin and integrin alpha5. The expression of fibronectin was higher than that of laminin in the early days (p < 0.05). Temporal and spatial immunostaining of integrin beta4 and alpha5 in the apical pole of luminal and glandular cells was observed as pregnancy progressed (p < 0.05). In vitro results showed that human chorionic gonadotropin (hCG) stimulated laminin expression, downregulated integrin beta4 expression, whereas estradiol decreased fibronectin expression by Ishikawa cells. hCG suppressed fibronectin expression in endometrial stromal cells in culture. Our results suggest that fibronectin is responsible for induction of decidual cell differentiation, and different temporal and spatial expression of the integrins may play a role in implantation. Our in vitro results suggest that regulation of extracellular matrix remodeling and integrin switching are at least partially regulated by reproductive hormones.     

Oestrogen receptor alpha and beta mRNA expression in human endometrium throughout the menstrual cycle.

We examined the localization of oestrogen receptor (ER) beta mRNA in the human endometrium throughout the menstrual cycle using non-radioactive in-situ hybridization with Brigati-tailed oligonucleotides. The findings were compared with those of ERalpha in order to examine the possible biological significance of ERbeta in the human endometrium. Both ERalpha and ERbeta mRNA expression were detected in all major human uterine cell types, including glandular epithelial cells, stromal cells and smooth muscle cells of the uterine wall, at every menstrual cycle stage. However, ERalpha mRNA expression was more prominent than that of ERbeta in all cell types throughout the menstrual cycle. In proliferative phase endometrium, ERalpha mRNA was expressed in both glandular epithelial and stromal cells, while ERbeta mRNA was expressed predominantly in glandular epithelial cells. Although the same pattern was observed in the secretory phase, both the ERalpha and ERbeta mRNA expression was relatively weaker. These results suggest that oestrogenic effects occur predominantly through ERalpha, but that ERbeta may also play a role in the modulation of oestrogenic action, especially on glandular epithelial cells in the human endometrium throughout the menstrual cycle.     

Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor of the liver; prognosis depends on the tendency to metastasize. Cancer cell invasion is regulated by proteolytic remodeling of extracellular matrix components and by integrin expression. We have shown that matrix metalloproteinase-2 (MMP-2) and membrane-type-1 matrix metalloproteinase (MT1-MMP) cleave Laminin-5 (Ln-5), stimulating cell migration. Here we report that all HCC cells express MT1-MMP, migrate on Ln-1 and Collagen IV, whereas only HCC cells that express alpha3beta1 integrin secrete detectable levels of gelatinases, migrate on Ln-5, and invade through a reconstituted basement membrane (BM). Migration on Ln-5 is blocked by BB-94, an MMP inhibitor, and by MIG1, a monoclonal antibody that hinders migration on MMP-2-cleaved Ln-5. Invasion through a reconstituted BM is also inhibited by BB-94. HCC alpha3beta1-negative cells migrate on Ln-1 and Collagen IV, but not on Ln-5, and do not invade through a reconstituted BM, although they express MT1-MMP. Anti-alpha3beta1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through MATRIGEL: In vivo, alpha3beta1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both alpha3beta1 integrin and gelatinase activity are required for HCC migration and invasion.     

Characterization of adhesive interactions between mast cells and laminin isoforms: evidence of a principal role for alpha 6 integrin.

Mast cells are known to adhere to laminin, although there is limited information on the characteristics of this event. To further examine this adhesive interaction, we thus determined the adherence of murine mast cell lines and primary bone marrow cultured mast cells (BMCMC) to murine laminin (mLN), human placental laminin-1 (hLN), merosin (laminin-2) and various laminin fragments, concentrating on activating stimuli, the involvement of integrins, and the effect on mast cell activation. Murine mast cells were found to adhere to both mLN and hLN and to merosin, not only following exposure to phorbol 12-myristate 13-acetate (PMA), but also after Fc epsilon RI aggregation or addition of stem cell factor (SCF). Adhesion to laminin was partially inhibited by soluble E8 and PA22-2, both fragments of laminin that promote mast-cell adhesion when bound on surfaces. Mast-cell lines and BMCMC consistently expressed high levels of alpha 6 integrin. Antibody to alpha 6 blocked spontaneous and inhibited activated mast-cell adhesion to hLN, and inhibited mast-cell adhesion to mLN and its fragment E8. Mast-cell adhesion to both laminin isoforms increased Fc epsilon RI-mediated mast-cell secretion. These observations demonstrate that mast-cell attachment to laminin is promoted by physiological stimuli, is mediated principally by alpha 6 integrin, and results in enhanced cell activation.     

Substrate architecture and fluid-induced shear stress during chondrocyte seeding: role of alpha5beta1 integrin

Chondrocyte behaviour has been shown previously to be influenced by the architecture of the substrate on which the cells are grown. Chondrocytes cultured on fully porous titanium alloy substrates showed greater spreading and more matrix accumulation when compared to cells grown on porous-coated substrates with solid bases. We hypothesized that these features developed because of differences in fluid-induced shear stresses due to substrate architecture and that integrins mediate these responses. Computational fluid dynamics analyses predicted that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on porous-coated substrates with solid bases where media flow-through is restricted. To validate this model, the seeding protocol was modulated to affect fluid flow and this affected cell spreading and matrix accumulation as predicted. Integrin blocking experiments revealed that alpha5beta1 integrins regulated cell shape under these two conditions and when cell spreading was prevented the increased accumulation of collagen and proteoglycans by chondrocytes seeded on fully porous substrates did not occur. Identifying the substrate-induced mechanical and molecular mechanisms that influence chondrocyte behaviour and tissue formation may ultimately lead to the formation of a tissue that more closely resembles natural articular cartilage.     

Invasion of human respiratory epithelial cells by Bordetella pertussis: possible role for a filamentous hemagglutinin Arg-Gly-Asp sequence and alpha5beta1 integrin

Bordetella pertussis, the agent of whooping cough, is capable of invading human respiratory epithelial cells. In this study, we investigated the mechanisms by which B. pertussis invades the human lung epithelial cell line A549 and normal human bronchial epithelial (NHBE) cells. In vitro adhesion and invasion assays using both cell types with a virulent B. pertussis strain and its isogenic mutants revealed profound defects in a mutant deficient in filamentous hemagglutinin (FHA) expression. In addition, a mutant in which an FHA Arg-Gly-Asp (RGD) site had been changed to Arg-Ala-Asp had significantly diminished invasiveness, although its adhesiveness was comparable to that of the parental strain. Furthermore, a synthetic RGD-containing hexapeptide inhibited invasion of both cell types by the virulent strain. These results demonstrate that an RGD sequence of FHA is involved in B. pertussis invasion of epithelial cells in vitro. Monoclonal antibodies directed against human alpha5beta1 integrin, but not other integrins, blocked invasion, indicating that this integrin is involved in B. pertussis invasion. Taken together, these findings suggest that B. pertussis FHA may promote invasion of human respiratory epithelial cells through the interaction of its RGD sequence with host cell alpha5beta1 integrin.     

Adhesion strength of human tenocytes to extracellular matrix component-modified poly(DL-lactide-co-glycolide) substrates

We report a direct measurement of the adhesion strength of human embryonic tenocytes (HETCs) and transformed human embryonic tenocytes (THETCs) to fibronectin (FN)- and type I collagen (CNI)- modified poly(DL-lactide-co-glycolide) (PLGA) substrates with a micropipette aspiration technique. PLGA substrates were first coated with poly-D-lysine (PDL), and then with various concentrations (1 microg/ml, 2 microg/ml, 5 microg/ml, and 10 microg/ml) of FN and CNI in serum-free F12 media. Anti-FN and Anti-CNI antibodies were used to inhibit attachment of tenocytes to FN- and CNI- modified substrates in a dilution range of 1:5000-1:500 and 1:1500-1:250, respectively. The substrates were employed for incubation of HETCs and THETCs for 30 min at 37 degrees C before the adhesion strength measurements. We found that the adhesion strengths showed a strong dependence on the seeding time and FN or CNI concentrations. Anti-FN and Anti-CNI antibodies significantly compromised adhesion of HETCs and THETCs to the corresponding modified substrates (P < 0.05). These findings show that FN- or CNI-modified polymer substrates offer significant advantages for tissue engineering tendon scaffolds concerning tenocyte adhesion. In addition, HETCs and THETCs bear similar biological behaviors in terms of adhesion, indicating the possibility of using THETCs in place of HETCs in tissue engineering construction of human tendons.     

Stage-specific alternative cplicing of CD44 and alpha 6 beta 1 integrin in colorectal tumorigenesis

Recent reports suggest that cancerogenesis induces changes in alternative processing of human genes. However, little is known about the regulation of alternative splicing during malignant transformation. Therefore, we examined changes in alternative splicing of two different adhesion molecules, alpha 6 beta 1 integrin and CD44, in multiple stages of colon tumorigenesis. Using semiquantitative RT-PCR it is shown that the alternatively spliced isoforms of both adhesion molecules, alpha 6A and -B and CD44v6, are significantly upregulated in colorectal adenoma (n = 20) compared to normal colon mucosa (n = 32) (P < 0.01). Although beta1 isoforms were expressed in almost all tissues, there was a significant increase in the intensity of gene expression of beta 1A compared to beta 1B (P <0.05) in adenoma tissue. Interestingly, CD44v6 and alpha 6 variant isoforms were downregulated in carcinoma tissue (n = 28) compared to adenoma. These results establish a link between neoplastic transformation and alternative splicing of cell adhesion molecules. Furthermore, these data suggest that colon epithelial cells carrying splice variants of adhesion molecules might acquire a selective growth advantage during early tumorigenesis.     

Aphthous stomatitis (canker sores): A consequence of high oral submucosal viscosity (the role of extracellular matrix and the possible role of lectins)

Recurrent aphthous stomatitis (RAS) or canker sores occur in 20-60% of all persons. The lesion occurs because of increased viscosity of oral submucosal extracellular matrix (ECM). The lesions begin in the second decade and peak in the third decade. Sex hormones are an important influence on fibroblasts, especially in the early phase of exposure. Sex hormones are known to concentrate, to a degree, in the bucal mucosa in animals. Lesions of RAS localize clinically and experimentally at sites of trauma. In the skin, edema is known to trigger early cellular inflammation. Increased viscosity of ECM heightens the response. The histopathology of the ulcerated lesions is similar to that which occurs under sites of acute inflammation in the skin. Systemic corticosteroids completely supress the lesions. Caustics, such as silver nitrate and phenol, stop the growth and pain of lesions. Irritants are known to break ECM viscosity. The oral mucosa exerts some control on underlying ECM. Substances such as lectins influencing the mucosa could influence ECM. Soluble substances in food or organisms could also penetrate to influence ECM. A number of different foods have been incriminated as trigger agents in individual cases. This includes gluten in patients with gluten sensitive enteropathy. Gluten is known to alter the mucosa of the small intestine in persons with celiac disease.     

Adhesion to fibronectin primes eosinophils via alpha 4 beta 1 (VLA-4).

Human peripheral blood eosinophils adhered specifically to microtitre plates coated with plasma fibronectin (Fn) in a dose- and time-dependent fashion. Adhesion was optimal at 60 min at a concentration of 100 micrograms/ml. Adherence to Fn was up-regulated by platelet-activating factor (PAF; optimum concentration of 10(-6) M) and was significantly inhibited by a polyclonal anti-Fn antibody (P < 0.05). The following evidence suggested that eosinophil adhesion to Fn was mediated by alpha 4 beta 1: (1) eosinophil adherence to Fn was not inhibited by an Arg-Gly-Asp-Ser (RGDS) synthetic peptide; (2) there was a dose-dependent adherence of eosinophils to microtitre plates coated with the 40,000 MW proteolytic fragment of Fn that contains the CS-1 alpha 4 beta 1 binding region, whereas adherence to the 120,000 MW chymotryptic fragment of Fn, which contains the RGD-dependent binding site, was weak and only observed at high concentrations (> 250 micrograms/ml); (3) significant inhibition of eosinophil adherence to Fn was achieved by monoclonal antibodies (mAb) against the alpha chain of VLA-4 but not by a mAb against CD45 or a mouse myeloma antibody as negative controls. After adhesion to Fn, eosinophils were investigated for their capacity to release leukotriene C4 in response to stimulation with a suboptimal concentration of calcium ionophore (2 x 10(-6) M). Significant enhancement of release was detected with Fn-coated plates but not with the control bovine serum albumin (BSA) (P < 0.01). Furthermore, this enhancement was significantly inhibited by the alpha 4 beta 1 mAb HP2/1 (P < 0.05) but not by an anti-CD45 mAb. From these studies we conclude that (1) alpha 4 beta 1 (VLA-4) integrin is a major receptor for Fn on human eosinophils and (2) adhesion to Fn may prime eosinophils for mediator release during allergic inflammation.     

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.     

Isolation and cultivation of integrin alpha(6)beta(1)-expressing salivary gland graft cells: a model for use with an artificial salivary gland

Regeneration of the salivary glands' (SGs) normal function for patients with cancer of the head and neck treated with irradiation would be a major contribution to their quality of life. This could be accomplished by re-implantation of autologous SG cells into the residual irradiated tissue or by implantation of tissue-engineered artificial SGs. Both methods depend on the isolation of cells able to propagate and differentiate into SG epithelial cells. Recently, it has been shown that SG integrin alpha(6)beta(1)-expressing (SGIE) cells have stem cell capabilities, but these cells could be isolated only after duct ligation insult requiring surgical intervention. Because such an invasive procedure is not clinically acceptable for these patients, our aim in the present study was to explore the use of immuno-magnetic separation of untreated and short heat stress-conditioned rats as a less-insulting methodology for enhancement of these cells. Our results show that submandibular SGIE cells could be isolated and cultivated from untreated animals. However, short heat stress (HS) increased the number of isolated SGIE cells 4.7-fold and their proliferation and clonal capability 4.6-fold and 3 fold, respectively. We believe that SGIE graft cells may be suitable candidates for future tissue-engineered SGs that have been damaged by irradiation in patients with head and neck cancer.     

Lack of integrin alpha1beta1 leads to severe glomerulosclerosis after glomerular injury

Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin alpha1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary alpha1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin alpha1beta1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin alpha1beta1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin alpha1beta1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis.     

Parenteral administration of an activating monoclonal antibody to the alpha1beta1 integrin in dogs

In mice, monoclonal antibody (mAb) to the alpha1 integrin abrogate gastro-intestinal damage during graft-versus-host-disease (GVHD), suggesting anti alpha1 mAb as candidates for treatment in humans as well. Our current data show that one such reagent, mAb 1B3.1, when immobilized to plastic wells via rabbit- anti murine (ram) immunoglobulin (Ig) induces a protein kinase-dependent spreading of activated human T cells. Furthermore, it significantly increases the proliferative response, and expression of interleukin-2 (IL-2) receptors (R) and CD69, of resting T cells, expressing minimal integrin on the cell surface, to sub-optimal stimulation by anti-CD3 mAb. We found, in addition, that mAb 1B3.1 a) immuno-precipitates alpha1beta1 integrins from cell-surface iodinated canine epithelial cells b) is highly reactive with canine T cells after their activation and c) inhibits adhesion of canine T cells to collagen IV. Despite the potential ability of the mAb to co-activate T cells in vitro, two dogs that received 4 injections of 0.5-0.3 mg/Kg of mAb 1B3.1 remained healthy, developing only marginal transient lymphopenia. Injection of 0.75mg/Kg in a third dog induced a more marked lymphopenia, and an additional dose of 1.0 mg/Kg 2 weeks later was followed by gastrointestinal hemorrhage. importantly, the lymphopenia was associated with a greater and more persistent decrease of CD8+ than of CD4+ T cells, leading to an increase in the CD4/CD8 ratio 24 hours after the injection. Thus, despite it's co-activating effects in vitro, administration of this mAb in vivo is feasible when appropriately dosed, and may have immuno-modulatory effects.     

实验性变态反应性神经炎中整合素α6β4的表达及意义

目的 研究实验性变态反应性神经炎(EAN)中整合素α6β4的mRNA表达变化,探讨α6β4与许旺细胞功能状态的关系,以及其表达变化与髓鞘损伤和修复过程的关系。方法 建立Lewis大鼠EAN动物模型,取18只模型鼠坐骨神经,用原位杂交和半定量逆转录-聚合酶链反应(RT-PCR)方法检测模型的不同时期Lewis大鼠神经局部的整合素α6β4表达,以正常鼠(3只)为空白对照。结果 原位杂交结果表明在EAN的发病过程中,α6的表达未见明显变化,而β4的表达在急性期明显减弱,恢复期或后期逐渐恢复。β4表达减弱主要体现为阳性信号的稀疏而不反映为单个信号的量的变化。以GAPDH为内参照,对整合素α6β4的mRNA水平做半定量分析,结果显示：EAN病变过程中,α6的mRNA表达变化不明显,而β4的mRNA呈现早期减少后期逐渐恢复进而高于正常的变化过程。结论 炎症因素造成了许旺细胞变性损伤,影响其整合素的表达,许旺细胞出现了与胚胎发育过程类似的表型转换。从这个意义上,可以把α6β4看作许旺细胞分化状态的一个标志,至少可以看作是标志EAN病变过程的一个重要分子。α6β4表达变化与髓鞘的损伤和修复过程密切相关。