Histochemistry of glycosaminoglycans in cartilage ground substance. Alcian-blue staining and lectin-binding affinities in semithin Epon sections

The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A I, Arachis hypogaea, Ulex europaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding sites (positive staining for all lectins tested except H. pomatia) observed corresponded to areas positive for keratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.     

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Lectin binding of surface epithelia and concomitant glands of gallbladder and biliary ducts in the guinea pig

Complex carbohydrate components of secretory granules and the glycocalix were analysed in surface epithelia, endoepithelial glands and exoepithelial tubuloalveolar glands of the biliary-ductular system (guinea pig). Brunner glands and pyloric glands were studied for comparison. The columnar epithelial cells of the gallbladder and biliary ducts displayed a well-developed PAS-positive apical glycocalix. These materials strongly bound Ricinus communis A I, Ulex europaeus I, Lotus tetragonolobus A and wheat-germ-A lectins. With the exception of Lotus A lectin which did not bind at all, the same lectins stained the basolateral cell surface. The secretory granules in the supranuclear regions of surface epithelia and in the exoepithelial glands strongly bound Ricinus A I, Ulex europaeus I, wheat-germ-A and Helix pomatia lectins. Concanavalin A was less intensively bound by the secretions of tubuloalveolar glands than by the secretory granules in surface epithelia. The luminal and basolateral cell surfaces of glandular cells in the exoepithelial glands were stained by the same spectrum of lectins as were the columnar cells of surface epithelia, but the staining was less distinct. In the guinea pig, the lectin-binding patterns of tubuloalveolar glands in the biliary ducts closely resembled those of Brunner glands and pyloric glands. The secretions of the tubuloalveolar glands were different from the secretion of surface epithelia, as they bound Concanavalin A less intensively.     

Histochemistry of glycosaminoglycans in cartilage ground substance

Summary The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A I, Arachis hypogaea, Ulex curopaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding site (positive staining for all lectins tested except H. pomatia) observed corresponded to areas positive forkeratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.Supported by the Hochschuljubiläumsstiftung der Stadt Wien     

Adhesion of small cell lung cancer cells to E- and P-Selectin under physiological flow conditions: implications for metastasis formation

Haematogenous metastasis of small cell lung cancer (SCLC) is still a poorly understood process and represents the life threatening event in this malignancy. In particular, the rate-limiting step within the metastatic cascade is not yet clearly defined although, many findings indicate, that extravasation of circulating tumour cells is crucially important as most tumour cells within the circulation undergo apoptosis. If extravasation of SCLC tumour cells mimics leukocyte–endothelial interactions, SCLC cells should adhere to E- and P-selectins expressed on the luminal surface of activated endothelium. The adhesion to E- and P-selectin under physiological shear stress with regard to adhesive events, rolling behaviour and rolling velocity was determined in the human SCLC cell lines SW2, H69, H82, OH1 and OH3. OH1 SCLC cells adhered best to recombinant human (rh) E-selectin FC-chimeras and human lung endothelial cells (HPMEC), H82 SCLC cells adhered best to activated human umbilical vein endothelial cells (HUVEC) under physiological shear stress. As OH1 cells had also produced by far the highest number of spontaneous lung metastases when xenografted into pfp/rag2 mice in previous experiments our findings implicate that adhesion of SCLC cells to E-selectin is of paramount importance in SCLC metastasis formation.     

Pancreatic duct glands. II. Lectin binding affinities of ductular epithelium,ductular glands,and Brunner glands

Summary Secretory epithelia and ductular glands were studied in main pancreatic ducts of guinea pig, rabbit, rat, and man. Brunner glands were studied for comparison. Semithin sections from Epon-embedded tissues were etched with sodium methylate and incubated with horseradish peroxidase-conjugated lectins. Columnar cells in the epithelium of pancreatic ducts are endowed with well-developed microvillar borders. These apical regions strongly stain with Lotus A-, wheat germ-, and Ricinus I-lectins. Basolateral plasma membranes bind Ricinus I-, Ulex europaeus I-, and wheat germ-lectins. Cytosomes in the supranuclear regions of epithelial cells are interpreted as secretory granules. These droplets are marked by wheat germ-lectin and to a lesser degree by Ricinus I- and Ulex europaeus I-lectins. Ductular glands of the main pancreatic ducts contain secretions that bind Helix-, wheat germ-, and Ulex europaeus I-lectins. Their apical and basolateral cell membranes deeply stain with wheat germ- and Ulex europaeus I-lectins. Secretions of Brunner glands bind Ricinus I-, Ulex europaeus I-, Helix-, and wheat germ lectins. Their apical and basolateral cell membranes stain with Ricinus I- and wheat germ-lectins.-Species differences in lectin-binding affinities of complex carbohydrates were observed and are described.Part of these results has been presented as a poster during the 4th Symposium on Lectins in Cell Biology and Medicine June 25, 1983, Cologne, Federal Republic of Germany