Liisa Laitinen

Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues

SummaryThe binding ofGriffonia simplicifolia agglutinin-I (GSA—I) and the isolectins GSA-I-AB3 and GSA-I-B4, having affinity for some α-d-galactosyl andN-acetyl galactosaminyl residues was studied in different mouse tissues. In brain, cardiac muscle and skeletal muscle, the GSA-I-lectin conjugates showed prominent binding only to blood vessel endothelia. Similarly, in the liver and kidney cortex the GSA-I-conjugates selectively reacted with endothelial cells of the sinusoids and with intertubular and glomerular capillaries, respectively. However, a strong reactivity with the GSA-I-conjugates was additionally seen in the acinar cells of the pancreas, in the stratified squamous epithelia of skin and tongue, and in transitional epithelium. SDS—PAGE electrophoresis combined with the lectin-blotting technique indicated that a similar set of glycoproteins are responsible for the GSA-I binding, even in different tissues. Another lectin with specificity for α-d-galactose, theMaclura pomifera agglutinin, displayed a distinctly different distribution of binding sites, mainly in the basement membranes, of all mouse tissues studied. The results suggest that some α-d-galactosyl residues, recognized by the binding of GSA-I lectins, are preferentially expressed in endothelial cells of mouse tissues, and also provide further evidence that endothelial cells can present a highly specific surface glycosylation pattern.

Changes in the glycosylation pattern during embryonic development of mouse kidney as revealed with lectin conjugates.

Distribution of lectin-binding sites in adult and developing mouse kidney was studied with fluorochrome- and peroxidase-coupled lectins. Effects of fixation methods on lectin-binding patterns were also compared. Un-induced mesenchymal cells and ureter bud of the early metanephros reacted with Concanavalin A, Lens culinaris, Ricinus communis I, and wheat germ agglutinins, whereas binding sites for both soybean and peanut (PNA) agglutinins were seen only in ureter bud tissue. On induction, PNA positivity rapidly appeared in the induced, condensed areas of the metanephrogenic mesenchyme. Early glomeruli expressed heterogeneously terminal galactosyl and N-acetylgalactosaminyl moieties in the podocytes. Later, these sites disappeared and were apparently covered by sialic acids. Endothelia also displayed a comparable sialylation of terminal saccharide moieties during maturation. Binding sites for many of the above lectins were also found in the developing proximal and distal tubules. Terminal fucosyl residues, characteristic of mature proximal tubules, appeared during day 13 of development. Dolichos biflorus agglutinin reactivity, typically seen in the collecting ducts, appeared by day 13. Griffonia simplicifolia-I-B4 isolectin reactivity was exclusively localized to endothelial in adult kidney cortex, but in embryonic kidneys reactivity with collecting duct and podocytes was also seen. These results suggest that the compartmentalized expression of cell glycoconjugates in adult mouse kidney is acquired in a sequential manner during development. Such sequential appearance of the mature glycosylation pattern probably reflects functional maturation of the nephron.

Differential expression of laminin polypeptides in developing and adult human kidney.

We studied the expression of laminin chains in embryonic and adult human kidney by indirect immunofluorescence with monoclonal antibodies (MAbs). In embryonic human kidney, immunoreactivity for laminin alpha 1, beta 1, and gamma 1 chains was found in basement membranes (BMs) of primary vesicles, in comma- and S-shaped bodies, and in more mature stages of glomeruli and in tubules. The beta 2 chain of laminin was absent in the early glomerular structures but was prominent in BMs of maturing glomeruli (GBMs) and Bowmans capsule (BCBMs) and was also detectable in some tubules. Both the beta 2 and alpha 2 chains were variably seen in medullary tubule BMs. In adult human kidney, laminin alpha 1 chain was seen in GBMs and all tubule BMs (TBMs) as well as in arterial smooth muscle BMs (SMBMs). Laminin beta 1 chain reactivity was found in all TBMs, but not in GBMs or SMBMs. In the glomerulus, a distinct mesangial type of reaction was revealed with the MAbs to beta 1 and alpha 2 chains. The GBMs and SMBMs reacted with MAbs to the beta 2 chain, but reactivity was lacking in BCBMs. Laminin gamma 1 chain immunoreactivity was weakly present in BCBMs, GBMs, and SMBMs. The alpha 3 and beta 3 chains could not be detected in developing or adult human nephron. The results show that during development the BMs in human nephron undergo distinct changes, laminin beta 1 chain being transiently co-expressed with alpha 1 chain during early glomerular development and then becoming replaced by the beta 2 chain, which, on the other hand, disappears from the BCBMs on maturation. The alpha 2 chain appears to emerge in the mesangium late during development.

Integrins in human cells and tumors

We have studied the distribution of the alpha- and beta-subunits of integrins in developing and adult human kidney as well as in selected other tissues and cultured cells. In cultured cells some of the integrin subunits (beta 1, alpha 1, alpha 2 and alpha 5) colocalize with talin at focal adhesions when plated on an appropriate ligand. Similarly, in tissues the polarization of beta 1-integrins in colocalization with talin appears to indicate adhesive complexes, as demonstrated in adult glomeruli. In human kidney, the alpha subunits of integrins were seen to be segment-specifically expressed already in fetal tissues. In glomeruli the integrin alpha 1 subunit characterized mesangial cells while the alpha 2 and alpha 3 subunits showed immunoreactivity in endothelial cells and podocytes, respectively. In renal tubuli, the alpha 6 subunit, complexed with the beta 1 subunit, showed a typical polarized distribution coaligning with the tubular basement membrane while the alpha 3 and alpha 2 subunits were expressed in distal tubular cells. These results suggested that in kidney the alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins can function as basement membrane receptors. The alpha 5 subunit was nearly lacking in the kidney and it appears to be mainly expressed in some smooth muscle cells. In other tissues distinct patterns in the expression of integrins were found. Thus, in many glandular epithelial cells the alpha 3 beta 1 integrin appeared to function as a basement membrane receptor while in various stratified epithelia and in the breast such a polarized localization could be found for the alpha 6 beta 4 integrin. Finally, although presenting a clearly polarized distribution for beta 1 integrins, none of the alpha subunits could be found in cardiac or skeletal muscle cells and none of the integrins could be revealed in neuronal cells of human developing and adult cerebrum or cerebellum, although neurons in peripheral tissues contained abundantly the alpha 6 beta 1 integrin complex. In human tumors, the tumor cells, including also metastastatic tumors, generally presented the same integrins as their tissues of origin. In some poorly differentiated tumors both a population heterogeneity and even a lack of expression or a disorganization of basement membrane receptor integrins was obvious.

Dolichos biflorus agglutinin (DBA) reacts selectively with mast cells in human connective tissues

Dolichos biflorus agglutinin (DBA) binds to N-acetyl-D-galactosamine (GalNAc) residues in glycoconjugates and agglutinates erythrocytes carrying blood group antigen A. In cryostat sections of various tissues from blood group-specified humans, fluorochrome-coupled DBA bound preferentially to fusiform connective tissue cells and to certain epithelial cells. The connective tissue cells were identified as mast cells by their typical metachromasia in consecutive staining with toluidine blue. Double labeling with DBA and conjugated avidin revealed two distinct populations of mast cells. In several tissues the DBA-reactive cells likewise displayed uniform avidin reactivity. In intestinal mucosa, however, morphologically distinct DBA-binding mast cells were found, which were labeled with the avidin conjugates only in specially fixed paraffin sections. DBA did not bind to vascular endothelial cells, which could be identified by double staining with antibodies to factor VIII-related antigen. Labeling with Helix pomatia agglutinin (HPA), another blood group A-reactive lectin, resulted in distinct blood group-dependent fluorescence of the endothelia. Sophora japonica agglutinin (SJA), a blood group B-reactive lectin, labeled vascular endothelial cells in tissues from blood group A, AB, and B donors. HPA and SJA reacted with small mast cells in the gastrointestinal mucosa but failed to label large mast cells in any of the tissues. These results indicate that the blood group reactivity of lectins, as determined by erythroagglutination, is not necessarily consistent with their reactivity with blood group determinants in tissue sections. Moreover, DBA conjugates appear to be a reliable probe for detection of mast cells in various human connective tissues.

Differentiation of metanephric tubules following a short transfilter induction pulse

SummaryUndifferentiated metanephric mesenchymes, when grown in transfilter contact with an inductor tissue, differentiate into epithelial kidney tubules. The segregation of these tubules into the different segments of the nephron was studied.In explants grown in continuous transfilter contact with the inductor, immunohistological and histochemical markers specific for the glomerular epithelial, proximal tubule, and distal tubule cells appeared by 4 1/2 to 5 days, 4 days, and 5 days of culture, respectively. Electron microscopy confirmed segmentation of the tubules: Avascular glomeruli with glomerular basement membrane material, proximal tubules with brush border formation, and distal tubules were revealed in the explants after 5 days of culture.A short (18 h) transfilter induction pulse, followed by a prolonged subculture in the absence of the inductor, resulted sulted in the formation of only a small number of tubules in about half of the explants while the rest remained undifferentiated. These scarce tubules showed the markers specific for the proximal tubules only. The segregation of all three aspects of the nephron seems to be programmed during the transfilter culture, but apparently the time needed for the induction of the different segments varies.

Psophocarpus tetragonolobus Agglutinin Reveals N-Acetyl Galactosaminyl Residues Confined to Endothelial Cells and Some Epithelial Cells in Human Tissues'

We studied the binding of Psophocarpus tetragonolobus agglutinin (PTA) conjugates to human adult tissues. In all kidney specimens studied, PTA bound in a blood group-independent way to endothelia in glomerular and intertubular capillaries as well as in larger vessels. In addition, a heterogeneous binding to collecting duct cells was seen. In specimens of human smooth, cardiac, and skeletal muscle, cerebellum, lung, thyroid gland, liver, proliferative endometrium, and placenta, PTA bound only to endothelial of capillaries and larger vessels. In epidermis and gingiva, PTA conjugates additionally revealed reactivity with keratinocytes. Similarly, in salivary gland, urinary bladder, gastrointestinal tract, mammary gland, and renal pelvis, PTA reacted with some epithelial cell layers. The PTA conjugates gave an even cell surface membrane staining of cultured umbilical vein endothelial cells. Lectin-affinity binding of radioactively surface-labeled endothelial cells showed that PTA and Ulex europaeus I agglutinin (UEA-I) recognized related major cell surface glycoproteins. The results with PTA conjugates show that certain N-acetyl galactosaminyl residues are, in addition to some epithelial cells, confined to endothelial cells in human tissues.