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L-selectin-dependent lymphocyte extravasation is a hallmark of acute heart allograft rejection in rats. On screening over 600 endomyocardial biopsies (EMBs), taken at different time points after heart transplantation in man, we identified 91 samples with histological signs of acute rejection. Rejection and nonrejection EMBs were analyzed for the presence of properly glycosylated, ie, sulfated sialyl Lewis-x (sLex) decorated L-selectin ligands. Two anti-sLex (2F3 and HECA-452) and one anti-6- or 6'-sulfated and/or 6, 6'-bisulfation (MECA-79) monoclonal antibodies were used. Nonrejecting heart endothelium did not express, or expressed only weakly, sulfated and or sLex decorations of L-selectin ligands. On the contrary, these epitopes were readily detectable on endothelium of capillaries and venules at the onset and during acute rejection episodes. The more intense the sulfated sLex expression was, the more severe the rejection episode was in histological grading. The endothelial expression of L-selectin ligands decreased to background levels as the rejection resolved. Our data demonstrate a complete correlation between the level of expression of the sulfated sLex-decorated ligands on the one hand and the histological severity of acute heart allograft rejection on the other hand. These data suggest that functionally active endothelial L-selectin ligands are instrumental in lymphocyte extravasation into the human heart allografts at the onset and during acute rejection episodes.

Purpose: E- and P-selectins, expressed on vascular endothelium, and their sialyl-Lewis(x )(sLe(x))- and/or sialyl-Lewis(a) (sLe(a))-containing ligands have a crucial role in extravasation and metastasis of circulating cells. We wanted to analyse the role of selectins and their ligands in head and neck tumours., Methods: A total of 40 consecutive biopsy specimens were collected from surgery performed at the Helsinki University Central Hospital between September 1995 and November 1996. The series of specimens contained both benign and malignant head and neck tumours of epithelial, lymphoid or mesenchymal origin. All these were analysed with immunohistochemistry for epithelial and endothelial expression of sLe(x) and sLe(a) glycans and E- and P-selectins., Results: Epithelial expression of sLe(x) and sLe(a) glycans was higher in benign than in malignant lesions in both epithelial and lymphoid tumours. On the other hand, endothelial expression of sLe(x), sLe(a), E- and P-selectin was lower in benign than in malignant lesions in both epithelial and lymphoid tumours., Conclusions: These data suggest that altered epithelial and endothelial expression of sLe(x) and sLe(a )glycans acting on selectin ligands is linked to the development of head and neck tumours.

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLe(x) and sLe(a) respectively) decorated ligands. Endothelial cells have been shown to express sLe(x) epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLe(x) on sialylated N-acetyllactosamine via the action of alpha(1,3)fucosyltransferase(s), endothelial cells can also degrade sLe(x) to Lewis x through the action of alpha(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLe(x), which facilitates their adhesion to endothelial E- and P-selectin.

Acute organ transplant rejection is characterized by a heavy lymphocyte infiltration. We have previously shown that alterations in the graft endothelium lead to increased lymphocyte traffic into the graft. Here, we demonstrate that lymphocytes adhere to the endothelium of rejecting cardiac transplants, but not to the endothelium of syngeneic grafts or normal hearts analyzed with the in vitro Stamper-Woodruff binding assay. Concomitant with the enhanced lymphocyte adhesion, the cardiac endothelium begins to de novo express sialyl Lewis(a) and sialyl Lewis(x) (sLea and sLex) epitopes, which have been shown to be sequences of L-selectin counterreceptors. The endothelium of allografts, but not that of syngeneic grafts or normal controls, also reacted with the L-selectin-immunoglobulin G fusion protein, giving further proof of inducible L-selectin counterreceptors. The lymphocyte adhesion to endothelium could be significantly decreased either by treating the lymphocytes with anti-L-selectin antibody HRL-1, or by treating the tissue sections with sialidase or anti-sLea or anti-sLex monoclonal antibodies. Finally, we synthetized enzymatically several members of the sLex family oligosaccharides and analyzed their ability to block lymphocyte adhesion to cardiac endothelium. The monovalent sLex (a tetramer), divalent sLex (a decamer), and tetravalent sLex (a 22-mer) could all significantly reduce lymphocyte binding, but the inhibition by the tetravalent sLex-construct was clearly superior to other members of the sLex family. The crucial control oligosaccharides, sialyl lactosamines lacking fucose but being otherwise similar to the members of sLex family, had no effect on lymphocyte binding.

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface protein. It is produced in large amounts in essentially all colon and several other adenocarcinomas. It has therefore been widely used as a clinical tumor marker. CEA is also a member of the immunoglobulin superfamily. Members of this family, such as ICAM-1, ICAM-2, VCAM-1 and NCAM, are known to participate in cell-cell adhesion. Similarly, the intercellular adhesion properties of CEA have been documented: it has been shown to mediate homotypic adhesion of cultured human colon adenocarcinoma cell lines. In this study we show for the first time that CEA is expressed on cultured human umbilical vein endothelial cells and on the endothelial cell line Ea.hy926. The expression of CEA on cultured endothelial cells can be enhanced by TNF-alpha or IFN-gamma, and decreased by IL-4. We demonstrate using immunohistochemistry that anti-CEA monoclonal antibody reacted with FVIII-positive endothelium in tissue sections prepared from lymph nodes. Finally, we were able to show that CEA-positive breast carcinoma cells bind to purified CEA protein, whereas CEA-negative breast carcinoma cells do not. These results reveal for the first time that endothelial cells express CEA on the cell surface and suggest that CEA-expressing adenocarcinomas could adhere to endothelial cells via CEA-CEA interaction, thus facilitating tumor cell extravasation and hematogenic metastasis.

We have analyzed the autopsy material of 11 cardiac allograft recipients, whose grafts became nonfunctional less than 1 year after transplantation. All patients had received routine triple-drug immunosuppression. Two of these patients showed accelerated transplant arteriosclerosis; the graft loss was associated with strong intimal proliferative response affecting the first-order and second-order intramuscular branches of the major coronary vessels. Histologic evidence showed only a slight inflammatory reaction in the vascular adventitia. The media was mostly intact, and the internal elastic lamina showed occasional breaks. A very prominent inflammatory reaction was noted in the subendothelial space in the intima. Immunohistochemistry with monoclonal antibodies revealed that the luminal part of the intima was invaded by anti-leukocyte common antigen and anti-UCHL1 positive T lymphocytes, whereas the abluminal part consisted mostly of anti-smooth muscle alpha-actin-positive smooth muscle cells. We call this subendothelial accumulation of lymphocytes endothelialitis. The suggestion has been made that accelerated arteriosclerosis may be associated with cyclosporine treatment. Correlation with the recipient's cytomegalovirus status in this study suggests that cytomegalovirus infection may contribute to the development of endothelialitis and accelerated arteriosclerosis in heart transplant recipients.

High endothelial venules (HEV) lined by the high endothelium are the sites where leukocytes enter into the lymph nodes from the blood. Lymphocyte homing into lymph nodes is organ-selective, i.e., different molecules are involved in the lymphocyte homing to peripheral nodes compared with mucosa associated lymphoid tissue. The traffic into peripheral nodes is regulated by the expression of L-selectin on leukocytes and its ligand on HEVs. The ligand for L-selectin is suggested to be a 50, 90, or 105 kd glycoprotein, which is sulfated, fucosylated, and sialylated. The two other members of the selectin family (E- and P-selectin) recognize sialyl-Lewis x and -Lewis a (sLex and sLea, respectively) carbohydrate motifs, and there is preliminary data suggesting that this would also be the case for L-selectin. We have initiated a study to identify the expression of these sialylated structures on endothelial surfaces. We present data that show that HEVs in peripheral nodes, but not in the mucosa-associated lymphoid tissue, express large quantities of sLex and sLea identified by MAbs in immunohistology. Endothelium in capillaries or larger vessels in non-lymphoid tissues do not react with anti-sLex or -Lea mAbs. Only 1-2% of the lymphocytes in the peripheral blood express sLex and so far only the skin-homing lymphocytes are known to be sLex positive in larger quantities. We show that in many occasions the B cells in the peripheral lymph-node germinal centers are also sLex-, but not sLea-positive, and provide evidence of the restricted pattern of sLex and sLea expression on peripheral lymph-node HEVs. We propose that they are at least parts of the ligand for L-selectin.

The authors have compared the reactivity of monoclonal antibodies (MAb) directed against endothelial adhesion molecules (ICAM-1, ICAM-2, VCAM-1, ELAM-1) in hyperplastic, nonmalignant, and malignant lymph nodes. The authors demonstrate that the reactivity with ICAM-1 MAb is stronger in the high endothelial venules (HEV) and other smaller vessels (SV) in lymphomas compared with hyperplastic lymph nodes. Similarly, the reactivity of an ICAM-2 MAb (6D5) was shown to be higher in malignant lymph nodes compared with nonmalignant ones. ICAM-2 MAb stained both the HEV and SV. VCAM-1 MAb reacted strongly with germinal centers and its endothelial reactivity was higher in the lymphoma nodes. ELAM-1 MAb stained only faintly some endothelial cells in malignant tissue. These data suggest that besides the known regulatable endothelial adhesion molecules ICAM-1 and VCAM-1, the expression of ICAM-2 can be modified.

Rat aortic allografts immunosuppressed with cyclosporin--but not with azathioprine or steroids--develop an early inflammatory lesion in the subendothelial space. This "endothelialitis" is followed by an influx of proliferating smooth muscle cells into the intima, resulting in intimal thickening and accelerated arteriosclerosis. Administration of azathioprine and steroids largely ameliorates the development of the accelerated lesion. Similar endothelialitis and accelerated arteriosclerosis have been observed previously in the autopsy material of cardiac transplant recipients. Our results confirm the suggestion that the development of accelerated allograft arteriosclerosis is most likely linked to cyclosporin administration.