High endothelial cells synthesize and degrade sLex. Putative implications for L-selectin-dependent recognition.

L-selectin guides lymphocytes into peripheral lymphoid tissues by recognizing glycoprotein ligands decorated with 6-sulfated sialyl Lewis x (sulfo sLex). Here we have used a rat peripheral lymph node high endothelial cell line (Ax) to study in detail the synthesis, expression and degradation of sLex epitope. We show here that Ax cells possess active alpha(1,3)fucosyltransferase Fuc-TVII, the enzyme responsible for the final fucosylation of sialyl-N-acetyllactosamine during sLex synthesis, and express sLex on the cell surface. Furthermore, these cells degrade sLex, primarily by desialylating it to neutral Lex epitopes by alpha(2,3)sialidase(s).

Expression and function of alpha 2,3-sialyl- and alpha 1,3/1,4-fucosyltransferases in colon adenocarcinoma cell lines: role in synthesis of E-selectin counter-receptors.

We show here that colon-carcinoma cell lines adhere to E-selectin via sialyl Lewis x and sialyl Lewis a (s(Lex) and s(Lea)) oligosaccharides and that this adhesion can be enhanced by TNF stimulation. To study in greater detail this endothelial binding, we analysed the mRNA expression and function of the enzymes participating in the generation of s(Lex) and s(Lea on cancer cells. These oligosaccharides are synthesized by sequential action of alpha 2,3 sialyl (alpha 2,3-ST) and alpha 1,3/1/4 fucosyltransferases (alpha 1,3/1,4-FT) on existing (poly)N-acetyllactosamine chains. We report here that mRNAs of 2 recently cloned alpha 2,3-STs and 4 alpha 1,3/1,4-FTs are expressed in adenocarcinoma cells. In functional assays alpha 2,3-ST and alpha 1,3- or 1,4-FT activities were observed in adenocarcinoma cell lysates to exogenous N-acetyllactosamine and lacto-N-biose acceptors and to their sialylated derivatives, leading to the synthesis of the sialyl-N-acetyllactosamine and s(Lex) or the sialyllacto-N-biose and s(Lea), respectively. Furthermore, the inflammatory cytokine TNF could enhance some alpha 2,3-ST and alpha 1,3/1,4-FT activities capable of generating E-selectin counter-receptors. Taken together, these data show that COLO 205 and HT-29 adenocarcinoma cell lines adhere to E-selectin in a TNF-inducible manner via their cell-surface s(Lex) and s(Lea). These cells also express mRNA as well as inducible enzyme activities of several alpha 2,3-STs and alpha 1,3/1,4-FTs responsible for the final steps in the synthesis of s(Lex) and s(Lea).(ABSTRACT TRUNCATED AT 250 WORDS)

De novo expression of endothelial sialyl Lewis(a) and sialyl Lewis(x) during cardiac transplant rejection: superior capacity of a tetravalent sialyl Lewis(x) oligosaccharide in inhibiting L-selectin-dependent lymphocyte adhesion.

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.

Alpha E beta 7 and alpha 4 beta 7 integrins associated with intraepithelial and mucosal homing, are expressed on macrophages.

The two beta 7 integrins alpha E beta 7 and alpha 4 beta 7 are the most recently described members of the integrins participating in intercellular binding. Their expression has been shown to be restricted to leukocytes and they have been suggested to be predominantly found in lymphocytes associating with the epithelium. Expression of beta 7 has mainly been studied on lymphocytes whereas macrophages have been reported not to express the beta 7 integrins. In this paper we have studied the expression of beta 7 integrins in monocytoid cells. The myelomonocytic cell lines HL-60 and THP-1 did not express beta 7 mRNA or protein, but differentiation of these cell lines to macrophages with phorbol 12-myristate 13-acetate (PMA) led to a strong induction of the beta 7 mRNA expression. A clear but less pronounced up-regulation of beta 7 mRNA-expression was also seen after treatment of HL-60 and THP-1 cells with interferon-gamma (IFN-gamma). However, its up-regulating effect on the surface expression of alpha 4 beta 7 and alpha E beta 7 complexes (detected by the monoclonal antibodies Act I and HML-1, respectively) exceeded that observed with PMA. To verify the in vitro cell line observations with normal cells, we also studied peripheral blood monocytes and tissue macrophages. Peripheral blood monocytes were Act I- and HML-1- in flow cytometry, but their expression was increased after a 72-h culture in the presence of PMA or IFN-gamma. Also, several Act I+ and HML-1+ macrophages were found in immunohistochemical stainings of both liver and edemic lung biopsies as well as in lymph node sinuses. We therefore conclude that while monocytes do not express beta 7 integrins the more differentiated cells of the monocyte-macrophage lineage do express both the alpha 4 beta 7 and alpha E beta 7 integrins, which might play a role in their intraepithelial homing.

Alpha 2,3-sialyl and alpha 1,3-fucosyltransferase-dependent synthesis of sialyl Lewis x, an essential oligosaccharide present on L-selectin counterreceptors, in cultured endothelial cells.

Sialyl Lewis x (sLex) oligosaccharides have been shown to be present in counterreceptors for L-selectin. We and others have previously shown that high endothelial cells in lymph nodes and at sites of inflammation express sLex. Here we show that also cultured human umbilical vein endothelial cells (HUVEC) express sLex on their cell surface. This oligosaccharide is formed by sequential action of alpha 2,3-sialyl- (alpha 2,3-ST) and alpha 1,3-fucosyltransferases (alpha 1,3-FT) on N-acetyllactosamine. At least two of the several alpha 2,3-ST and four of the several alpha 1,3-FT are present in HUVEC. In functional assays both alpha 2,3-ST and alpha 1,3-FT activities were observed in HUVEC lysates with exogenous lactosamine and sialyllactosamine acceptors, leading to the generation of the sialyllactosamine and sLex sequences, respectively. TNF stimulation increased the level of mRNA expression of FT VI, and the alpha 1,3-FT activity in HUVEC. Taken together these data show that endothelial cells express sLex and that they possess mRNA as well as enzyme activities of several alpha 2,3-ST and alpha 1,3-FT necessary in the final steps of sLex synthesis. Furthermore, inflammatory cytokines such as TNF can enhance transferase activities relevant in generating putative L-selectin counterreceptors.

Genistein enhances the ICAM-mediated adhesion by inducing the expression of ICAM-1 and its counter-receptors.

Binding of circulating cells to endothelium is mediated by recognition between endothelial adhesion molecules and their counter-receptors. The beta 2 integrins are a group of adhesion molecules, mainly expressed on leukocytes, that mediate intercellular binding by recognizing their counterparts on endothelial cells, among others ICAM-1. In this study we have studied the regulation of this interaction in myelomonocytic cells treated with genistein, a tyrosine kinase inhibitor with several other biological functions. We show that genistein upregulates the surface expression of the beta 2-integrins in the monoblastic THP-1 and to a lesser extent in the promyelocytic HL-60 leukemia cell lines. This upregulation leads to an increase in the adherence of THP-1 cells to ICAM-1. Genistein also modulates the expression of ICAM-1 on endothelial cells by potentiating the upregulating effect of TNF and IFN-gamma. Genistein may thus enhance intercellular binding by affecting both the endothelium and the circulating cells.

In vivo evidence of the role of alpha 4 beta 1-VCAM-1 interaction in sarcoma, but not in carcinoma extravasation.

Tumor-cell invasion can occur via either lymphatics or blood vessels. When in the blood circulation, tumor cells have to adhere to endothelium lining the blood vessels before they can extravasate. Several families of adhesion molecules have been recognized: selectins and their oligosaccharide-containing ligands and integrins and their counter-receptors belonging to the immunoglobulin superfamily. Besides their essential role in leukocyte extravasation, these adhesion molecules have been proposed by vitro experiments to be involved in tumor-cell invasion by facilitating the adhesion of malignant cells to endothelium leading to extravasation and metastasis. We have previously shown that, in vitro, several sarcoma cell lines adhere strongly to cultured endothelial cells via alpha 4 beta 1-VCAM-1 interaction. Here we show that sarcoma cells, especially in the metastatic lesions, were strongly alpha 4 beta 1 positive but did not express alpha 4 beta 7, which is another receptor for VCAM-I. Furthermore, we demonstrate that the capillary endothelium within metastatic sarcoma lesions reacted strongly with anti-VCAM-I antibody and very often the alpha 4 beta 1-expressing sarcoma cells were localized in the close vicinity of VCAM-I-expressing vessels. As control material we analyzed carcinoma specimens, but could not detect any alpha 4-integrin expression on malignant cells even though the endothelial cells were often VCAM-I positive. These results suggest that carcinomas do not use alpha 4 beta 1-VCAM-I in extravasation and, taken together, provide circumstantial evidence that in vitro findings of alpha 4 beta1-VCAM-I-dependent sarcoma cell adhesion to endothelium can be extended to in vivo situations.

Sialyl Lewis(x)- and L-selectin-dependent site-specific lymphocyte extravasation into renal transplants during acute rejection.

Kidney allograft rejection is an inflammatory process dominated by lymphocytes. During rejection lymphocytes preferentially adhere to the peritubular capillary endothelium (PTCE), which acquires morphological features common to high endothelium. These observations indicate that PTCE is the site of lymphocyte entry into the rejecting renal allograft. Of the identified endothelial adhesion molecules, ICAM-1 was already expressed on the endothelium of normal kidneys, and its expression was strongly enhanced during rejection without site-specific restriction. VCAM-1 was not expressed on the endothelium of normal or syngeneic kidneys, but its expression was induced during allograft rejection not only in PTCE, but occasionally also on the endothelium of larger vessels. Sialyl Lewisx (sLex) showed a very restricted pattern of expression; endothelium was sLex-negative both in control and syngeneic kidneys. On the other hand, PTCE reacted strongly with anti-sLex antibody in allografts. When kidney frozen sections were treated with sialidase the binding of lymphocytes decreased by 70%. Low-dose chymotrypsin treatment of lymphocytes, known to remove L-selectin from the lymphocyte surface, decreased their binding to PTCE by 60%. Likewise lymphocyte adhesion to PTCE was inhibited by 70% by anti-sLex- and anti-L-selectin-antibodies and by sLex tetrasaccharide. Finally PTCE in the allografts, but not in syngeneic grafts or normal kidneys, bound an L-selectin-IgG fusion protein, indicating that ligands for L-selectin were induced during rejection.

Characterization of the E-selectin ligand on NK cells.

In this study we demonstrate that human CD56+CD16+/CD3- NK cells adhere to the E-selectin expressed by stimulated HUVEC in a sialidase- and Ca(2+)-dependent manner, and express a silylated Lex adhesion structure. We have characterized this sLe(x) epitope on NK cell in detail and show here that the sLe(x) on NK cells was not recognized by the CSLEX1 Ab, but was readily identified by two anti-di-sLe(x) Abs, KM-93 and FH-6. Furthermore, cleaving sialic acid with a sialidase treatment revealed a pool of Le(x) epitopes on the NK cells surface, providing further proof that NK cells express sLe(x) epitopes. Extensive protease treatments did not cleave the sLe(x) epitope from NK cells, which suggests that it could be linked to a lipid backbone. This di-sLe(x) was able to mediate adhesion to E-selectin, suggesting that it represents an essential part or is closely related to a selectin ligand on NK cells. We were also able to show that NK cells possess several alpha 2,3 sialyltransferases and alpha 1,3 or alpha 1,3/4 fucosyltransferases. These enzymes are crucial in the synthesis of sLe(x) epitopes on cell surfaces. Taken together, we provide evidence that NK cells have a di-sLe(x) oligosaccharide capable of adhesion to E-selectin, and NK cells have the machinery (i.e., relevant transferases) to generate these sialylated Lewis oligosaccharides.

Enhanced ICAM-1-dependent adhesion of myelomonocytic cells expressing increased levels of beta 2-integrins and CD43.

Interaction of ICAM-1 and its ligands plays an important role in the leukocyte binding to endothelium. The best characterized ICAM-ligands belong to the family of beta 2-integrins (CD11/CD18), but recently it has been suggested that CD43, a molecule with no structural resemblance to integrins binds ICAM-1 also. On the leukocytes the main regulatory pathway for ICAM-mediated binding is believed to be a short-term regulation of the avidity of CD11/CD18. In this study the authors investigated whether a quantitative increase in the surface expression of ICAM-ligands also can lead to enhanced binding to purified ICAM-1. PMA-treatment differentiates myelomonocytic cell lines into macrophages with a concomitant increase in the surface expression and mRNA-levels of the beta 2-integrin alpha- and beta-chains as well as that of CD43, another ICAM-ligand. The binding of the PMA-treated THP-1 cells to ICAM-1 was increased simultaneously compared to non-treated cells. The binding was blocked completely with antibodies to CD18 and ICAM-1. It is concluded that in addition to the transient qualitative regulation, a long-term quantitative regulation of ICAM-1 ligands also plays a role in increasing the adhesiveness of myelomonocytic cells. This may be relevant in chronic inflammation episodes.

Human NK cells expressing alpha 4 beta 1/beta 7 adhere to VCAM-1 without preactivation.

The authors demonstrate that resting CD56+/CD3- NK cell adhesion to the endothelial VCAM-1 is over three-fold higher than CD56-/CD3+ T-cell adhesion. T-cell, but not NK-cell adhesion, to VCAM-1 is enhanced significantly by stimulation. The expression of VCAM-1 receptor subunits alpha 4 and beta 1 on both effector cells remains unchanged upon stimulation. A subpopulation of NK cells, as well as of T cells, was found to express beta 7, whose expression was not altered upon stimulation. The authors conclude that the adhesive properties of the same receptor structures on these distinct cell populations are regulated in a different manner, according to the specific functions of the effector cells of the immune system.

Expression of six protein kinase C isotypes in endothelial cells.

Protein kinase C (PKC) family is an important regulatory element in signal transduction, cellular regulation and tumor promotion. The classical PKC isotypes (alpha, beta and gamma) are Ca(2+)-dependent and can be activated by diacylglycerol. The novel isotypes, PKC delta, PKC epsilon, PKC eta (L) and PKC theta, are Ca(2+)-independent, whereas the two atypical PKCs (zeta and lambda) lack the Ca(2+)-binding region and are not activated by diacylglycerol. Here we show that cultured human endothelial cell line EA.hy926 as well as freshly isolated human umbilical vein endothelial cells express members of all PKC subfamilies. No traces of PKC gamma or delta were detected in endothelial cells. On the contrary the classical PKCs (alpha and beta), the novel PKC epsilon, as well as the atypical PKC zeta are present at the mRNA level in human endothelial cells and the corresponding proteins are also detected by immunoblotting.

Down-regulation of monocytic VLA-4 leads to a decreased adhesion to VCAM-1.

The alpha 4 beta 1 integrin VLA-4 is expressed on practically all leukocytes, except on mature granulocytes. Here we show that in vitro treatment of monocytic cells with phorbol-12-myristate-13-acetate (PMA) leads to a selective decrease in the VLA-4 alpha-chain expression, both at the RNA and protein level. Meanwhile the expression of beta 1 and that of alpha 5, another alpha-chain associating with beta 1, was seen to increase. The decrease of alpha 4 expression was restricted to monocytic cells, and was not observed on other VLA-4-positive cells tested (MOLT-4 T cells and HOS sarcoma cells). The down-regulation of the VLA-4 alpha-chain was followed by a decreased binding capacity of the cells to recombinant VCAM-1. This data indicates that while previous findings show that the integrin-dependent adhesion may rapidly be regulated by altering the avidity of the interacting molecules, their quantitative modulation also has a clear impact on adhesion.

Chronic rejection of rat aortic allografts. III. Synthesis of major eicosanoids by vascular wall components and effect of inhibition of the thromboxane cascade.

We have previously demonstrated that rat aortic allografts from the DA (RT1a) to the WF (RT1v) strain develop chronic arteriosclerotic changes in the vascular wall after a short spontaneously reversible acute rejection episode. These changes, which are lacking in syngeneic DA-to-DA control grafts, are virtually identical with those observed in human allografts during chronic rejection. In this study we have investigated whether eicosanoids are involved in the generation of arteriosclerotic changes. Incubation of aortic wall rings in vitro and immunochemical assays demonstrated that the arteriosclerotic allografts synthesize significantly more thromboxane B2 (TxB2) but not 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) or leukotriene B4. The increased synthesis of TxB2 in the allografts persisted for at least 5 months after transplantation. Separate incubation of the two major components of the vascular wall, after microdissection of the intima and (media plus) adventitia, demonstrated that most of the synthesis of TxB2 during chronic rejection was due to the outer layer of aorta, presumably the inflammatory cells of the adventitia. In contrast, most of the 6-keto-PGF1 alpha was synthesized by the inner layer of the aorta, presumably the endothelial cells and the smooth muscle cells of the intima. Administration of 1 mg.kg-1 x day-1 of a specific TxA2 receptor inhibitor, GR32191B, to the recipient rat reduced the proliferative response of inflammatory cells in the adventitia by 30%, as detected by in vivo tritiated-thymidine (3H-TdR) labeling and autoradiography, but did not reduce the proliferative response of smooth muscle cells in the media and intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic rejection of rat aortic allograft. II. Administration of cyclosporin induces accelerated allograft arteriosclerosis.

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.

Chronic rejection in rat aortic allografts. An experimental model for transplant arteriosclerosis.

Chronic rejection has several histological appearances, depending on the type of organ graft. Common to all of them is transplant arteriosclerosis associated with an ongoing inflammatory response in the transplanted graft. To the contrary of classical atherosclerosis, in which the manifestations are mostly focal, proximal, and asymmetric, transplant arteriosclerosis is generalized, and the intimal thickening is concentric. In this article, we describe an experimental animal model whereby transplant arteriosclerosis may be investigated in the inbred rat. Aortic allografts were transplanted from DA (RTIa) to major histocompatibility complex-incompatible WF (RTIv) rats or, for control, to rats of the DA strain. Transplantation was followed by an acute inflammation episode in the aortic adventitia of the allograft, largely lacking in the syngeneic graft, with a prominence of lymphoid activation markers (Cd25) in the cells of the inflammatory infiltrate. The inflammation episode peaked at 2 months after transplantation, became attenuated, and was followed by a proliferative response of myocytes in the allograft media. An increase in the migration of myocytes to the subendothelial space (presumably through small breaks generated in the internal elastic lamina) was observed thereafter, and myocyte proliferation continued in the intima with some intermingled macrophages. Finally, necrosis and disappearance of myocytes and their replacement by fibrous tissue were observed in the media. These alterations are virtually identical with the vascular lesion of chronically rejecting parenchymal organ transplants in human subjects. We suggest that aortic allografts exchanged between histoincompatible rat strains may be used as an experimental model for transplant arteriosclerosis.

Properties of B cells and Thy-1-antigen-expressing cells infiltrating rat renal allografts.

We have examined (1) the frequency of B cells secreting antibodies against donor major histocompatibility complex (MHC) antigens and (2) the properties of Thy-1-antigen-expressing leukocytes in rats rejecting renal allografts. Our results show that B cells secreting antibodies are present in the inflammatory cell population at the frequency of 1:850. Among them only 1 out of 2-150 is engaged in production of antibodies directed to the graft MHC antigens, depending on the method of assay. This suggests that despite the observed significant production of nonspecific immunoglobulin in situ, only a minority of the B-cell population is specifically committed to the graft MHC antigens. This finding is concordant with the described previously low frequencies of the T cells specifically directed toward the graft MHC antigen. The role of the "immunologically noncommitted" cells in graft rejection is unknown. We have found that a substantial part (up to 60%) of inflammatory cells invading a rat kidney allograft express the Thy-1 antigen. This suggests that they might be immature (progenitor?) cells and, therefore, unable to respond to the graft antigens. Progenitor-like properties of these cells have been confirmed by their ability to reconstitute lethally irradiated syngeneic rat. Finally, these immature cells are of lymphoid, not of myeloid, linkage, because they do not proliferate in the presence of GM-CSF.

The frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting renal allografts.

We have estimated the frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting histoincompatible renal allografts. In a major plus minor antigen-incompatible DA-to-WF combination on day 4 post-transplantation, reverse protein A plaque assay demonstrated that in the graft the frequency of lymphoid cells secreting Ig was 1:850. A major locus-incompatible and minor locus-compatible, congeneic LBN-to-Lewis strain combination was then applied to estimate the specificity of the secreted antibody. The lymphoid inflammatory cells were fused with mouse myeloma cells, cultured under limiting dilution conditions, and assayed by ELISA to donor and irrelevant strain spleen cells. Among cells infiltrating the graft, the fusion frequency was 1:172 x 10(3) and the frequency of Ig-producing hybrids 1:400 x 10(3) (i.e., this assay was approximately three log orders less sensitive than the reverse pA assay). The frequency of hybridomas secreting specifics antibodies against donor MHC antigens was 1:720 x 10(3) (i.e., every second hybridoma deriving from inflammatory population produced specific Ig). In addition, there was at least one obviously polyspecific population of hybridomas, detectable only in the spleen and reactive with all rat strains tested with a frequency of 1:700 x 10(3). The inflammatory cells were also cultured directly under limiting dilution conditions, and the frequency of Ig-secreting cells was determined by ELISA. The frequency of inflammatory lymphocytes secreting detectable amounts of immunoglobulin in the supernatant was 1:14 x 10(3) in the graft (i.e., this assay was approximately one log order less sensitive than the reverse protein A plaque assay).(ABSTRACT TRUNCATED AT 250 WORDS)