- Email: [email protected]
Increased adhesion of lymphoid cells to glycated proteins
The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804 www.elsevier.com/locate/ijbcb
Increased adhesion of lymphoid cells to glycated proteins Georgi I. Ivanov a, Todor A. Chaushev a, Lilia N. Dakovska b, Stanimir D. Kyurkchiev a,* a
Department of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shosse, 1113 So®a, Bulgaria b University Hospital of Endocrinology and Gerontology, Medical University, 6 Blvd. Damian Gruev, 1303 So®a, Bulgaria Received 25 November 1998
Abstract Background and aims: the advanced glycation end-products are involved in the pathogenesis of vascular damages and other clinical complications in diabetic patients. The aim of this study was to investigate the adhesion of lymphoid cells to nonenzymatically glycated proteins in comparison with the unmodi®ed substances. Methods: two cell lines (monocyte-macrophage line U937 and the T-cell line Jurkat) were used throughout the experiments. The cells were left to adhere to nonenzymatically glycated and native proteins coated on a 96-well ¯atbottom plates and the cellular adhesion was registered as absorption at 550 nm following the method described by Ivanov and Kyurkchiev [G. Ivanov, S. Kyurkchiev, Eect of advanced glycosylation end-products on the activity of integrins expressed on U937 cells, Hum. Immunol. 59 (1998) 325±330.]. Results: it was found that the monocytes had increased adhesion to nonenzymatically glycated proteins such as collagen, ®bronectin and bovine serum albumin, whereas the T-cells had increased adhesion to the glycated collagen and bovine serum albumin but reduced adhesion to advanced glycated ®bronectin. Experiments with dierent stimulating agents showed that phorbol-myriastate, acetate (A550=0.672 2 0.068, S.E.M., n = 40), glucose (A550=0.593 2 0.051, S.E.M., n = 40) and TNF-a (A550=0.580 2 0.042, S.E.M., n = 40) increased the adhesion of U937 cells to advanced glycated bovine serum albumin in comparison with the adhesion of the untreated cells (A550=0.260 2 0.046, S.E.M., n = 40). This is probably due to an upregulation of the expression or the activity of the receptors for the advanced glycation end-products. Conclusion: based on the results obtained it is concluded that the receptors for nonenzymatically glycated proteins expressed on the surface of lymphoid cells could act also as cell adhesion molecules. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Nonenzymatic glycation; Diabetes; Cell adhesion; Receptors; Atherogenesis
Abbreviations: AGEs, advanced glycation end-products; RAGE, receptor for advanced glycation end-products; PBS, phosphate buered saline; PMA, phorbol myriastate acetate; BSA, bovine serum albumin; HSA, human serum albumin; FCS, fetal calf serum; TNF-a, tumor necrosis factor a,; IFN-g, interferon g; LPS, lipopolysaccharide; RGID, arginine±glycine±aspartic acid. * Corresponding author. Fax: +35-92-720-925. E-mail address: [email protected] (Stanimir D. Kyurkchiev) 1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 2 5 - 4
798
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
1. Introduction The chronic maintenance of high glucose levels in the blood leads to a drastic modi®cation of the structure, function and properties of many biomolecules. It is due to a multistep nonenzymatic reaction between the glucose molecule and the a- or e-amino-groups of the proteins, called glycation. The products formed during the early stage of the nonenzymatic glycation, named Amadori-products, undergo complex rearrangements and are converted further into advanced glycation end-products (AGEs). It has been shown that the latter are the major cause for the clinical complications in diabetes mellitus patients such as nephropathy, retinopathy, neuropathy and atherosclerosis [1,2]. AGE-structures have been found in long living proteins like hemoglobin and extracellular matrix proteins [3]. These highly reactive AGE-compounds accumulated in the basement membrane of the blood vessel walls can further link covalently soluble proteins such as IgG, albumin or immune complexes [4]. Much progress in understanding the signi®cance of the nonenzymatic glycation has been made after the identi®cation of speci®c AGEbinding proteins on the surface of many cell types. AGE-binding proteins of 60 and 90 kDa were isolated from rat liver membranes and it has been shown that they were expressed on rat monocyte/macrophage surfaces [5]. Another AGE-binding protein, a member of the immunogobulin superfamily was isolated from bovine lung [6] and was found to be expressed on endothelial cells, mononuclear phagocytes and vascular smooth muscle cells [7]. This 35-kDa protein, called receptor for advanced glycation end-products (RAGE) was further cloned and expressed [8]. Another family of AGE-binding proteins of 30, 40 and 50 kDa was identi®ed using ligand blotting with radioiodinated AGE-albumin [9]. The interaction of AGEs with their binding proteins on the surface of the corresponding cell types is thought to induce multiple pathogenic responses, like vascular hyperpermeabillity [10], proliferation of smooth muscle cells [11], expression of adhesion molecules [12], production
of cytokines and growth factors [3,13], induction of oxidative stress in vasculature [14,15], transendothelial monocyte hemotaxis [16,17]. Most of the listed eects are strongly related to the early atherogenesis and contribute to the micro- and macroangiopathy in diabetes. Less is known about the interference of AGEs with the cellular adhesion. Gilcrease and Hoover have observed an increased adhesion of endotoxin activated human monocytes on advanced glycated proteins [18,19], which was explained by an AGE±RAGE interaction. Advanced glycated vitronectin was found to cause impaired endothelial cell adhesion and spreading [20]. Wautier et al. [14] reported an enhanced binding of diabetic erythrocytes to endothelial cells causing vascular dysfunction. This study is focused on the eect of the dierent stages of the nonenzymatic glycation reaction on the adhesion of lymphoid cells. 2. Materials and methods 2.1. Cell lines Two human cell lines were used throughout these experiments. A monocyte-macrophage-like cell line U937 was kindly donated by INSERM U435, Paris, France and the T-cell lymphoma line Jurkat was supplied by the National Bank for Microbial Strains and Tissue Cultures, Bulgaria. 2.2. Glycation of protein The nonenzymatic glycation of human serum albumin (Behringwerke, Germany) as well as the measurement of AGE-formation were carried out as already described [21]. Bovine serum albumin (BSA, purchased from Serva, Germany) free of LPS was glycated in vitro by dissolving 0.15 g protein in 5 ml 10 mM phosphate buered saline (PBS, pH 7.4) containing 200 mM glucose-6-phosphate (Sigma, USA) and 0.05% sodium azide. The reaction mixture was incubated at 378C for 25 days and thereafter extensively dialysed against PBS at 48C. Another
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
batch of BSA was incubated in PBS without glucose-6-phosphate at 378C for 25 days and was used as a control. Sheep collagen type-ill and human ®bronectin (free of LPS), kindly donated by Dr. G. Altankov, Institute of Biophysics, Bulgarian Academy of Sciences were glycated as described by others [20]. Brie¯y, the proteins (100 mg/ml in PBS) were coated on a 96-well ¯at-bottom plate (Nunc, Denmark) for 1 h at 378C (100 ml per well). Wells were washed three times with PBS and incubated with 100 ml per well glucose-6phosphate (200 mmol/l), glucose (200 mmol/l) or PBS for 10 days at 378C. In each experiment eight independent wells were treated with each of the corresponding agents. Glucose-6-phosphate was used to obtain extensively glycated products (AGEs) and glucose was used to reduce the AGE-levels and obtain mildly glycated products (most probably Amadori-adducts). 2.3. Cell adhesion assay Plates coated with native and glycated collagen, ®bronectin or BSA (100 ml per well) were used for the adhesion of U937 and Jurkat cells according to the following protocol. Wells were washed three times with PBS and the residual protein binding sites were blocked with 1% BSA in PBS (200 ml per well) at 378C for 1 h. Cells grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), Lglutamine, sodium pyruvate and antibiotics (all purchased from Sigma, USA) were collected and their viability was measured by trypan blue exclusion. The cell suspension was washed two times with serum free medium. Samples of 1.105 cells in 200 ml were transferred into each well and incubated at 378C for 2 h. The nonadherent cells were removed by double washing with the same medium and the adherent cells were ®xed with 200 ml per well 0.5% glutaraldehyde for 30 min at room temperature. After that 200 ml per well L-glycine (0.2 M) was added and incubated for 30 min at 378C. After washing with PBS the adherent cells were stained with 0.5% methylviolet in 20% ethanol for 20 min at room temperature followed by three washings of 1 min with
799
destilled H2O. Then they were treated with 10% acetic acid (200 ml per well) and the absorption was measured at 550 nm in a MR 700 microplate reader (Dynatech, USA). Where indicated cells were incubated for 2 h with AGE±BSA (200 mg/ ml), PMA (20 ng/ml, Sigma Co.), TNF-a (20 ng/ ml, Sigma Co.), glucose (30 mM, Sigma Co.) or recombinant IFN-g (200 IU/ml) kindly donated by Professor I. Ivanov, Institute of Molecular Biology, Bulgarian Academy of Sciences. 2.4. Biotinylation of human serum albumin (HSA) Native and nonenzymatically glycated HSA were dialyzed against 0.1 M NaHCO3 to a ®nal concentration of 1 mg/ml. To each ml of protein solution were added 125 ml of biotin-N-hydroxysuccinimide ester (1 mg/ml) dissolved in dimethylsulphoxid. The reaction mixture was then left at RT for 4 h on a magnetic stirrer and thereafter extensively dialyzed against PBS. 2.5. Bindinq assay of biotinylated AGE±HSA by U937 cells U937 cells were collected and washed twice with PBS. The cell pellet was resuspended in PBS containing 2% FCS to a ®nal concentration of 5.105 cells/ml. One ml of cell suspension was transferred into each of three dierent glass tubes and supplemented with either PBS, biotinylated native or glycated HSA to a ®nal concentration of 10 mg/ml. The tubes were chilled in ice, shaken for 1 h and washed twice with PBS. The cell pellet was resuspended in PBS containing 2% FCS and 5 mg/ml avidin-peroxidase (Sigma, USA) and shaken on ice for another 1 h. After double washing with PBS the cells were resuspended in citrate buer (pH 5.0) containing 0.05% orthophenylenediamine (Sigma, USA) and 0.03% H2O2. The colour reaction was stopped with 4 N H2SO4 and the optical density was read at 492 nm with Microplate Reader. The tube which contained just cells supplemented with avidin-peroxidase and any biotinylated ligand was used as a blank by the recording.
800
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804 Table 1 Adhesion of PMA-stimulated U937 and Jurkat cells to unmodi®ed and nonenzymatically glycated collagen. Each value represents the mean 2S.E.M. of 32 independent measurements in four independent experiments
Fig. 1. Adhesion of: (a) nonstimulated U937 cells to native BSA; (b) nonstimulated U937 cells to AGE±BSA; (c) PMAstimulated U937 cells to native BSA; (d) PMA-stimulated U937 cells to AGE±BSA; (e) nonstimulated Jurkat cells to native BSA; (f) nonstimulated Jurkat cells to AGE±BSA; (g) PMA-stimulated Jurkat cells to native BSA; (h) PMA-stimulated Jurkat cells to AGE±BSA. Each value represents the mean2S.E.M. of 40 independent measurements in ®ve independent experiments.
2.6. Statistical analysis All data are presented as mean 2 S.E.M. values. Nonparametric statistical analysis (Mann±Whitney U-test) was used to determine the signi®cance of the dierences. 3. Results To study the adhesion of lymphoid cells to nonenzymatically glycated and unmodi®ed proteins we have measured the adhesion of U937 and Jurkat cells to AGE±BSA and BSA. A very low increase in the adhesion of the U937 cells (A550=0.2602 0.046) to AGE±BSA (Fig. 1(b)) was registered in comparison with the adhesion
Adhesive ligand
U937 cells
Jurkat cells
Collagen Mildly glycated collagen AGE±collagen
0.1072 0.028 0.2552 0.032 0.4832 0.038
0.09620.017 0.32620.041 0.25820.029
of the cells to unmodi®ed BSA (A550=0.165 2 0.021, Fig. 1(a)) but the dierence was not statistically signi®cant ( p>0.05). However, a drastic increase in cell adhesion was found after stimulating the cells with PMA, which is known to activate the functions of some cell-adhesion molecules [22,23]. In the latter case the adhesion of the monocytes (A550=0.199 2 0.023) to BSA (Fig. 1(c)) was not changed but the adhesion to AGE±BSA (Fig. 1(d)) increased signi®cantly since the absorption value was A550=0.6162 0.085. Similar adhesion was recorded with the T-cell line Jurkat (Fig. 1(e±h)). To check whether the adhesion was speci®c for the AGE±BSA or it was due to AGEs independently of the nature of the protein we have studied also the adhesion of the cells to the extracellular matrix proteins collagen and ®bronectin upon stimulation with PMA. The results obtained with collagen were quite similar to the binding of PMA-stimulated cells to glycated secreted proteins. The adhesion of both cell lines was higher to the modi®ed protein in comparison
Table 2 Adhesion of PMA-stimulated U937 and Jurkat cells to unmodi®ed and nonenzymatically glycated ®bronectin. Each value represents the mean 2S.E.M. of 32 independent measurements in four independent experiments Adhesive ligand
U937 cells
Jurkat cells
Fibronectin Mildly glycated ®bronectin AGE±®bronectin
0.26520.021 0.38720.034 0.50220.029
0.64520.062 0.49120.048 0.37220.031
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
Fig. 2. Adhesion of U937 cells to AGE±BSA after stimulation with: (a) no stimulating agent; (b) PMA (20 ng/ml); (c) AGE± BSA (200 mg/ml); (d) glucose (30 mM); (e) IFN-g (200 IU/ ml); (f) TNF-a (20 ng/ml). Each value represents the mean2S.E.M. of 40 independent measurements in ®ve independent experiments.
with the native protein. It was demonstrated that for U937 cells this phenomenon was dependent on the extent of glycation since the adhesion was stronger to AGE±collagen in comparison with the mildly glycated collagen (Table 1). When ®bronectin was used in the cell adhesion experiments the monocytes adhered also stronger to the AGE±®bronectin than to the unmodi®ed ®bronectin (Table 2). The adhesion to the mildly glycated ®bronectin was higher in comparison with the native ®bronectin, but lower than that with the AGE±®bronectin (Table 2) as the dierences were statistically signi®cant. Dierent results were obtained when the adhesion of T-cells to ®bronectin was studied (Table 2). Under similar experimental conditions it was shown that the adhesion of Jurkat cells to AGE±®bronectin was much lower in comparison with the adhesion to the native ®bronectin. This
801
Fig. 3. Binding of biotinylated native and advanced glycated HSA by living un®xed U937 cells. Each value represents the mean 2S.E.M. of 11 independent experiments.
eect was found to be speci®c for the AGE± ®bronectin since the mildly glycated product of the same protein did not cause statistically signi®cant change ( p>0.05). Modulation eect of cytokines and other substances on cell adhesion to glycated proteins was also studied. As shown in Fig. 2 a stimulating eect on the adhesion of U937 cells to AGE± BSA was observed upon incubation with PMA Fig. 2(b)), glucose (A550=0.672 2 0.068, (A550=0.593 2 0.051, Fig. 2(d)) or TNF-a (A550=0.580 2 0.042, Fig. 2(f)) compared to A550=0.2602 0.046, corresponding to the adhesion of the nonstimulated cells (Fig. 2(a)). Contrary to that the treatment of the cells with AGE±BSA (Fig. 2(c)) or IFN-g (Fig. 2(e)) did not cause an increase of the binding to glycated proteins. The values recorded in these experand iments were A550=0.1992 0.028 A550=0.3112 0.024, respectively. In attempts to check the presence of AGEbinding proteins on the surface of U937 cells bio-
802
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
tinylated AGE±HSA (native HSA) was allowed to react with living un®xed cells. As illustrated in Fig. 3, U937 cells bound more avidly biotinylated AGE±HSA (A492=0.752 2 0.035) in comparison with the nonglycated biotinylated protein (A492=0.2852 0.026) since the dierence was statistically signi®cant ( p < 0.05). These results clearly demonstrated that U937 cells expressed functionally active AGE-binding proteins.
4. Discussion It has been generally accepted that the altered cellular adhesion is a sign of endothelial dysfunction related to severe pathological changes. The increased adhesion and subsequent migration of monocytes through the endothelia are known to be the very early events in atherogenesis. In that context it is important to de®ne and study the factors aecting the adhesion of cells to vascular walls. The nonenzymatic glycation is a process aecting the long lived proteins of the extracellular matrix, which are involved in the adhesion of leukocytes. To study the eect of the nonenzymatic glycation on cell adhesion we have prepared both mildly and extensively glycated collagen, ®bronectin and BSA. Glucose-6-phosphate was used to accelerate the glycation reaction in vitro, since it is several times more reactive then the glucose molecule. It has been shown that the incubation of proteins with glucose-6-phosphate for short periods of time results in remarkable accumulation of AGE-structures [11,20,24,25], while at the same incubation glucose causes only minor protein modi®cations [20]. In this way we have prepared mildly glycated products by incubation of the proteins with glucose and extensively glycated products by incubation with glucose-6-phosphate. Using these glycated proteins we registered an increased cellular adhesion of U937 and Jurkat cells to glycated collagen and bovine serum albumin. It is worth mentioning that even minor alterations of the protein molecule led to a signi®cant increase in the adhesion of these cells. Since both cell lines are negative for the integrins
VLA-1, VLA-2 and VLA-3 (which are also collagen receptors [26]) we assume that the observed adhesion is probably due to a speci®c interaction between the AGEs receptors (known to be expressed in high numbers on the surface of monocytes [3,12,13]) and the corresponding AGE±protein. Apparently, this interaction does not depend on the nature of the protein but on the AGE-structures themselves. The results obtained with the Jurkat cells suggests that AGE-binding proteins might be also expressed on the surface of T-cells. The increased cellular adhesion to mildly glycated proteins could be a sign for the presence of another type of receptors speci®c for the early glycation products. An alternative explanation might be that the early products are also able to interact with the AGE-receptors although at lower anity in comparison with the original AGE-ligands. The adhesion of U937 cells to ®bronectin was quite similar to that observed with collagen. The mildly and extensively glycated products of ®bronectin showed dierential eects on T-cells adhesion. A reduced adhesion of Jurkat cells was registered only to the advanced glycated ®bronectin, but not to the mildly glycated protein. Although the reason for the interference of AGEs with cellular adhesion is obscure, we assume that it can be due to an impaired interaction between the cell surface integrins and ®bronectin. Some integrins of b1-type recognize the RGD-sequence (arginine±glycine±aspartic acid) in ®bronectin whose arginine residue could be modi®ed by glycation [27]. This modi®ed ligand would lead to a reduced cell adhesion which could not be compensated by the interaction between AGEs and their receptors because of the lower expression of the AGE-binding proteins in the T-cells. It was also demonstrated that PMA, elevated glucose levels and TNF-a increased the adhesion of U937 cells to AGE±BSA. Depper et al. [28] have shown that PMA employs its eect on lymphocytes by activating protein kinase C, thus mimicking the action of diacylglycerols and ligand±receptor interaction, respectively. This event is essential for the activation of the leuko-
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
cytes since it results in an increase of the number of certain receptors on cells surface [29]. Our results are also consistent with published data concerning the stimulating eect of cachectin/TNF on the binding, endocytosis and degradation of AGE-modi®ed albumin by murine peritoneal macrophages and human blood monocytes in vitro [30], which is explained by an upregulation of some AGE-receptors. Shaw and Crabbe [31] have observed a nonspeci®c binding of AGEs to the surface of macrophages. Based on these and our results we can speculate that the increased adhesion to AGE± proteins is due to an interaction of AGEs with AGE-binding proteins expressed on the cells surface. The latter proteins might not be AGEreceptors, since these molecules have not been shown to transduce signals within the cell. In spite of that the interaction between these proteins and AGEs could have an enhancing eect on cell adhesion. The results reported in this study give us reason to postulate a new function for the AGEbinding proteins which could act as cell adhesion molecules. Concerning the biological signi®cance of this phenomenon it could be speculated that the increased adhesion of monocytes to glycated proteins of the basement membrane of the blood vessels would favor their arrest and subsequent proliferation in the subendothelial space, which might be an early event in the atherogenesis. On the other hand, a reduced adhesion of the T-cells to AGE±®bronectin would prevent their extravasation from the blood vessels and migration to the side of in¯ammation. The results presented here further support the idea that AGEs might play an important role in the mechanisms of accelerated micro- and macroangiopathy and might be one of the reasons for the reduced capability of the immune system for ®ghting bacterial and viral infections in patients with diabetes mellitus. Acknowledgements This work was supported by grants MU-BM26 and K-611 of the National Fund ``Scienti®c
803
Investigations'' of the Ministry of Education and Science, So®a, Bulgaria.
References [1] J.T. Wu, Advanced glycation end-products: a new disease marker for diabetes and aging, J. Clin. Lab. Anal. 7 (1993) 252±255. [2] J.T. Wu, Review of diabetes: identi®cation of markers for early detection, glycemic control, and monitoring clinical complications, J. Clin. Lab. Anal. 7 (1993) 293± 300. [3] H. Vlassara, R. Bucala, L. Striker, Pathogenic eects of advanced glycation: biochemical, biologic and clinical implications for diabetes and aging, Lab. Invest. 70 (1994) 138±151. [4] M. Brownlee, The pathological implications of protein glycation, Clin. Invest. Med. 18 (1995) 275±281. [5] Z. Yang, Z. Makita, Y. Horii, S. Brunelle, A. Cerami, P. Sehajpel, Two novel rat liver membrane proteins that bind advanced glycation end-products: relationship to macrophage receptor for glucose-modi®ed proteins, J. Exp. Med. 174 (1991) 515±524. [6] A.M. Schmidt, M. Vianna, M. Gerlach, J. Brett, J. Ryan, J. Kao, C. Esposito, H. Hegarty, H. Hurley, M. Clauss, F. Wang, Y. Pan, T. Tsang, D. Stern, Isolation and characterization of two binding proteins for advanced glycation end-products from bovine lung which are present on the endothelial cell surface, J. Biol. Chem. 267 (1992) 14987±14997. [7] A.M. Schmidt, O. Hori, R. Cao, S. Yan, J. Brett, J. Wautier, S. Ogawa, K. Kuwabara, M. Matsumoto, RAGE-A novel cellular receptor for advanced glycation end-products, Diabetes 45 (Suppl. 3) (1996) 77±80. [8] M. Neeper, A.M. Schmidt, J. Brett, S. Yang, F. Wang, Y. Pan, K. Elliston, D. Stern, A. Shaw, Cloning and expression of a cell surface receptor for advanced glycation end-products of proteins, J. Biol. Chem. 267 (1992) 14998±15004. [9] E.Y. Skolnik, Z. Yang, S. Rado, M. Kirstein, H. Vlassara, Human and rat mesangial cell receptors for glucose-modi®ed proteins: potential role in kidney tissue remodeling and diabetic nephropathy, J. Exp. Med. 174 (1991) 931±939. [10] J. Wautier, M. Wautier, A.M. Schmidt, G. Anderson, O. Hori, C. Zoukourian, L. Capron, O. Chappey, S. Yan, J. Brett, P. Guillausseau, D. Stern, Receptormediated endothelial cell dysfunction in diabetic vasculopathy, J. Clin. Invest. 97 (1996) 238±243. [11] K. Iino, M. Yoshinari, M. Yamamoto, K. Kaku, Y. Doi, K. Ichikawa, M. Iwase, Fujishima, Eect of glycated collagen on proliferation of human smooth muscle cells in vitro, Diabetologia 39 (1996) 800±806. [12] H. Vlassara, H. Fuh, T. Donnelly, M. Cybulsky, Advanced glycation endproducts promote adhesion mol-
804
[13]
[14]
[15] [16]
[17]
[18]
[19] [20]
[21]
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804 ecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits, Mol. Med. 1 (1995) 447±456. H. Vlassara, L. Moldawer, B. Chan, Cachectin/TNF and IL-1 induced by glucose-modi®ed proteins: role in normal tissue remodeling, Science 240 (1988) 1546± 1548. J. Wautier, C. Zoukourian, O. Chappey, M. Wautier, P. Guillausseau, R. Cao, O. Hori, D. Stern, A.M. Schmidt, Advanced glycation end-products (AGEs) on the surface of diabetic erythrocytes bind to the vessel wall via a speci®c receptor inducing oxidant stress in the vasculature: a link between surface-associated AGEs and diabetic complications, Proc. Natl. Acad. Sci. USA 91 (1994) 7742±7746. N. Taniguchi, et al., Involvement of glycation and oxidative stress in diabetic macroangiopathy, Diabetes 45 (Suppl. 3) (1996) 81±83. M. Kirstein, J. Brett, S. Rado, S. Ogawa, D. Stern, H. Vlassara, Advanced protein glycation induces transendothelial human monocyte chemotaxis and secretion of platelet-derived growth factor: Role in vascular disease of diabetes and aging, Proc. Natl. Acad. Sci. USA 87 (1990) 9010±9014. M.L. Yong, G. Baviello, H. Vlassara, T. Mitsuhashi, Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages, J. Immunol. Methods 201 (1996) 183±188. M.Z. Gilcrease, R.L. Hoover, Activated human monocytes exhibit receptor-mediated adhesion to a nonenzymatically glycated protein substrate, Diabetologia 33 (1990) 329±333. M.Z. Gilcrease, R.L. Hoover, Human monocyte interactions with nonenzymatically glycated collagen, Diabetologia 35 (1992) 160±164. I.W. Bobbink, H.C. Boer, W.L. Tekelenburg, J.D. Banga, P.G. Groot, Eect of extracellular matrix glycation on endothelial cell adhesion and spreading, Diabetes 46 (1997) 87±89. S. Kyurkchiev, G. Ivanov, V. Manolova, Advanced glycated end-products activate the functions of cell adhesion molecules on lymphoid cells, Cell. Mol. Life Sci. 53 (1997) 911±916.
[22] R. Rothlein, T. Springer, The requirement for lymphocyte function associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester, J. Exp. Med. 163 (1986) 1132±1149. [23] G. Ivanov, S. Kyurkchiev, Eect of advanced glycosylation end-products on the activity of integrins expressed on U937 cells, Hum. Immunol. 59 (1998) 325±330. [24] H. Vlassara, R. Bucala, Advanced glycation and diabetes complications: an update, Diabetes Annu. 9 (1995) 227±244. [25] R. Bucala, P. Model, M. Russel, A. Cerami, Modi®cation of DNA rearrangements in an Escherichia coli plasmid, Proc. Natl. Acad. Sci. USA 82 (1985) 8439±8442. [26] S.F. Schlossman, L. Baumsell, W. Gilks, J. Harlan, T. Kishimoto, C. Morimoto, J. Ritz, S. Shaw, R. Silverstein, T. Springer, T. Tedder, R. Todd, White cell dierentiation antigens, in Proceedings of the ®fth international workshop and conference held in Boston, USA, 3±7 November, 1993, Oxford University Press (1995). [27] M.E. Westwood, P.J. Thornalley, Molecular characteristics of methylglyoxal-modi®ed bovine and human serum albumins: comparison with glucose-derived advanced glycation endproduct-modi®ed serum albumins, J. Prot. Chem. 14 (1995) 359±372. [28] J.M. Depper, L. Warren, M. Kronke, P. Noguchi, R. Cunningham, T. Waldmann, W. Creene, Regulation of interleukin 2 receptor expression: eects of phorbol diester, phospholipase C and reexposure to lectin or antigen, J. Immunol. 133 (1984) 3054±3061. [29] Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science 258 (1992) 607±613. [30] H. Vlassara, L. Moldawer, B. Chan, Macrophage/ monocyte receptor for nonenzymatically glycated proteins is upregulated by cachectin/turnor necrosis factor, Clin. Invest. 84 (1989) 1813±1820. [31] S. Shaw, J. Crabbe, Non-speci®c binding of advancedglycosylation endproducts to macrophages outweighs speci®c receptor-mediated interactions, Biochem. J. 304 (1994) 121±129.
Increased adhesion of lymphoid cells to glycated proteins Georgi I. Ivanov a, Todor A. Chaushev a, Lilia N. Dakovska b, Stanimir D. Kyurkchiev a,* a
Department of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shosse, 1113 So®a, Bulgaria b University Hospital of Endocrinology and Gerontology, Medical University, 6 Blvd. Damian Gruev, 1303 So®a, Bulgaria Received 25 November 1998
Abstract Background and aims: the advanced glycation end-products are involved in the pathogenesis of vascular damages and other clinical complications in diabetic patients. The aim of this study was to investigate the adhesion of lymphoid cells to nonenzymatically glycated proteins in comparison with the unmodi®ed substances. Methods: two cell lines (monocyte-macrophage line U937 and the T-cell line Jurkat) were used throughout the experiments. The cells were left to adhere to nonenzymatically glycated and native proteins coated on a 96-well ¯atbottom plates and the cellular adhesion was registered as absorption at 550 nm following the method described by Ivanov and Kyurkchiev [G. Ivanov, S. Kyurkchiev, Eect of advanced glycosylation end-products on the activity of integrins expressed on U937 cells, Hum. Immunol. 59 (1998) 325±330.]. Results: it was found that the monocytes had increased adhesion to nonenzymatically glycated proteins such as collagen, ®bronectin and bovine serum albumin, whereas the T-cells had increased adhesion to the glycated collagen and bovine serum albumin but reduced adhesion to advanced glycated ®bronectin. Experiments with dierent stimulating agents showed that phorbol-myriastate, acetate (A550=0.672 2 0.068, S.E.M., n = 40), glucose (A550=0.593 2 0.051, S.E.M., n = 40) and TNF-a (A550=0.580 2 0.042, S.E.M., n = 40) increased the adhesion of U937 cells to advanced glycated bovine serum albumin in comparison with the adhesion of the untreated cells (A550=0.260 2 0.046, S.E.M., n = 40). This is probably due to an upregulation of the expression or the activity of the receptors for the advanced glycation end-products. Conclusion: based on the results obtained it is concluded that the receptors for nonenzymatically glycated proteins expressed on the surface of lymphoid cells could act also as cell adhesion molecules. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Nonenzymatic glycation; Diabetes; Cell adhesion; Receptors; Atherogenesis
Abbreviations: AGEs, advanced glycation end-products; RAGE, receptor for advanced glycation end-products; PBS, phosphate buered saline; PMA, phorbol myriastate acetate; BSA, bovine serum albumin; HSA, human serum albumin; FCS, fetal calf serum; TNF-a, tumor necrosis factor a,; IFN-g, interferon g; LPS, lipopolysaccharide; RGID, arginine±glycine±aspartic acid. * Corresponding author. Fax: +35-92-720-925. E-mail address: [email protected] (Stanimir D. Kyurkchiev) 1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 2 5 - 4
798
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
1. Introduction The chronic maintenance of high glucose levels in the blood leads to a drastic modi®cation of the structure, function and properties of many biomolecules. It is due to a multistep nonenzymatic reaction between the glucose molecule and the a- or e-amino-groups of the proteins, called glycation. The products formed during the early stage of the nonenzymatic glycation, named Amadori-products, undergo complex rearrangements and are converted further into advanced glycation end-products (AGEs). It has been shown that the latter are the major cause for the clinical complications in diabetes mellitus patients such as nephropathy, retinopathy, neuropathy and atherosclerosis [1,2]. AGE-structures have been found in long living proteins like hemoglobin and extracellular matrix proteins [3]. These highly reactive AGE-compounds accumulated in the basement membrane of the blood vessel walls can further link covalently soluble proteins such as IgG, albumin or immune complexes [4]. Much progress in understanding the signi®cance of the nonenzymatic glycation has been made after the identi®cation of speci®c AGEbinding proteins on the surface of many cell types. AGE-binding proteins of 60 and 90 kDa were isolated from rat liver membranes and it has been shown that they were expressed on rat monocyte/macrophage surfaces [5]. Another AGE-binding protein, a member of the immunogobulin superfamily was isolated from bovine lung [6] and was found to be expressed on endothelial cells, mononuclear phagocytes and vascular smooth muscle cells [7]. This 35-kDa protein, called receptor for advanced glycation end-products (RAGE) was further cloned and expressed [8]. Another family of AGE-binding proteins of 30, 40 and 50 kDa was identi®ed using ligand blotting with radioiodinated AGE-albumin [9]. The interaction of AGEs with their binding proteins on the surface of the corresponding cell types is thought to induce multiple pathogenic responses, like vascular hyperpermeabillity [10], proliferation of smooth muscle cells [11], expression of adhesion molecules [12], production
of cytokines and growth factors [3,13], induction of oxidative stress in vasculature [14,15], transendothelial monocyte hemotaxis [16,17]. Most of the listed eects are strongly related to the early atherogenesis and contribute to the micro- and macroangiopathy in diabetes. Less is known about the interference of AGEs with the cellular adhesion. Gilcrease and Hoover have observed an increased adhesion of endotoxin activated human monocytes on advanced glycated proteins [18,19], which was explained by an AGE±RAGE interaction. Advanced glycated vitronectin was found to cause impaired endothelial cell adhesion and spreading [20]. Wautier et al. [14] reported an enhanced binding of diabetic erythrocytes to endothelial cells causing vascular dysfunction. This study is focused on the eect of the dierent stages of the nonenzymatic glycation reaction on the adhesion of lymphoid cells. 2. Materials and methods 2.1. Cell lines Two human cell lines were used throughout these experiments. A monocyte-macrophage-like cell line U937 was kindly donated by INSERM U435, Paris, France and the T-cell lymphoma line Jurkat was supplied by the National Bank for Microbial Strains and Tissue Cultures, Bulgaria. 2.2. Glycation of protein The nonenzymatic glycation of human serum albumin (Behringwerke, Germany) as well as the measurement of AGE-formation were carried out as already described [21]. Bovine serum albumin (BSA, purchased from Serva, Germany) free of LPS was glycated in vitro by dissolving 0.15 g protein in 5 ml 10 mM phosphate buered saline (PBS, pH 7.4) containing 200 mM glucose-6-phosphate (Sigma, USA) and 0.05% sodium azide. The reaction mixture was incubated at 378C for 25 days and thereafter extensively dialysed against PBS at 48C. Another
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
batch of BSA was incubated in PBS without glucose-6-phosphate at 378C for 25 days and was used as a control. Sheep collagen type-ill and human ®bronectin (free of LPS), kindly donated by Dr. G. Altankov, Institute of Biophysics, Bulgarian Academy of Sciences were glycated as described by others [20]. Brie¯y, the proteins (100 mg/ml in PBS) were coated on a 96-well ¯at-bottom plate (Nunc, Denmark) for 1 h at 378C (100 ml per well). Wells were washed three times with PBS and incubated with 100 ml per well glucose-6phosphate (200 mmol/l), glucose (200 mmol/l) or PBS for 10 days at 378C. In each experiment eight independent wells were treated with each of the corresponding agents. Glucose-6-phosphate was used to obtain extensively glycated products (AGEs) and glucose was used to reduce the AGE-levels and obtain mildly glycated products (most probably Amadori-adducts). 2.3. Cell adhesion assay Plates coated with native and glycated collagen, ®bronectin or BSA (100 ml per well) were used for the adhesion of U937 and Jurkat cells according to the following protocol. Wells were washed three times with PBS and the residual protein binding sites were blocked with 1% BSA in PBS (200 ml per well) at 378C for 1 h. Cells grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), Lglutamine, sodium pyruvate and antibiotics (all purchased from Sigma, USA) were collected and their viability was measured by trypan blue exclusion. The cell suspension was washed two times with serum free medium. Samples of 1.105 cells in 200 ml were transferred into each well and incubated at 378C for 2 h. The nonadherent cells were removed by double washing with the same medium and the adherent cells were ®xed with 200 ml per well 0.5% glutaraldehyde for 30 min at room temperature. After that 200 ml per well L-glycine (0.2 M) was added and incubated for 30 min at 378C. After washing with PBS the adherent cells were stained with 0.5% methylviolet in 20% ethanol for 20 min at room temperature followed by three washings of 1 min with
799
destilled H2O. Then they were treated with 10% acetic acid (200 ml per well) and the absorption was measured at 550 nm in a MR 700 microplate reader (Dynatech, USA). Where indicated cells were incubated for 2 h with AGE±BSA (200 mg/ ml), PMA (20 ng/ml, Sigma Co.), TNF-a (20 ng/ ml, Sigma Co.), glucose (30 mM, Sigma Co.) or recombinant IFN-g (200 IU/ml) kindly donated by Professor I. Ivanov, Institute of Molecular Biology, Bulgarian Academy of Sciences. 2.4. Biotinylation of human serum albumin (HSA) Native and nonenzymatically glycated HSA were dialyzed against 0.1 M NaHCO3 to a ®nal concentration of 1 mg/ml. To each ml of protein solution were added 125 ml of biotin-N-hydroxysuccinimide ester (1 mg/ml) dissolved in dimethylsulphoxid. The reaction mixture was then left at RT for 4 h on a magnetic stirrer and thereafter extensively dialyzed against PBS. 2.5. Bindinq assay of biotinylated AGE±HSA by U937 cells U937 cells were collected and washed twice with PBS. The cell pellet was resuspended in PBS containing 2% FCS to a ®nal concentration of 5.105 cells/ml. One ml of cell suspension was transferred into each of three dierent glass tubes and supplemented with either PBS, biotinylated native or glycated HSA to a ®nal concentration of 10 mg/ml. The tubes were chilled in ice, shaken for 1 h and washed twice with PBS. The cell pellet was resuspended in PBS containing 2% FCS and 5 mg/ml avidin-peroxidase (Sigma, USA) and shaken on ice for another 1 h. After double washing with PBS the cells were resuspended in citrate buer (pH 5.0) containing 0.05% orthophenylenediamine (Sigma, USA) and 0.03% H2O2. The colour reaction was stopped with 4 N H2SO4 and the optical density was read at 492 nm with Microplate Reader. The tube which contained just cells supplemented with avidin-peroxidase and any biotinylated ligand was used as a blank by the recording.
800
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804 Table 1 Adhesion of PMA-stimulated U937 and Jurkat cells to unmodi®ed and nonenzymatically glycated collagen. Each value represents the mean 2S.E.M. of 32 independent measurements in four independent experiments
Fig. 1. Adhesion of: (a) nonstimulated U937 cells to native BSA; (b) nonstimulated U937 cells to AGE±BSA; (c) PMAstimulated U937 cells to native BSA; (d) PMA-stimulated U937 cells to AGE±BSA; (e) nonstimulated Jurkat cells to native BSA; (f) nonstimulated Jurkat cells to AGE±BSA; (g) PMA-stimulated Jurkat cells to native BSA; (h) PMA-stimulated Jurkat cells to AGE±BSA. Each value represents the mean2S.E.M. of 40 independent measurements in ®ve independent experiments.
2.6. Statistical analysis All data are presented as mean 2 S.E.M. values. Nonparametric statistical analysis (Mann±Whitney U-test) was used to determine the signi®cance of the dierences. 3. Results To study the adhesion of lymphoid cells to nonenzymatically glycated and unmodi®ed proteins we have measured the adhesion of U937 and Jurkat cells to AGE±BSA and BSA. A very low increase in the adhesion of the U937 cells (A550=0.2602 0.046) to AGE±BSA (Fig. 1(b)) was registered in comparison with the adhesion
Adhesive ligand
U937 cells
Jurkat cells
Collagen Mildly glycated collagen AGE±collagen
0.1072 0.028 0.2552 0.032 0.4832 0.038
0.09620.017 0.32620.041 0.25820.029
of the cells to unmodi®ed BSA (A550=0.165 2 0.021, Fig. 1(a)) but the dierence was not statistically signi®cant ( p>0.05). However, a drastic increase in cell adhesion was found after stimulating the cells with PMA, which is known to activate the functions of some cell-adhesion molecules [22,23]. In the latter case the adhesion of the monocytes (A550=0.199 2 0.023) to BSA (Fig. 1(c)) was not changed but the adhesion to AGE±BSA (Fig. 1(d)) increased signi®cantly since the absorption value was A550=0.6162 0.085. Similar adhesion was recorded with the T-cell line Jurkat (Fig. 1(e±h)). To check whether the adhesion was speci®c for the AGE±BSA or it was due to AGEs independently of the nature of the protein we have studied also the adhesion of the cells to the extracellular matrix proteins collagen and ®bronectin upon stimulation with PMA. The results obtained with collagen were quite similar to the binding of PMA-stimulated cells to glycated secreted proteins. The adhesion of both cell lines was higher to the modi®ed protein in comparison
Table 2 Adhesion of PMA-stimulated U937 and Jurkat cells to unmodi®ed and nonenzymatically glycated ®bronectin. Each value represents the mean 2S.E.M. of 32 independent measurements in four independent experiments Adhesive ligand
U937 cells
Jurkat cells
Fibronectin Mildly glycated ®bronectin AGE±®bronectin
0.26520.021 0.38720.034 0.50220.029
0.64520.062 0.49120.048 0.37220.031
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
Fig. 2. Adhesion of U937 cells to AGE±BSA after stimulation with: (a) no stimulating agent; (b) PMA (20 ng/ml); (c) AGE± BSA (200 mg/ml); (d) glucose (30 mM); (e) IFN-g (200 IU/ ml); (f) TNF-a (20 ng/ml). Each value represents the mean2S.E.M. of 40 independent measurements in ®ve independent experiments.
with the native protein. It was demonstrated that for U937 cells this phenomenon was dependent on the extent of glycation since the adhesion was stronger to AGE±collagen in comparison with the mildly glycated collagen (Table 1). When ®bronectin was used in the cell adhesion experiments the monocytes adhered also stronger to the AGE±®bronectin than to the unmodi®ed ®bronectin (Table 2). The adhesion to the mildly glycated ®bronectin was higher in comparison with the native ®bronectin, but lower than that with the AGE±®bronectin (Table 2) as the dierences were statistically signi®cant. Dierent results were obtained when the adhesion of T-cells to ®bronectin was studied (Table 2). Under similar experimental conditions it was shown that the adhesion of Jurkat cells to AGE±®bronectin was much lower in comparison with the adhesion to the native ®bronectin. This
801
Fig. 3. Binding of biotinylated native and advanced glycated HSA by living un®xed U937 cells. Each value represents the mean 2S.E.M. of 11 independent experiments.
eect was found to be speci®c for the AGE± ®bronectin since the mildly glycated product of the same protein did not cause statistically signi®cant change ( p>0.05). Modulation eect of cytokines and other substances on cell adhesion to glycated proteins was also studied. As shown in Fig. 2 a stimulating eect on the adhesion of U937 cells to AGE± BSA was observed upon incubation with PMA Fig. 2(b)), glucose (A550=0.672 2 0.068, (A550=0.593 2 0.051, Fig. 2(d)) or TNF-a (A550=0.580 2 0.042, Fig. 2(f)) compared to A550=0.2602 0.046, corresponding to the adhesion of the nonstimulated cells (Fig. 2(a)). Contrary to that the treatment of the cells with AGE±BSA (Fig. 2(c)) or IFN-g (Fig. 2(e)) did not cause an increase of the binding to glycated proteins. The values recorded in these experand iments were A550=0.1992 0.028 A550=0.3112 0.024, respectively. In attempts to check the presence of AGEbinding proteins on the surface of U937 cells bio-
802
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
tinylated AGE±HSA (native HSA) was allowed to react with living un®xed cells. As illustrated in Fig. 3, U937 cells bound more avidly biotinylated AGE±HSA (A492=0.752 2 0.035) in comparison with the nonglycated biotinylated protein (A492=0.2852 0.026) since the dierence was statistically signi®cant ( p < 0.05). These results clearly demonstrated that U937 cells expressed functionally active AGE-binding proteins.
4. Discussion It has been generally accepted that the altered cellular adhesion is a sign of endothelial dysfunction related to severe pathological changes. The increased adhesion and subsequent migration of monocytes through the endothelia are known to be the very early events in atherogenesis. In that context it is important to de®ne and study the factors aecting the adhesion of cells to vascular walls. The nonenzymatic glycation is a process aecting the long lived proteins of the extracellular matrix, which are involved in the adhesion of leukocytes. To study the eect of the nonenzymatic glycation on cell adhesion we have prepared both mildly and extensively glycated collagen, ®bronectin and BSA. Glucose-6-phosphate was used to accelerate the glycation reaction in vitro, since it is several times more reactive then the glucose molecule. It has been shown that the incubation of proteins with glucose-6-phosphate for short periods of time results in remarkable accumulation of AGE-structures [11,20,24,25], while at the same incubation glucose causes only minor protein modi®cations [20]. In this way we have prepared mildly glycated products by incubation of the proteins with glucose and extensively glycated products by incubation with glucose-6-phosphate. Using these glycated proteins we registered an increased cellular adhesion of U937 and Jurkat cells to glycated collagen and bovine serum albumin. It is worth mentioning that even minor alterations of the protein molecule led to a signi®cant increase in the adhesion of these cells. Since both cell lines are negative for the integrins
VLA-1, VLA-2 and VLA-3 (which are also collagen receptors [26]) we assume that the observed adhesion is probably due to a speci®c interaction between the AGEs receptors (known to be expressed in high numbers on the surface of monocytes [3,12,13]) and the corresponding AGE±protein. Apparently, this interaction does not depend on the nature of the protein but on the AGE-structures themselves. The results obtained with the Jurkat cells suggests that AGE-binding proteins might be also expressed on the surface of T-cells. The increased cellular adhesion to mildly glycated proteins could be a sign for the presence of another type of receptors speci®c for the early glycation products. An alternative explanation might be that the early products are also able to interact with the AGE-receptors although at lower anity in comparison with the original AGE-ligands. The adhesion of U937 cells to ®bronectin was quite similar to that observed with collagen. The mildly and extensively glycated products of ®bronectin showed dierential eects on T-cells adhesion. A reduced adhesion of Jurkat cells was registered only to the advanced glycated ®bronectin, but not to the mildly glycated protein. Although the reason for the interference of AGEs with cellular adhesion is obscure, we assume that it can be due to an impaired interaction between the cell surface integrins and ®bronectin. Some integrins of b1-type recognize the RGD-sequence (arginine±glycine±aspartic acid) in ®bronectin whose arginine residue could be modi®ed by glycation [27]. This modi®ed ligand would lead to a reduced cell adhesion which could not be compensated by the interaction between AGEs and their receptors because of the lower expression of the AGE-binding proteins in the T-cells. It was also demonstrated that PMA, elevated glucose levels and TNF-a increased the adhesion of U937 cells to AGE±BSA. Depper et al. [28] have shown that PMA employs its eect on lymphocytes by activating protein kinase C, thus mimicking the action of diacylglycerols and ligand±receptor interaction, respectively. This event is essential for the activation of the leuko-
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804
cytes since it results in an increase of the number of certain receptors on cells surface [29]. Our results are also consistent with published data concerning the stimulating eect of cachectin/TNF on the binding, endocytosis and degradation of AGE-modi®ed albumin by murine peritoneal macrophages and human blood monocytes in vitro [30], which is explained by an upregulation of some AGE-receptors. Shaw and Crabbe [31] have observed a nonspeci®c binding of AGEs to the surface of macrophages. Based on these and our results we can speculate that the increased adhesion to AGE± proteins is due to an interaction of AGEs with AGE-binding proteins expressed on the cells surface. The latter proteins might not be AGEreceptors, since these molecules have not been shown to transduce signals within the cell. In spite of that the interaction between these proteins and AGEs could have an enhancing eect on cell adhesion. The results reported in this study give us reason to postulate a new function for the AGEbinding proteins which could act as cell adhesion molecules. Concerning the biological signi®cance of this phenomenon it could be speculated that the increased adhesion of monocytes to glycated proteins of the basement membrane of the blood vessels would favor their arrest and subsequent proliferation in the subendothelial space, which might be an early event in the atherogenesis. On the other hand, a reduced adhesion of the T-cells to AGE±®bronectin would prevent their extravasation from the blood vessels and migration to the side of in¯ammation. The results presented here further support the idea that AGEs might play an important role in the mechanisms of accelerated micro- and macroangiopathy and might be one of the reasons for the reduced capability of the immune system for ®ghting bacterial and viral infections in patients with diabetes mellitus. Acknowledgements This work was supported by grants MU-BM26 and K-611 of the National Fund ``Scienti®c
803
Investigations'' of the Ministry of Education and Science, So®a, Bulgaria.
References [1] J.T. Wu, Advanced glycation end-products: a new disease marker for diabetes and aging, J. Clin. Lab. Anal. 7 (1993) 252±255. [2] J.T. Wu, Review of diabetes: identi®cation of markers for early detection, glycemic control, and monitoring clinical complications, J. Clin. Lab. Anal. 7 (1993) 293± 300. [3] H. Vlassara, R. Bucala, L. Striker, Pathogenic eects of advanced glycation: biochemical, biologic and clinical implications for diabetes and aging, Lab. Invest. 70 (1994) 138±151. [4] M. Brownlee, The pathological implications of protein glycation, Clin. Invest. Med. 18 (1995) 275±281. [5] Z. Yang, Z. Makita, Y. Horii, S. Brunelle, A. Cerami, P. Sehajpel, Two novel rat liver membrane proteins that bind advanced glycation end-products: relationship to macrophage receptor for glucose-modi®ed proteins, J. Exp. Med. 174 (1991) 515±524. [6] A.M. Schmidt, M. Vianna, M. Gerlach, J. Brett, J. Ryan, J. Kao, C. Esposito, H. Hegarty, H. Hurley, M. Clauss, F. Wang, Y. Pan, T. Tsang, D. Stern, Isolation and characterization of two binding proteins for advanced glycation end-products from bovine lung which are present on the endothelial cell surface, J. Biol. Chem. 267 (1992) 14987±14997. [7] A.M. Schmidt, O. Hori, R. Cao, S. Yan, J. Brett, J. Wautier, S. Ogawa, K. Kuwabara, M. Matsumoto, RAGE-A novel cellular receptor for advanced glycation end-products, Diabetes 45 (Suppl. 3) (1996) 77±80. [8] M. Neeper, A.M. Schmidt, J. Brett, S. Yang, F. Wang, Y. Pan, K. Elliston, D. Stern, A. Shaw, Cloning and expression of a cell surface receptor for advanced glycation end-products of proteins, J. Biol. Chem. 267 (1992) 14998±15004. [9] E.Y. Skolnik, Z. Yang, S. Rado, M. Kirstein, H. Vlassara, Human and rat mesangial cell receptors for glucose-modi®ed proteins: potential role in kidney tissue remodeling and diabetic nephropathy, J. Exp. Med. 174 (1991) 931±939. [10] J. Wautier, M. Wautier, A.M. Schmidt, G. Anderson, O. Hori, C. Zoukourian, L. Capron, O. Chappey, S. Yan, J. Brett, P. Guillausseau, D. Stern, Receptormediated endothelial cell dysfunction in diabetic vasculopathy, J. Clin. Invest. 97 (1996) 238±243. [11] K. Iino, M. Yoshinari, M. Yamamoto, K. Kaku, Y. Doi, K. Ichikawa, M. Iwase, Fujishima, Eect of glycated collagen on proliferation of human smooth muscle cells in vitro, Diabetologia 39 (1996) 800±806. [12] H. Vlassara, H. Fuh, T. Donnelly, M. Cybulsky, Advanced glycation endproducts promote adhesion mol-
804
[13]
[14]
[15] [16]
[17]
[18]
[19] [20]
[21]
G. I. Ivanov et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 797±804 ecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits, Mol. Med. 1 (1995) 447±456. H. Vlassara, L. Moldawer, B. Chan, Cachectin/TNF and IL-1 induced by glucose-modi®ed proteins: role in normal tissue remodeling, Science 240 (1988) 1546± 1548. J. Wautier, C. Zoukourian, O. Chappey, M. Wautier, P. Guillausseau, R. Cao, O. Hori, D. Stern, A.M. Schmidt, Advanced glycation end-products (AGEs) on the surface of diabetic erythrocytes bind to the vessel wall via a speci®c receptor inducing oxidant stress in the vasculature: a link between surface-associated AGEs and diabetic complications, Proc. Natl. Acad. Sci. USA 91 (1994) 7742±7746. N. Taniguchi, et al., Involvement of glycation and oxidative stress in diabetic macroangiopathy, Diabetes 45 (Suppl. 3) (1996) 81±83. M. Kirstein, J. Brett, S. Rado, S. Ogawa, D. Stern, H. Vlassara, Advanced protein glycation induces transendothelial human monocyte chemotaxis and secretion of platelet-derived growth factor: Role in vascular disease of diabetes and aging, Proc. Natl. Acad. Sci. USA 87 (1990) 9010±9014. M.L. Yong, G. Baviello, H. Vlassara, T. Mitsuhashi, Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages, J. Immunol. Methods 201 (1996) 183±188. M.Z. Gilcrease, R.L. Hoover, Activated human monocytes exhibit receptor-mediated adhesion to a nonenzymatically glycated protein substrate, Diabetologia 33 (1990) 329±333. M.Z. Gilcrease, R.L. Hoover, Human monocyte interactions with nonenzymatically glycated collagen, Diabetologia 35 (1992) 160±164. I.W. Bobbink, H.C. Boer, W.L. Tekelenburg, J.D. Banga, P.G. Groot, Eect of extracellular matrix glycation on endothelial cell adhesion and spreading, Diabetes 46 (1997) 87±89. S. Kyurkchiev, G. Ivanov, V. Manolova, Advanced glycated end-products activate the functions of cell adhesion molecules on lymphoid cells, Cell. Mol. Life Sci. 53 (1997) 911±916.
[22] R. Rothlein, T. Springer, The requirement for lymphocyte function associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester, J. Exp. Med. 163 (1986) 1132±1149. [23] G. Ivanov, S. Kyurkchiev, Eect of advanced glycosylation end-products on the activity of integrins expressed on U937 cells, Hum. Immunol. 59 (1998) 325±330. [24] H. Vlassara, R. Bucala, Advanced glycation and diabetes complications: an update, Diabetes Annu. 9 (1995) 227±244. [25] R. Bucala, P. Model, M. Russel, A. Cerami, Modi®cation of DNA rearrangements in an Escherichia coli plasmid, Proc. Natl. Acad. Sci. USA 82 (1985) 8439±8442. [26] S.F. Schlossman, L. Baumsell, W. Gilks, J. Harlan, T. Kishimoto, C. Morimoto, J. Ritz, S. Shaw, R. Silverstein, T. Springer, T. Tedder, R. Todd, White cell dierentiation antigens, in Proceedings of the ®fth international workshop and conference held in Boston, USA, 3±7 November, 1993, Oxford University Press (1995). [27] M.E. Westwood, P.J. Thornalley, Molecular characteristics of methylglyoxal-modi®ed bovine and human serum albumins: comparison with glucose-derived advanced glycation endproduct-modi®ed serum albumins, J. Prot. Chem. 14 (1995) 359±372. [28] J.M. Depper, L. Warren, M. Kronke, P. Noguchi, R. Cunningham, T. Waldmann, W. Creene, Regulation of interleukin 2 receptor expression: eects of phorbol diester, phospholipase C and reexposure to lectin or antigen, J. Immunol. 133 (1984) 3054±3061. [29] Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science 258 (1992) 607±613. [30] H. Vlassara, L. Moldawer, B. Chan, Macrophage/ monocyte receptor for nonenzymatically glycated proteins is upregulated by cachectin/turnor necrosis factor, Clin. Invest. 84 (1989) 1813±1820. [31] S. Shaw, J. Crabbe, Non-speci®c binding of advancedglycosylation endproducts to macrophages outweighs speci®c receptor-mediated interactions, Biochem. J. 304 (1994) 121±129.