40 1
ISOLATION AND CULTURE OF SYNOVIAL MICROVASCULAR ENDOTHELIAL CELLS Characterization and Assessment of Adhesion Molecule Expression S. E. ABBOT, A. KAUL, C. R. STEVENS, and D. R. BLAKE
Objective. In vitro studies investigating the role of the synovial endothelium in the pathogenesis of rheumatoid arthritis (RA)have, until recently, been performed using cultured endothelial cells of nonsynovial macrovascular origin. In an attempt to more correctly model in vivo conditions, a method for the isolation and culture of synovial microvascular endothelial cells (SMEC) has been developed. Methods. SMEC were isolated, primarily, by the use of lectin-coated (Ulex europaeus agglutinin type I), magnetizable polystyrene beads. Results. Isolated cells exhibit classic endothelial “cobblestone” morphology, express von Willebrand factor, metabolize acetylated low-density lipoprotein, and exhibit a cytokine (interleukin-lkmediated expression of endothelial leukocyte adhesion molecule type 1 (ELAM-1) and intercellular adhesion molecule type 1 (ICAM-1). ELAM-1 levels were significantly elevated in SMEC, compared with human umbilical vein endothelial cells, over a range of interleukin-1 concentrations. Conclusion. This increased expression of From the Inflammation Group, Bone and Joint Research Unit, The London Hospital Medical College, London, United Kingdom. Supported by the Arthritis and Rheumatism Council of Great Britain. S. E. Abbot, MSc; A. Kaul, BDs; C. R. Stevens, PhD; D. R. Blake, MB, FRCP. Address reprint requests to S. E. Abbot, MSc, The London Hospital Medical College, Arthritis and Rheumatism Council Building, 25-29 Ashfield Street, London El 2AD. UK. Submitted for publication April 19, 1991; accepted in revised form December 26, 199 I. Arthritis and Rheumatism, Vol. 35, No. 4 (April 1992)
ELAM-1 by SMEC may be a potentiating step in the pathogenesis of RA. The etiology of rheumatoid arthritis (RA) remains unknown; however, recent studies have suggested a central role for the synovial microvascular endothelium in its pathogenesis. Persistent inflammation is characteristic of the rheumatoid joint. Inflammation is modulated by endothelial cell control of leukocyte chemotaxis, adhesion, and infiltration (1). The endothelium is known to locally regulate vascular tone and permeability via the antagonistic actions of endothelium-derived relaxing factor and endothelin. The eventual destruction of the rheumatoid joint is a consequence of synovial membrane proliferation and pannus invasion of the articular cartilage. These processes are associated with uncontrolled endothelial cell migration and proliferation (angiogenesis). More recently, the synovial endothelium has been directly implicated in contributing to the erosive process through its ability to generate proinflammatory reactive oxygen species (2,3). Investigations of these processes have, until recently, used endothelial cells isolated from the large blood vessels of various species (4). It has been recognized that there is heterogeneity between endothelial cells from different species, as well as from different tissues within a species (5,6). This would imply that modeling the synovial microvasculature by the in vitro use of endothelial cells of nonsynovial origin might be invalid. In an attempt to more correctly model endothelial responses in the synovium, we devised a method for isolating and culturing microvas-
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cular endothelial cells of synovial origin. Initial studies have indicated that there are differences in the expression of endothelial leukocyte adhesion molecule type 1 (ELAM-1) by synovial microvascular endothelial cells (SMEC) and human umbilical vein endothelial cells (HUVEC).
MATERIALS AND METHODS Cell isolation. HUVEC were isolated from fresh umbilical cords by the method of Jaffe et al(7). Isolated cells were cultured in MI99 in a manner similar to that used for SMEC, except that HUVEC were maintained initially with a lower concentration of growth factor (30 CLg/ml). Microvascular preparations were obtained from synovium excised from rheumatoid hip or knee joints obtained at the time of joint replacement surgery. The methods described here were modified from several micro- and macrovascular isolation procedures (5,7-10). Excised tissue was immediately placed in ice-cold Ca2+ and Mgzf-free Hanks’ balanced salt solution (HBSS; Sigma, Poole, Dorset, UK). The HBSS was exchanged 3 times to remove excess blood from the sample prior to dissecting the synovial membrane free of capsular material. At this stage, the microvascular nature of endothelial cells is confirmed according to vessel size. With the aid of a dissecting microscope, vessels with a lumen diameter >50 pm were removed from the dissected tissue and discarded. The remaining tissue was then minced into -3-mm3 pieces, using crossed scalpels. The minced tissue was incubated in HBSS containing 0.1% type I1 collagenase (Sigma) and 0.1% crude collagenase (Sigma) for 60 minutes, with continuous stirring. Following collagenase digestion, the minced preparation was compressed with the flat face of a microspatula. The compressive action of the spatula expresses capillary segments from the cut edges of the tissue. Waste tissue, capillary segments, and single connective cells were separated by sequential filtering through sieves of various pore sizes. All material was passed through a 250-pm steel mesh (Sigma), which trapped large segments of waste tissue. Capillary segments were trapped on a nylon screen (50-pm pore size; Cavendish, London, UK). Segments were then removed by backwashing the filter with HBSS. Samples were centrifuged and resuspended in culture medium M199 (Gibco, Paisley, Scotland) supplemented with 40% fetal calf serum (FCS; Gibco), 100 unitdm1 penicillin and 100 pdml streptomycin (Sigma), 100 pg/ml endothelial cell growth factor (ECGF; Sigma), 17 units/ml heparin (Sigma), 2 mM L-glutamine (Sigma), and 1.2% NaHCO, (Flow; Irvine, Scotland). The resulting isolated cell suspension contains a heterogeneous cell population, from which the endothelial cells were purified. Cell purification. Cell populations, extracted by collagenase digest, contain various proportions of endothelial cells, usually in the range of 1-10%. Further cell purification
steps require an accurate estimate of endothelial cell number. To facilitate this, the mixed cell population obtained from the collagenase digest was plated onto 35-mm Primaria (Falcon, Newcastle-Upon-Tyne, UK) dishes. Following 48 hours growth, endothelial cells were distinguished from other contaminating cells by morphologic features, i.e., those cells growing in discrete colonies of 10-50 cells exhibiting a “cobblestone-like” pattern. An initial enrichment of the endothelial cell proportions was achieved by manually removing contaminating nonendothelial cell colonies from the dish with a flame-drawn pasteur pipette, followed by aspiration of the culture medium. Once an endothelial cell :contaminating cell ratio of approximately 1 : 1 has been achieved, all cells were removed from the plate using a plastic cell scraper (Falcon). A single-cell suspension was then prepared by repeated transfer of the cells through a fire-polished siliconized pasteur pipette. For the purpose of further purification, the total cell number was assessed using a hemacytometer, the endothelial cell proportion being assumed to be 50% of the total. Further purification was achieved by a magnetic cellseparation technique. This employs lectin-coated magnetizable polystyrene beads (Dynabeads, Dynal; Dynatech, Alexandria, VA). Ulex europaeus agglutinin type I (UEA I) lectin binds specifically to many glycoproteins and glycolipids containing a-linked fucose residues and has been established as an excellent marker for human endothelial cells (1 1). Tosyl-activated Dynabeads were washed in distilled water. UEA I was covalently coupled to the beads by vortex suspension in 0.5M borate solution, pH 9.5, containing 10 &ml UEA I. Equal volumes of washed Dynabeads and UEA I solution were mixed for 24 hours at 22°C by slow rotation. The coated beads were collected using a magnelic particle concentrator (MPC). The beads were washed in 0.01M phosphate buffered saline, pH 7.4, containing 0.1% bovine serum albumin for 10 minutes, then for a further 30 minutes, followed by an overnight wash at 4°C. Dynabeads were added to the cell suspension at an approximate concentration of 3 per endothelial cell. Cells and beads were gently mixed at 4°C for 10 minutes, after which endothelial cells that were bound to the Dynabeads were isolated using the MCP. Unbound cells were removed by washing with HBSS. Cell-bead complexes were then resuspended in HBSS, and the procedure was repeated two more times. Isolated synovial microvascular endothelial cells were plated onto 0.1% gelatin-coated dishes. The cells were grown to confluence and passaged with a split ratio of 1 :3. Isolated SMEC were used for assay purposes at passage 3; however, we have found it possible to passage these cells as many as 7 times without altering the endothelial morphology. Characterization of SMEC. Isolated cells were characterized using 4 criteria, as follows. 1) Development of a classic “cobblestone” morphology of the cell monolayer at confluence (7). 2) Immunohistochemical localization of von Willebrand factor (vWF) in a granular cytoplasmic pattern. Localization of vWF was carried out on cells grown to confluence on 0.1% gelatin-coated Thermanox coverslips,
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using a Vectastain elite ABC, peroxidase, reagent kit (Vector, Burlingame, CA). Rabbit anti-human vWF (Dakopatts, Copenhagen, Denmark) primary antibody was used at 1 : 1,500 dilution. 3) Uptake of acetylated low-density lipoprotein (LDL). Endothelial cells possessing a “scavenger pathway” selectively ingest modified LDL (12). Confluent monolayers of HUVEC, SMEC, and mouse fibroblasts were incubated with 10 pg/ml of 1,l’-dioctadecyl-1-(3,3,3’,3’tetramethyl)-indocarboxyamine perchlorate-labeled acetylated LDL (Dil-Ac-LDL; Biogenesis, Bournemouth, UK) for 4 hours, after which the monolayers were examined for fluorescence at 480 nm. 4) Expression of ELAM-1 and intercellular adhesion molecule type 1 (ICAM- I ) , as determined by enzyme-linked immunosorbent assay (ELISA). ELISAs were carried out on 4 occasions, using SMEC and HUVEC grown to confluence in SMEC culture medium on 0. I% gelatin-coated 96-well plates. Confluent cell density was -9,000 cells/well; assays were performed using 3 wells per concentration of interleukin-I (IL-I). Briefly, culture medium from the plates was replaced either with fresh medium alone or with fresh medium containing recombinant IL-la (Roche, Welwyn Garden City, UK) at 2.5, 5 , 25, 50, 250, and 500 D10 unitslml. Plates were incubated at 37°C in an atmosphere of 5% CO, for 4-6 hours. Subsequently, cells were fixed and stained for ELAM-1 and ICAM-I using an ELISA as previously described (13).
RESULTS
B
C Figure 1. Immunohistochemical localization of von Willebrand factor as discrete cytoplasmic granules in A, human umbilical vein
endothelial cells and B, synovial microvascular endothelial cells, but not C, mouse fibroblasts. Bar = 25 pm.
Isolated SMEC grew to confluence on a variety of gelatin-coated substrates. Contamination by nonendothelial cells was not a major problem since the few contaminating cells could be removed manually in a manner similar to that detailed in the initial isolation. Confluent SMEC appeared as closely packed hexagonal cells which overlapped little with adjacent cells. The monolayer exhibited a strict contact-inhibited growth pattern which could be maintained for several weeks with little evident morphologic change. Immunohistochemical localization of vWF showed a discrete granular cytoplasmic staining pattern in SMEC, similar to that observed in HUVEC. No staining was observed on mouse fibroblasts (Figures 1A-C). Following incubation with Dil-Ac-LDL, both SMEC and HUVEC, but not the mouse fibroblasts, fluoresced brightly (Figures 2A-C). ELISAs carried out on passage-4 cultures demonstrated that SMEC express low levels of ELAM-1 constitutively. This expression rose in a dosedependent manner when cells were preincubated with various concentrations of IL-] ELAM-l expression was significantly higher (P< 0.05) than basal levels at
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B A
Figure 2. Fluorescence of ingested 1 ,l’-dioctadecyl-l-(3,3,3’,3’tetramethyl)-indocarboxyamine perchlorate-labeled acetylated lowdensity lipoprotein metabolized by A, human umbilical vein endothelial cells and B, synovial microvascular endothelial cells, but not C, mouse fibroblasts. Bar = 25 pm.
C all IL- 1 concentrations (Figure 3). Constitutive ICAM-1 expression was moderate on SMEC, and this basal expression was significantly increased ( P < 0.05) in response to IL-1 at concentrations of 25 units/ml and 250 units/ml. Comparison of the results of assays performed under the same conditions using HUVEC showed that in response to IL-I, SMEC expressed significantly higher levels of ELAM-I at all concentrations (P < 0.05). These differences may, however, reflect similar increases in both cell types, with SMEC having a higher basal level. There was little difference in the expression of ICAM-1 by the two cell types in response to 1L-1 (Figures 3A and B). Identical assays
performed on HUVEC, grown to confluence in either HUVEC or SMEC culture medium, showed that the FCS or ECGF levels in growth medium had no significant effect on either ICAM-I or ELAM-I expression.
DISCUSSION The in vitro use of isolated large-vessel endothelial cells, particularly HUVEC, to model microvascular-associated events is a technique that is used in many laboratories. Extrapolation of the results obtained using HUVEC to the conditions within a microvascular bed, in this case the synovium, has been called into question because of potential heterogeneity
ISOLATION AND CULTURE OF SMEC Optical Density
,,I
+
U.J
0.25 0.2
+
0.15
+
11-1 Concentration (units/ml)
~ ~ 0 . 0 5
A Optical hnsity 0.25 0.2 0.15
T
T
0.1 0.05
0
0
0
0.05 0.05
5
5
50
1 50
500 500
IL-1 Concentration (unitdml)
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Figure 3. Expression of A, endothelial leukocyte adhesion molecule type 1 (ELAM-1) and B, intercellular adhesion molecule type I (ICAM-I) by synovial microvascular endothelial cells (SMEC) (solid bars) and by human umbilical vein endothelial cells (HUVEC) (open bars) in the presence of various concentrations of interleukin-1 (IL-I). There was a significant increase in basal expression of ELAM-1 by SMEC compared with HUVEC; this increase was maintained at all IL-1pretreatment levels. There were no significant differences in ICAM-1 expression by SMEC compared with HUVEC, either basally or in response to IL-1 pretreatment.
of the cells. To facilitate more appropriate modeling of the synovium in our studies of inflammation, a method for the isolation and culture of synovial microvascular endothelial cells was devised. The method described yields high numbers of cells which have been characterized, by several standard techniques, as being endothelial and of synovial microvascular origin. These cells retained all endothelial characteristics through 6 subcultures over the course of several weeks in culture. All of the characterization procedures, with the exception of ELAWICAM expression, are procedures that employ features documented as being retained by both micro- and macrovascular endothelial cells after isolation and culture. Thus, the similarity of
405
HUVEC to SMEC with respect to these criteria was anticipated. The main difference found in the characterization of the two cell types related to the expression of ELAM-1, which will be discussed below. Central to the method is a sorting and purification step utilizing an endothelial-specific lectin (UEA-1) covalently coupled to magnetizable polystyrene beads. Similar methods have previously been employed to successfully isolate a variety of cell types including SMEC (14). It has been suggested by Lea et a1 (15) that Dynabead adherence to cells prevents cell adhesion to tissue culture plastic, but this was disputed in the isolation of SMEC (14). We found that Lea and colleagues’ observations held true, since a significant inhibition of cell adhesion to plastic was observed when the bead-to-cell ratio exceeded 10: 1. It is presumed that the inhibition is due to attached beads physically preventing cell spreading. Jackson et al(l4) reportedly avoided this problem by removing attached beads from isolated cells by competitive binding of free O.1M fucose to the beads at 4°C. This technique, however, did not prove to be reproducible. We have shown that a successful alternative is to use fewer beads. To achieve this, we employed a manual weeding process, as previously described (3, which would give an accurate estimate of endothelial cell numbers prior to the addition of the beads. This allows the calculation of the correct number of beads to add in order to achieve a low bead-to-cell ratio. We found that a ratio of -3: 1 gave a good harvest of endothelial cells without interfering with adhesion to plastic and subsequent proliferation. The long-term culture of SMEC, at present, requires the use of high levels of ECGF (100 pg/ml) and FCS (40%). Apart from the high cost of these additions, the effects of such high levels on endothelial cell behavior are unknown. While the method is effective, it still requires a constant supply of fresh synovial tissue, since isolated cells have a finite usable life in culture. In the future, it may be possible to immortalize the cell line, thus overcoming this problem to some extent. The present study was, in part, undertaken to assess the suitability of extrapolating the cytokine responses of macrovascular cells, particularly HUVEC, to the microvasculature of the synovium. Similar trends were observed in ELAM-I and ICAM-1 expression on HUVEC and SMEC. While the basic morphologic markers of endothelial cells show similar patterns, our results suggest that SMEC may show an enhanced basal expression and response to IL-1, compared with HUVEC, particularly in their expression of ELAM-1. Synovial tissue sections immunohistochem-
ABBOT ET AL
406 ically stained using antibodies to vWF and ELAM-I have shown, respectively, a patchy distribution and localization to postcapillary venules. This is paradoxical considering the almost universal staining of the isolated microvascular cells. This suggests that either vWF- and ELAM-l-expressing cells have been preferentially selected during isolation, which is possible with regard to ELAM-1 expression, or that culture conditions may either have favored the universal induction of vWF and ELAM-1 expression or have lost an inhibitor that is present in vivo. It is unclear at this time whether these differences represent differences between large- and small-vessel endothelia or a specific response of synovium as an inflamed tissue. Again, further work comparing SMEC isolated from normal tissue and other capillary endothelial cells will be needed to ascertain whether this increased ELAM-1 expression on cultured synovial cells represents a potentiating step in inflammatory joint disease. The cytokine dependence of ELAM-1 expression suggests that its expression in vivo by a minority of the vessels in rheumatoid synovial tissue sections may be cytokine mediated. There is good evidence that cytokines such as IL-1 are present in synovial exudates and are produced by cells within the synovial lining. These cytokines are responsible for neutrophil and lymphocyte binding to endothelial cells in vitro, and thus have a direct relevance to the pathogenesis of RA. Of particular interest is the comparative responses of SMEC and HUVEC. These suggest that SMEC show an enhanced response to IL-1, as measured by the induction of the neutrophil-specific adhesion molecule, ELAM-1. Preliminary studies have shown SMEC to be functionally capable of binding neutrophils; however, experimental variation has precluded our being able to assess the significance of the differences between HUVEC and SMEC. Using the methods detailed here, we have shown that synovial microvascular endothelial cells exhibit a higher ELAM-1 expression, compared with that of HUVEC in the same conditions, a difference that may potentiate the chronic inflammation in the rheumatoid joint.
ACKNOWLEDGMENTS We gratefully acknowledge the generous gift of antiELAM-1 and anti-ICAM-1 antibodies by Dr. Dorian Haskard. Thanks are also extended to Michael Freeman and Dr.Andrew Waterfield for providing the synovial specimens.
REFERENCES 1. Pearson CM, Paulus HE, Machleder HI: The role of the lymphocyte and its products in the propagation of joint disease. Ann N Y Acad Sci 256:lSO-157, 1975
2. McCord JM: Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed Proc 46:24022406, 1987 3. Blake DR, Merry P, Unsworth J, Kidd BL, Outhwaite JM, Ballard R, Moms CJ, Gray L , Lunec J: Hypoxic reperfusion injury in the inflamed human joint. Lancet I:289-292, 1989 4. Yu CL, Haskard 0, Cavender D, Johnson AR: Human gamma interferon increases the binding of T lymphocytes to endothelial cells. Clin Exp Immunol62:554-560, 1985 5. Folkman J, Haudenschild CC, Zetter BR: Long term culture of capillary endothelial cells. Proc Natl Acad Sci USA 76:5217-5221, 1979 6. Zetter BR: The endothelial cells of large and small blood vessels. Diabetes 30 (suppl 2):24-28, 1981 7. JaEe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 52:2745-2756, 1973 8. Jackson CJ, Garbett PK, Marks RM, Chapman G, Sonnabend DH, Potter SR, Brooks PM, Schreiber L: Isolation and propagation of endothelial cells derived from rheumatoid synovial microvasculature. Ann Rheum Dis 48:733-736, 1989 9. Diglio CA, Grammas P, Giscomelli F, Wiener J: Primary culture of rat microvascular endothelial cells, isolation, growth, and characterization. Lab Invest 46554-563, 1982 10. Marks RM, Czerniecki M, Penny R: Human microvascular endothelial cells: an improved method for tissue culture and a description of some singular properties in culture. In Vitro Cell Dev Biol 21:627435, 1985 11. Holthofer H, Virtamen I, Karmiemi A-L, Hormia M, Linder E, Miettinen A: Ulex europaeus-1 lectin as a marker for vascular endothelium in human tissues. Lab Invest 47:60-66, 1982 12. Voyta JC, Via DP, Butterfield CE, Zetter BR: Identification and isolation of endothelial cells based on their increased uptake of acetylated low density lipoprotein. J Cell BioI99:203&2040, 1984 13. Wellicome SM, Thornhill MH, Pitzalis C, Thomas DS, Laachbury JSS, Panayi GS, Haskard DO: A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide. J Immunol 144:2558-2565, 1990 14. Jackson CJ,Garbett B, Nissen B, Schrieber L: Binding of human endothelium to Ulex europaeus-1 coated Dynabeads: application to the isolation of microvascular endothelium. J Cell Sci 96:257-262, 1990 15. Lea T, Smeland E, Funderud S, Vartel F, Davies C, Beiske K, Ugelstad J: Characterization of human mononuclear cells after positive selection with immunomagnetic particles. Scand J Immunol 23509419, 1986
ISOLATION AND CULTURE OF SYNOVIAL MICROVASCULAR ENDOTHELIAL CELLS Characterization and Assessment of Adhesion Molecule Expression S. E. ABBOT, A. KAUL, C. R. STEVENS, and D. R. BLAKE
Objective. In vitro studies investigating the role of the synovial endothelium in the pathogenesis of rheumatoid arthritis (RA)have, until recently, been performed using cultured endothelial cells of nonsynovial macrovascular origin. In an attempt to more correctly model in vivo conditions, a method for the isolation and culture of synovial microvascular endothelial cells (SMEC) has been developed. Methods. SMEC were isolated, primarily, by the use of lectin-coated (Ulex europaeus agglutinin type I), magnetizable polystyrene beads. Results. Isolated cells exhibit classic endothelial “cobblestone” morphology, express von Willebrand factor, metabolize acetylated low-density lipoprotein, and exhibit a cytokine (interleukin-lkmediated expression of endothelial leukocyte adhesion molecule type 1 (ELAM-1) and intercellular adhesion molecule type 1 (ICAM-1). ELAM-1 levels were significantly elevated in SMEC, compared with human umbilical vein endothelial cells, over a range of interleukin-1 concentrations. Conclusion. This increased expression of From the Inflammation Group, Bone and Joint Research Unit, The London Hospital Medical College, London, United Kingdom. Supported by the Arthritis and Rheumatism Council of Great Britain. S. E. Abbot, MSc; A. Kaul, BDs; C. R. Stevens, PhD; D. R. Blake, MB, FRCP. Address reprint requests to S. E. Abbot, MSc, The London Hospital Medical College, Arthritis and Rheumatism Council Building, 25-29 Ashfield Street, London El 2AD. UK. Submitted for publication April 19, 1991; accepted in revised form December 26, 199 I. Arthritis and Rheumatism, Vol. 35, No. 4 (April 1992)
ELAM-1 by SMEC may be a potentiating step in the pathogenesis of RA. The etiology of rheumatoid arthritis (RA) remains unknown; however, recent studies have suggested a central role for the synovial microvascular endothelium in its pathogenesis. Persistent inflammation is characteristic of the rheumatoid joint. Inflammation is modulated by endothelial cell control of leukocyte chemotaxis, adhesion, and infiltration (1). The endothelium is known to locally regulate vascular tone and permeability via the antagonistic actions of endothelium-derived relaxing factor and endothelin. The eventual destruction of the rheumatoid joint is a consequence of synovial membrane proliferation and pannus invasion of the articular cartilage. These processes are associated with uncontrolled endothelial cell migration and proliferation (angiogenesis). More recently, the synovial endothelium has been directly implicated in contributing to the erosive process through its ability to generate proinflammatory reactive oxygen species (2,3). Investigations of these processes have, until recently, used endothelial cells isolated from the large blood vessels of various species (4). It has been recognized that there is heterogeneity between endothelial cells from different species, as well as from different tissues within a species (5,6). This would imply that modeling the synovial microvasculature by the in vitro use of endothelial cells of nonsynovial origin might be invalid. In an attempt to more correctly model endothelial responses in the synovium, we devised a method for isolating and culturing microvas-
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cular endothelial cells of synovial origin. Initial studies have indicated that there are differences in the expression of endothelial leukocyte adhesion molecule type 1 (ELAM-1) by synovial microvascular endothelial cells (SMEC) and human umbilical vein endothelial cells (HUVEC).
MATERIALS AND METHODS Cell isolation. HUVEC were isolated from fresh umbilical cords by the method of Jaffe et al(7). Isolated cells were cultured in MI99 in a manner similar to that used for SMEC, except that HUVEC were maintained initially with a lower concentration of growth factor (30 CLg/ml). Microvascular preparations were obtained from synovium excised from rheumatoid hip or knee joints obtained at the time of joint replacement surgery. The methods described here were modified from several micro- and macrovascular isolation procedures (5,7-10). Excised tissue was immediately placed in ice-cold Ca2+ and Mgzf-free Hanks’ balanced salt solution (HBSS; Sigma, Poole, Dorset, UK). The HBSS was exchanged 3 times to remove excess blood from the sample prior to dissecting the synovial membrane free of capsular material. At this stage, the microvascular nature of endothelial cells is confirmed according to vessel size. With the aid of a dissecting microscope, vessels with a lumen diameter >50 pm were removed from the dissected tissue and discarded. The remaining tissue was then minced into -3-mm3 pieces, using crossed scalpels. The minced tissue was incubated in HBSS containing 0.1% type I1 collagenase (Sigma) and 0.1% crude collagenase (Sigma) for 60 minutes, with continuous stirring. Following collagenase digestion, the minced preparation was compressed with the flat face of a microspatula. The compressive action of the spatula expresses capillary segments from the cut edges of the tissue. Waste tissue, capillary segments, and single connective cells were separated by sequential filtering through sieves of various pore sizes. All material was passed through a 250-pm steel mesh (Sigma), which trapped large segments of waste tissue. Capillary segments were trapped on a nylon screen (50-pm pore size; Cavendish, London, UK). Segments were then removed by backwashing the filter with HBSS. Samples were centrifuged and resuspended in culture medium M199 (Gibco, Paisley, Scotland) supplemented with 40% fetal calf serum (FCS; Gibco), 100 unitdm1 penicillin and 100 pdml streptomycin (Sigma), 100 pg/ml endothelial cell growth factor (ECGF; Sigma), 17 units/ml heparin (Sigma), 2 mM L-glutamine (Sigma), and 1.2% NaHCO, (Flow; Irvine, Scotland). The resulting isolated cell suspension contains a heterogeneous cell population, from which the endothelial cells were purified. Cell purification. Cell populations, extracted by collagenase digest, contain various proportions of endothelial cells, usually in the range of 1-10%. Further cell purification
steps require an accurate estimate of endothelial cell number. To facilitate this, the mixed cell population obtained from the collagenase digest was plated onto 35-mm Primaria (Falcon, Newcastle-Upon-Tyne, UK) dishes. Following 48 hours growth, endothelial cells were distinguished from other contaminating cells by morphologic features, i.e., those cells growing in discrete colonies of 10-50 cells exhibiting a “cobblestone-like” pattern. An initial enrichment of the endothelial cell proportions was achieved by manually removing contaminating nonendothelial cell colonies from the dish with a flame-drawn pasteur pipette, followed by aspiration of the culture medium. Once an endothelial cell :contaminating cell ratio of approximately 1 : 1 has been achieved, all cells were removed from the plate using a plastic cell scraper (Falcon). A single-cell suspension was then prepared by repeated transfer of the cells through a fire-polished siliconized pasteur pipette. For the purpose of further purification, the total cell number was assessed using a hemacytometer, the endothelial cell proportion being assumed to be 50% of the total. Further purification was achieved by a magnetic cellseparation technique. This employs lectin-coated magnetizable polystyrene beads (Dynabeads, Dynal; Dynatech, Alexandria, VA). Ulex europaeus agglutinin type I (UEA I) lectin binds specifically to many glycoproteins and glycolipids containing a-linked fucose residues and has been established as an excellent marker for human endothelial cells (1 1). Tosyl-activated Dynabeads were washed in distilled water. UEA I was covalently coupled to the beads by vortex suspension in 0.5M borate solution, pH 9.5, containing 10 &ml UEA I. Equal volumes of washed Dynabeads and UEA I solution were mixed for 24 hours at 22°C by slow rotation. The coated beads were collected using a magnelic particle concentrator (MPC). The beads were washed in 0.01M phosphate buffered saline, pH 7.4, containing 0.1% bovine serum albumin for 10 minutes, then for a further 30 minutes, followed by an overnight wash at 4°C. Dynabeads were added to the cell suspension at an approximate concentration of 3 per endothelial cell. Cells and beads were gently mixed at 4°C for 10 minutes, after which endothelial cells that were bound to the Dynabeads were isolated using the MCP. Unbound cells were removed by washing with HBSS. Cell-bead complexes were then resuspended in HBSS, and the procedure was repeated two more times. Isolated synovial microvascular endothelial cells were plated onto 0.1% gelatin-coated dishes. The cells were grown to confluence and passaged with a split ratio of 1 :3. Isolated SMEC were used for assay purposes at passage 3; however, we have found it possible to passage these cells as many as 7 times without altering the endothelial morphology. Characterization of SMEC. Isolated cells were characterized using 4 criteria, as follows. 1) Development of a classic “cobblestone” morphology of the cell monolayer at confluence (7). 2) Immunohistochemical localization of von Willebrand factor (vWF) in a granular cytoplasmic pattern. Localization of vWF was carried out on cells grown to confluence on 0.1% gelatin-coated Thermanox coverslips,
ISOLATION AND CULTURE OF SMEC
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using a Vectastain elite ABC, peroxidase, reagent kit (Vector, Burlingame, CA). Rabbit anti-human vWF (Dakopatts, Copenhagen, Denmark) primary antibody was used at 1 : 1,500 dilution. 3) Uptake of acetylated low-density lipoprotein (LDL). Endothelial cells possessing a “scavenger pathway” selectively ingest modified LDL (12). Confluent monolayers of HUVEC, SMEC, and mouse fibroblasts were incubated with 10 pg/ml of 1,l’-dioctadecyl-1-(3,3,3’,3’tetramethyl)-indocarboxyamine perchlorate-labeled acetylated LDL (Dil-Ac-LDL; Biogenesis, Bournemouth, UK) for 4 hours, after which the monolayers were examined for fluorescence at 480 nm. 4) Expression of ELAM-1 and intercellular adhesion molecule type 1 (ICAM- I ) , as determined by enzyme-linked immunosorbent assay (ELISA). ELISAs were carried out on 4 occasions, using SMEC and HUVEC grown to confluence in SMEC culture medium on 0. I% gelatin-coated 96-well plates. Confluent cell density was -9,000 cells/well; assays were performed using 3 wells per concentration of interleukin-I (IL-I). Briefly, culture medium from the plates was replaced either with fresh medium alone or with fresh medium containing recombinant IL-la (Roche, Welwyn Garden City, UK) at 2.5, 5 , 25, 50, 250, and 500 D10 unitslml. Plates were incubated at 37°C in an atmosphere of 5% CO, for 4-6 hours. Subsequently, cells were fixed and stained for ELAM-1 and ICAM-I using an ELISA as previously described (13).
RESULTS
B
C Figure 1. Immunohistochemical localization of von Willebrand factor as discrete cytoplasmic granules in A, human umbilical vein
endothelial cells and B, synovial microvascular endothelial cells, but not C, mouse fibroblasts. Bar = 25 pm.
Isolated SMEC grew to confluence on a variety of gelatin-coated substrates. Contamination by nonendothelial cells was not a major problem since the few contaminating cells could be removed manually in a manner similar to that detailed in the initial isolation. Confluent SMEC appeared as closely packed hexagonal cells which overlapped little with adjacent cells. The monolayer exhibited a strict contact-inhibited growth pattern which could be maintained for several weeks with little evident morphologic change. Immunohistochemical localization of vWF showed a discrete granular cytoplasmic staining pattern in SMEC, similar to that observed in HUVEC. No staining was observed on mouse fibroblasts (Figures 1A-C). Following incubation with Dil-Ac-LDL, both SMEC and HUVEC, but not the mouse fibroblasts, fluoresced brightly (Figures 2A-C). ELISAs carried out on passage-4 cultures demonstrated that SMEC express low levels of ELAM-1 constitutively. This expression rose in a dosedependent manner when cells were preincubated with various concentrations of IL-] ELAM-l expression was significantly higher (P< 0.05) than basal levels at
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B A
Figure 2. Fluorescence of ingested 1 ,l’-dioctadecyl-l-(3,3,3’,3’tetramethyl)-indocarboxyamine perchlorate-labeled acetylated lowdensity lipoprotein metabolized by A, human umbilical vein endothelial cells and B, synovial microvascular endothelial cells, but not C, mouse fibroblasts. Bar = 25 pm.
C all IL- 1 concentrations (Figure 3). Constitutive ICAM-1 expression was moderate on SMEC, and this basal expression was significantly increased ( P < 0.05) in response to IL-1 at concentrations of 25 units/ml and 250 units/ml. Comparison of the results of assays performed under the same conditions using HUVEC showed that in response to IL-I, SMEC expressed significantly higher levels of ELAM-I at all concentrations (P < 0.05). These differences may, however, reflect similar increases in both cell types, with SMEC having a higher basal level. There was little difference in the expression of ICAM-1 by the two cell types in response to 1L-1 (Figures 3A and B). Identical assays
performed on HUVEC, grown to confluence in either HUVEC or SMEC culture medium, showed that the FCS or ECGF levels in growth medium had no significant effect on either ICAM-I or ELAM-I expression.
DISCUSSION The in vitro use of isolated large-vessel endothelial cells, particularly HUVEC, to model microvascular-associated events is a technique that is used in many laboratories. Extrapolation of the results obtained using HUVEC to the conditions within a microvascular bed, in this case the synovium, has been called into question because of potential heterogeneity
ISOLATION AND CULTURE OF SMEC Optical Density
,,I
+
U.J
0.25 0.2
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0.15
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11-1 Concentration (units/ml)
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A Optical hnsity 0.25 0.2 0.15
T
T
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0
0
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0.05 0.05
5
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1 50
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IL-1 Concentration (unitdml)
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Figure 3. Expression of A, endothelial leukocyte adhesion molecule type 1 (ELAM-1) and B, intercellular adhesion molecule type I (ICAM-I) by synovial microvascular endothelial cells (SMEC) (solid bars) and by human umbilical vein endothelial cells (HUVEC) (open bars) in the presence of various concentrations of interleukin-1 (IL-I). There was a significant increase in basal expression of ELAM-1 by SMEC compared with HUVEC; this increase was maintained at all IL-1pretreatment levels. There were no significant differences in ICAM-1 expression by SMEC compared with HUVEC, either basally or in response to IL-1 pretreatment.
of the cells. To facilitate more appropriate modeling of the synovium in our studies of inflammation, a method for the isolation and culture of synovial microvascular endothelial cells was devised. The method described yields high numbers of cells which have been characterized, by several standard techniques, as being endothelial and of synovial microvascular origin. These cells retained all endothelial characteristics through 6 subcultures over the course of several weeks in culture. All of the characterization procedures, with the exception of ELAWICAM expression, are procedures that employ features documented as being retained by both micro- and macrovascular endothelial cells after isolation and culture. Thus, the similarity of
405
HUVEC to SMEC with respect to these criteria was anticipated. The main difference found in the characterization of the two cell types related to the expression of ELAM-1, which will be discussed below. Central to the method is a sorting and purification step utilizing an endothelial-specific lectin (UEA-1) covalently coupled to magnetizable polystyrene beads. Similar methods have previously been employed to successfully isolate a variety of cell types including SMEC (14). It has been suggested by Lea et a1 (15) that Dynabead adherence to cells prevents cell adhesion to tissue culture plastic, but this was disputed in the isolation of SMEC (14). We found that Lea and colleagues’ observations held true, since a significant inhibition of cell adhesion to plastic was observed when the bead-to-cell ratio exceeded 10: 1. It is presumed that the inhibition is due to attached beads physically preventing cell spreading. Jackson et al(l4) reportedly avoided this problem by removing attached beads from isolated cells by competitive binding of free O.1M fucose to the beads at 4°C. This technique, however, did not prove to be reproducible. We have shown that a successful alternative is to use fewer beads. To achieve this, we employed a manual weeding process, as previously described (3, which would give an accurate estimate of endothelial cell numbers prior to the addition of the beads. This allows the calculation of the correct number of beads to add in order to achieve a low bead-to-cell ratio. We found that a ratio of -3: 1 gave a good harvest of endothelial cells without interfering with adhesion to plastic and subsequent proliferation. The long-term culture of SMEC, at present, requires the use of high levels of ECGF (100 pg/ml) and FCS (40%). Apart from the high cost of these additions, the effects of such high levels on endothelial cell behavior are unknown. While the method is effective, it still requires a constant supply of fresh synovial tissue, since isolated cells have a finite usable life in culture. In the future, it may be possible to immortalize the cell line, thus overcoming this problem to some extent. The present study was, in part, undertaken to assess the suitability of extrapolating the cytokine responses of macrovascular cells, particularly HUVEC, to the microvasculature of the synovium. Similar trends were observed in ELAM-I and ICAM-1 expression on HUVEC and SMEC. While the basic morphologic markers of endothelial cells show similar patterns, our results suggest that SMEC may show an enhanced basal expression and response to IL-1, compared with HUVEC, particularly in their expression of ELAM-1. Synovial tissue sections immunohistochem-
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406 ically stained using antibodies to vWF and ELAM-I have shown, respectively, a patchy distribution and localization to postcapillary venules. This is paradoxical considering the almost universal staining of the isolated microvascular cells. This suggests that either vWF- and ELAM-l-expressing cells have been preferentially selected during isolation, which is possible with regard to ELAM-1 expression, or that culture conditions may either have favored the universal induction of vWF and ELAM-1 expression or have lost an inhibitor that is present in vivo. It is unclear at this time whether these differences represent differences between large- and small-vessel endothelia or a specific response of synovium as an inflamed tissue. Again, further work comparing SMEC isolated from normal tissue and other capillary endothelial cells will be needed to ascertain whether this increased ELAM-1 expression on cultured synovial cells represents a potentiating step in inflammatory joint disease. The cytokine dependence of ELAM-1 expression suggests that its expression in vivo by a minority of the vessels in rheumatoid synovial tissue sections may be cytokine mediated. There is good evidence that cytokines such as IL-1 are present in synovial exudates and are produced by cells within the synovial lining. These cytokines are responsible for neutrophil and lymphocyte binding to endothelial cells in vitro, and thus have a direct relevance to the pathogenesis of RA. Of particular interest is the comparative responses of SMEC and HUVEC. These suggest that SMEC show an enhanced response to IL-1, as measured by the induction of the neutrophil-specific adhesion molecule, ELAM-1. Preliminary studies have shown SMEC to be functionally capable of binding neutrophils; however, experimental variation has precluded our being able to assess the significance of the differences between HUVEC and SMEC. Using the methods detailed here, we have shown that synovial microvascular endothelial cells exhibit a higher ELAM-1 expression, compared with that of HUVEC in the same conditions, a difference that may potentiate the chronic inflammation in the rheumatoid joint.
ACKNOWLEDGMENTS We gratefully acknowledge the generous gift of antiELAM-1 and anti-ICAM-1 antibodies by Dr. Dorian Haskard. Thanks are also extended to Michael Freeman and Dr.Andrew Waterfield for providing the synovial specimens.
REFERENCES 1. Pearson CM, Paulus HE, Machleder HI: The role of the lymphocyte and its products in the propagation of joint disease. Ann N Y Acad Sci 256:lSO-157, 1975
2. McCord JM: Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed Proc 46:24022406, 1987 3. Blake DR, Merry P, Unsworth J, Kidd BL, Outhwaite JM, Ballard R, Moms CJ, Gray L , Lunec J: Hypoxic reperfusion injury in the inflamed human joint. Lancet I:289-292, 1989 4. Yu CL, Haskard 0, Cavender D, Johnson AR: Human gamma interferon increases the binding of T lymphocytes to endothelial cells. Clin Exp Immunol62:554-560, 1985 5. Folkman J, Haudenschild CC, Zetter BR: Long term culture of capillary endothelial cells. Proc Natl Acad Sci USA 76:5217-5221, 1979 6. Zetter BR: The endothelial cells of large and small blood vessels. Diabetes 30 (suppl 2):24-28, 1981 7. JaEe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 52:2745-2756, 1973 8. Jackson CJ, Garbett PK, Marks RM, Chapman G, Sonnabend DH, Potter SR, Brooks PM, Schreiber L: Isolation and propagation of endothelial cells derived from rheumatoid synovial microvasculature. Ann Rheum Dis 48:733-736, 1989 9. Diglio CA, Grammas P, Giscomelli F, Wiener J: Primary culture of rat microvascular endothelial cells, isolation, growth, and characterization. Lab Invest 46554-563, 1982 10. Marks RM, Czerniecki M, Penny R: Human microvascular endothelial cells: an improved method for tissue culture and a description of some singular properties in culture. In Vitro Cell Dev Biol 21:627435, 1985 11. Holthofer H, Virtamen I, Karmiemi A-L, Hormia M, Linder E, Miettinen A: Ulex europaeus-1 lectin as a marker for vascular endothelium in human tissues. Lab Invest 47:60-66, 1982 12. Voyta JC, Via DP, Butterfield CE, Zetter BR: Identification and isolation of endothelial cells based on their increased uptake of acetylated low density lipoprotein. J Cell BioI99:203&2040, 1984 13. Wellicome SM, Thornhill MH, Pitzalis C, Thomas DS, Laachbury JSS, Panayi GS, Haskard DO: A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide. J Immunol 144:2558-2565, 1990 14. Jackson CJ,Garbett B, Nissen B, Schrieber L: Binding of human endothelium to Ulex europaeus-1 coated Dynabeads: application to the isolation of microvascular endothelium. J Cell Sci 96:257-262, 1990 15. Lea T, Smeland E, Funderud S, Vartel F, Davies C, Beiske K, Ugelstad J: Characterization of human mononuclear cells after positive selection with immunomagnetic particles. Scand J Immunol 23509419, 1986
