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VARIABILITY IN E-SELECTIN EXPRESSION, mRNA LEVELS AND sE-SELECTIN RELEASE BETWEEN ENDOTHELIAL CELL LINES AND PRIMARY ENDOTHELIAL CELLS
Cell Biology International 2000, Vol. 24, No. 2, 91–99 doi:10.1006/cbir.1999.0455, available online at http://www.idealibrary.com on
VARIABILITY IN E-SELECTIN EXPRESSION, mRNA LEVELS AND sE-SELECTIN RELEASE BETWEEN ENDOTHELIAL CELL LINES AND PRIMARY ENDOTHELIAL CELLS H. F. GALLEY*, M. G. BLAYLOCK, A. M. DUBBELS and N. R. WEBSTER Academic Unit of Anaesthesia and Intensive Care, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, U.K. Received 11 June 1999; accepted 30 August 1999
Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor (TNF-) and interleukin-1 (IL-1), median (range) percentage of E-selectinpositive HUVECs increased from 1.6(0.9–6.2)% to 91.4(83.0–96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-/IL-1. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of 2000 Academic Press adhesion. K: endothelial cells; E-selectin; adhesion molecule; leucocyte.
INTRODUCTION E-selectin is an inducible endothelial cell surface glycoprotein of the selectin family of adhesion molecules (Bevilacqua et al., 1989). It mediates the binding of neutrophils, eosinophils, monocytes, T lymphocytes and tumour cells to endothelium. E-selectin can be induced by tumour necrosis factor (TNF-), interleukin-1 (IL-1), lipopolysaccharide (LPS), interleukin-3 (IL-3), and interferon- (IFN-) (Pober et al., 1986). Although human umbilical vein endothelial cells (HUVECs) are widely used as a model of normal endothelium, the isolation procedure is cumbersome and the success rate can be low. In addition, cell activity and phenotype may vary between donor cords and at differing passages (Heurkens *To whom correspondence [email protected] 1065–6995/00/020091+09 $35.00/0
should
be
addressed.
E-mail:
et al., 1991) and primary cell cultures may require exogenous growth factors. Transformed cell lines, in contrast, can provide non-varying endothelial cells with a high proliferative potential, stable antigenicity and no growth factor requirement. Such cell lines express common endothelial cell markers such as von Willebrand factor (Edgell et al., 1983; Takahashi and Sawasaki, 1992) and are thus often assumed to exhibit other physiological responses identical to endothelial cells in vivo. Although some cell lines are anecdotally preferred over others for certain studies, there is little evidence in the literature relating specifically to functional E-selectin expression in endothelial cell lines. We investigated various measures of E-selectin expression and neutrophil adherence in three potential sources of endothelial cells as models of human endothelium: primary endothelial cells from umbilical cords, the spontaneously transformed 2000 Academic Press
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ECV304 endothelial cell line and the hybrid line EA.hy926. MATERIALS AND METHODS Cell lines The cell line ECV304 was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, U.K.). The cell line EA.hy926 was obtained as a gift from Zeneca Pharmaceuticals (Cheshire, U.K.), with permission from Dr Cora-Jean Edgell of the University of South Carolina at Chapel Hill. EA.hy926 cells were maintained in DMEM-glutamax-1 (Life Technologies, Paisley, U.K.), while ECV304 cells were maintained in M199-glutamax-1 (Life Technologies), both media were supplemented with 10% fetal calf serum (Seralab, Crawley, Sussex, U.K.) and 100 U/ml penicillin/100 g/ml streptomycin (Life Technologies). Cell lines were maintained at 37C in a humidified atmosphere of 5% CO2/95% air in 25 cm2 or 75 cm2 filter top tissue culture flasks. Primary endothelial cells Endothelial cells (HUVECs) were isolated from human umbilical cords. A minimum 20 cm length of cord was placed in sterile ice-cold buffer (HBSS without phenol red, containing 4.6 g/l HEPES and 100 U/ml penicillin) and stored at 4C for up to 3 days. Isolation of the cells was carried out in a Class II laminar flow hood. Cords were removed from buffer and sprayed with 70% v/v ethanol. Damaged areas of the cords were discarded and blood clots were removed by gentle squeezing. The vein was then cannulated and flushed with phosphate buffered saline (PBS, Dulbecco’s A, pH 7.4; Sigma Chemical Co Ltd, Poole, Dorset, U.K.) to remove all traces of blood. The vein was refilled with warm 20 ml M199-glutamax-1 (Life Technologies) containing 0.5 g/ml collagenase (type H; Boehringer-Mannheim, Lewes, East Sussex, U.K.) and incubated for 13 min at 37C. The cord was massaged, washed twice with PBS, the washes pooled and centrifuged at 200g for 5 min. The cell pellet was resuspended in 10 ml 20% M199glutamax-1 medium containing 20% FCS, 20 g/ml endothelial cell growth factor (Sigma), 5 U/ml heparin (Leo Laboratories Ltd, Princes Risborough, Kent, U.K.), 100 U/ml penicillin/100 g/ml streptomycin and seeded onto 25 cm2 flasks which had been previously coated with 0.1% w/v gelatin (type B from bovine skin; Sigma). After incubation
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at 37C in a humidified atmosphere of 5% CO2/95% air for 24 h, cells were washed in PBS and the medium was then replaced every 2–3 days. Expression of the endothelial cell antigen, von Willebrand factor, demonstrated by immunofluorescence, was also used to confirm the presence of a pure endothelial cell population. Flow cytometry Both cell lines and HUVECs were maintained in appropriate media containing supplements as described, except that HUVEC medium contained only 10% FCS to ensure standardized experimental conditions. Confluent cells were seeded onto sixwell tissue culture plates at 1106/well in a volume of 3 ml and incubated overnight at 37C. Cells were then exposed to 100 U/ml tumour necrosis factor (TNF-, human recombinant; R&D Systems Europe, Cowley, Oxon, U.K.) and 25 U/ml interleukin-1 (IL-1, human recombinant; R&D Systems) for up to 24 h. For flow cytometry cells were suspended in PBS containing 0.1% bovine serum albumin (BSA) and stained with 10 g/ml of either mouse anti-E-selectin (R&D Systems), mouse anti-ICAM-1 (R&D Systems) or mouse isotype control antibody (Sigma) for 30 min at 4C. Cells were then washed twice in PBS/BSA at 500g for 5 min and incubated with fluoresceinlabelled goat anti-mouse antibody for 30 min at 4C in the dark. Cells were analysed using a FACScan flow cytometer with a 488 nm argon laser (Becton Dickinson, Cowley, Oxon, U.K.), with filter settings for fluorescein isothiocyanate at 530 nm. Ten thousand cells were analysed from each tube and analysis gates were set according to both forward and side scatter profiles. Immunofluorescence Cell were grown to confluence on gelatin-coated chamber slides and incubated with 100 U/ml TNF- and 25 U/ml IL-1 for 6 h, prior to fixing in 0.03 citrate buffer pH 4.5/60% (v/v) acetone for 5 min at room temperature. Cells were then incubated with mouse anti-E-selectin (R&D Systems) or mouse isotype control antibody (Sigma) for 30 min at room temperature, washed in PBS-BSA and stained with fluorescein-conjugated goat anti-mouse antibody for a further 30 min at room temperature, protected from light. For von Willebrand factor, unstimulated fixed cells were incubated with rabbit-anti-Von Willebrand factor antibody for 30 min at room temperature prior to staining with rhodamine-conjugated anti-rabbit
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antibody for a further 30 min at room temperature, protected from light. Stained cells were mounted in Vectashield mountant (Vector Laboratories, Peterborough, U.K.) and viewed under a fluorescence microscope.
Non-adherent cells were gently washed off and discarded and adherent cells were lysed with 500 l 10% Triton X-100 (Sigma) prior to counting. Results were expressed as counts/min in the adherent cell population.
mRNA
Statistical analysis
Total RNA was isolated using guanidinium thiocyanate and phenol extraction (Totally RNA kit, Ambion Inc., Austin, TX, U.S.A.). Quantity and quality of RNA was determined by differential UV absorbance. The ratio of absorbance at 260 nm:280 nm was typically between 1.7–2.0 for pure RNA samples. Total RNA (15 g/lane) was electrophoresed for 4 h at 60 V under denaturing conditions on a 1% agarose gel containing formaldehyde, and blotted onto hybond N + hybridization membrane (Amersham International) overnight. RNA was then bound to the membrane by UV crosslinking and hybridized at 42C overnight to a digoxigenin-labelled E-selectin, IL-8 or -actin probe (R&D Systems). Following stringency washes, chemiluminescent detection was performed using CDP Star (Boehringer Mannheim) according to manufacturer’s instructions. mRNA was visualized by autoradiography and quantified by phosphorimaging.
Data are expressed as median (range) and represent results from a minimum of six separate experiments. Differences were assessed using Kruskal Wallis analysis of variance or Mann–Whitney U test as appropriate. A P value of c0.05 was taken as statistically significant.
Soluble E-selectin Confluent cells were seeded onto 24-well tissue culture plates at 0.4106/well in a volume of 0.5 ml and incubated overnight at 37C. To each well was then added 100 U/ml TNF- and 25 U/ml IL-1, and cells were incubated for a further 24 h. Soluble E-selectin was measured in culture medium using an enzyme immunoassay kit (R&D Systems). Neutrophil adherence HUVECs, EAhy.926 and ECV 304 were seeded into gelatin-coated six-well plates to a density of 5104 cells/cm2 and incubated overnight, prior to activation with TNF- (100 U/ml) and IL-1 (25 U/ml) for 6 h. Human polymorphonuclear leucocytes (PMN) were isolated from healthy donors using a single density gradient procedure as previously described (Goode et al., 1994). Isolated PMN (1107) were incubated with 250 Ci 51Cr (Amersham) for 90 min at 37C. Following incubation, cells were washed and resuspended at 3106 cells/ml and 500 l of the labelled PMN were added to washed and dried endothelial monolayers and incubated for 60 min at 37C.
RESULTS Flow cytometry E-selectin expression increased significantly in all endothelial cells after exposure to TNF-/IL-1 for 24 h (P=0.0001) but expression was approximately 100-fold higher in HUVEC’s by 3 h (Fig. 1). The median percentage of E-selectin -positive HUVECs increased from 1.6(0.9–6.2)% to 91.4(83.0–96.1)% following 3 h incubation with TNF-/IL-1 (P=0.001) and was still elevated at 24 h (57.3(40.9– 73.5)%, P=0.001, Fig. 1). The levels of TNF-/ IL-1 -induced ICAM-1 expression was similar in all three cell types, but basal expression in the absence of cytokine stimulation was significantly higher in ECV304 cells than either HUVECs (P=0.004) or EA.hy926 cells (P=0.004) and the kinetics of the expression was markedly different (Fig. 1B). Immunofluorescence Fluorescence microscopy revealed that staining for von Willebrand factor was present in all cell types but was less intense in the cell lines than in primary endothelial cells (Fig. 2A). None of the endothelial cells showed basal E-selectin expression in the absence of activation (not shown). However, after 6 h exposure of HUVECs to TNF-/IL-1, cell surface expression of E-selectin was seen as intense green staining (Fig. 2B). Minimal staining could be seen in the cell lines (Fig. 2B). mRNA Northern blotting showed that mRNA for E-selectin was not detectable in ECV304 and EA.hy926 cell lines, even after exposure to TNF-/ IL-1 for up to 8 h (Fig. 3). However in HUVECs,
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Fig. 1. The effect of exposure of human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for up to 24 h, on E-selectin (A) and intercellular adhesion molecule-1 (ICAM-1) (B) expression by flow cytometry. Bar and whisker graphs show median, 25th and 75th percentiles with ranges as vertical lines. Data are results obtained from six separate experiments. P value refers to Kruskal Wallis analysis of variance. *Significantly higher than in HUVECs and EA.hy926 cells (P=0.05), Mann–Whitney U test.
mRNA for E-selectin increased after 2 h, and was maximal after 6 h exposure to TNF-/IL-1 (Fig. 3). The ‘housekeeping’ mRNA probe for actin showed equal RNA loading (Fig. 3). All cell types showed TNF-/IL-1-mediated IL-8 mRNA expression, but maximal expression was later for ECV304 cells than both HUVECs and EA.hy926 cells (Fig. 3).
both primary endothelial cells and cell lines, following exposure to TNF-/IL-1 for 6 h, compared to adherence without TNF-/IL-1 (Fig. 5). However, TNF-/IL-1 stimulated adherence of PMN was markedly higher in HUVECs compared to ECV304 (P=0.004) and EA.hy926 (P=0.004) cell lines (Fig. 5).
sE-selectin
DISCUSSION
Basal unstimulated 24 h soluble E-selectin accumulation was significantly higher from HUVECs compared to both of the other cell lines. In medium from HUVECs, median (range) sE-selectin concentration was 131.7(95.7–155.7) pg/ml compared to 47.7(17.1–95.7) pg/ml from EA.hy926 cells (P=0.05) and 17.7(6.1–83.7) pg/ml from ECV304 cells (P=0.01, Fig. 4). When cells were exposed to TNF-/IL-1, sE-selectin accumulation from HUVECs increased significantly to 347(281.8– 395.8) pg/ml, (P=0.004, Fig. 4), but failed to increase in the cell lines (Fig. 4). Adherence The number of adherent PMN, as represented by the counts/min of 51Cr, increased significantly in
We have shown that E-selectin expression is negligible by flow cytometry, immunofluorescence, mRNA analysis or as soluble E-selectin in the human vascular endothelial cell lines ECV304 and EA.hy926, and is considerably less than in primary cell cultures. In addition, the expression of von Willebrand factor, ICAM-1 and IL-8 by these cell lines is different to primary vascular endothelial cells from human umbilical cords. E-selectin is an endothelial cell-specific 110 KDa glycoprotein receptor for sialyl Lewisx and sialyl Lewisa carbohydrate ligands on circulating neutrophils, eosinophils and monocytes and tumour cells. It also targets memory T lymphocytes to sites of cutaneous inflammation. E-selectin is expressed on the surface of endothelial cells in response to
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Fig. 2. (A) Photomicrograph of von Willebrand expression in (i) primary human umbilical cord endothelial cells (ii) ECV304 cells and (iii) EA.hy926 cells. (B) Photomicrograph of E-selectin expression on human umbilical cord endothelial cells following exposure to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for 24 h in (i) primary human umbilical cord endothelial cells (ii) ECV304 cells and (iii) EA.hy926 cells.
Fig. 3. Representative northern blot of mRNA for E-selectin, interleukin-8 (IL-8) and actin in human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, following 0–8 h exposure to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1). Numbers are arbitrary phosphorimager counts/mm2.
inflammatory stimuli and video microscopy has shown the importance of E-selectin in the support of neutrophil rolling under conditions of flow (Abbassi et al., 1993). Adhesion is an important part of the host response to infection and inflammation, enabling the margination of circulating cells. E-selectin also has a role in the adherence of monocytes and the formation of atherosclerotic
plaques. Induction of E-selectin expression is stimulated by endotoxin (LPS), TNF-, IL-1, IL-4, oxidized low-density lipoproteins, hyperthermia, chemotactic peptides, phorbol ester and lipid mediators (reviewed by Tonnesen, 1989). N-glycosylation of the E-selectin molecule is obligatory for endothelial cell surface expression (Pahlsson et al., 1995) and a lower molecular
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400 8 6 4 2 ** 0 – + – + – + TNF-α and IL-1β TNF-α and IL-1β TNF-α and IL-1β HUVECs ECV304 EA.hy926
0 – – + – + + TNF-α and IL-1β TNF-α and IL-1β TNF-α and IL-1β HUVECs ECV304 EA.hy926
Fig. 4. The effect of exposure of human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for 24 h, on soluble E-selectin accumulation in culture medium. Bar and whisker graphs show median, 25th and 75th percentiles with ranges as vertical lines. Data are results obtained from six separate experiments. P value refers to Mann–Whitney U test in HUVECs in the absence and presence of TNF-/IL-1. *Significantly lower than in HUVECs (P=0.05) Mann– Whitney U test; **significantly lower than in HUVECs (P=0.01) Mann–Whitney U test.
Fig. 5. The effect of exposure of human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for 24 h, on adherence of human polymorphonuclear leucocytes to endothelial monolayers. Bar and whisker graphs show median, 25th and 75th percentiles with ranges as vertical lines. Data are results obtained from six separate experiments. P value refers to Mann–Whitney U test in the absence and presence of TNF-/IL-1. *Significantly lower than in HUVECs (P=0.004) Mann–Whitney U test.
weight form of the molecule, termed soluble E-selectin (sE-selectin), which is thought to be a biologically active shed form of E-selectin (Newman et al., 1993) is released from the cell surface. Elevated circulating sE-selectin concentrations have been linked to several inflammatory conditions and atherosclerosis (Kaneko et al., 1996; Smith and Wayne, 1997). ECV304 is a spontaneously transformed cell line which originated from the umbilical cord of a Japanese infant. Expression of the endotheliumspecific marker, von Willebrand factor, has been shown previously (Takahashi and Sawasaki, 1992), which we have confirmed in the present study, although we found that expression was less than that in primary endothelial cells. Factor VIII expression was negative, however (Takahashi and Sawasaki, 1992), and hyaluronic acid metabolism was elevated compared to primary cell cultures (Amanuma and Mitsui, 1991). ECV304 cells also express colony stimulating factor receptor chains, in contrast to HUVECs (Colotta et al., 1993). Quantitative analysis of cell surface molecules in a comprehensive study of HUVECs and two cell lines (Mutin et al., 1997) revealed that ECV304 express tissue factor antigen (CD142) but not
platelet-endothelial cell adhesion molecule (PECAM,CD31) or vascular endothelial cadherin (VE cadherin, CD144), in addition to no detectable expression of E selectin under resting conditions. However, stimulated antigen expression in these cells was not studied. EA.hy926 is a hybrid cell line resulting from fusion of primary human umbilical vein endothelial cells with cells selected from the continuous human cell line A549, originally derived from a human lung carcinoma (Edgell et al., 1990). We found positive staining for von Willebrand factor in this cell line, as previously described (Edgell et al., 1983; van Oost et al., 1986; Wilbourn et al., 1992), but again staining was less than in primary cells. The cell line has been shown to be positive for factor VIII (Edgell et al., 1983), endothelin-converting enzyme (Walkden and Turner, 1995; Ahn et al., 1995), PECAM and VE cadherin (Mutin et al., 1997) but not E-selectin, under resting conditions. We found that E-selectin expression as shown by flow cytometry, immunofluorescence, mRNA levels and detection of sE-selectin, was upregulated in primary umbilical vein endothelial cells in response to TNF- and IL-1 after 2 h and was maximal at 6–8 h, declining by 24 h, as previously
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described. However, E-selectin cell surface expression was much lower under these conditions using the endothelial cell lines ECV304 and EA.hy926. Analysis of cell surface expression of E-selectin in HUVECs in response to TNF- and IL-1 has previously been shown to be maximal at 4–8 h (Sepp et al., 1994; Mutin et al., 1997). Northern analysis of mRNA for E-selectin in HUVECs in previous studies was maximal at 3–6 h (Kaneko et al., 1996; Moers et al., 1997) in accordance with the present study. Increased sE-selectin accumulation from HUVECs after 4–24 h exposure to LPS or cytokines have also been previously described (Sepp et al., 1994; Kaneko et al., 1996; Moers et al., 1997). Despite the relevance to the use of cell lines in studies of regulation of antigen expression in pathophysiological conditions, particularly regulation of the response to inflammation or infection, where the use of activated cells is required, there is little previous work on stimulated E selectin expression by the commonly used cell lines, EA.hy926 and ECV304. Thornhill et al. (1993) reported similar upregulation of cell surface E selectin expression and adhesion in EA.hy926 cells to that found in HUVECs. We also showed that E selectin expression by flow cytometry was upregulated in both EA.hy926 and ECV304 cells, but the magnitude of the increase was much less than in primary endothelial cells. However, closer examination of Thornhill’s data reveals some differences between primary endothelial cells and EA.hy926 cells. E selectin expression, measured using an ELISA technique on paraformaldehyde-fixed cell monolayers, rather than by flow cytometry as in the present study, was noticeably lower in EA.hy926 cells than HUVEC’s at both 6 and 20 h after stimulation with TNF (Thornhill et al., 1993), but, these authors did not perform statistical comparisons between the cell types. Both VCAM-1 and ICAM-1 expression in EA.hy926 were similar to that in HUVECs in response to TNF (Thornhill et al., 1993) but EA.hy926 cells did not respond to IL-4 or IFN- in terms of upregulation of ICAM-1 or VCAM-1, unlike HUVECs (Thornhill and Haskard, 1990; Thornhill et al., 1990). There have been no previous studies describing soluble E selectin, mRNA for E selectin or histochemical studies of endothelial antigens in cell lines compared to HUVECs. The mechanism of the reduced E-selectin expression in the cell lines ECV304 and EH.hy926 is unclear. We have shown that both cell lines constitutively express ICAM-1, confirming previous work on ICAM-1 (Burke-Gaffney and Hellewell 1996a,b; Wheller and Perretti, 1997) and ICAM-2
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(Nortamo et al., 1991). However, basal unstimulated expression was higher in ECV304 cells than either HUVECs or EA.hy926, in the present study, although the level of the response to cytokine stimulation with upregulation of ICAM-1 was similar, despite different kinetics. The cell lines also exhibit other inflammatory responses upon cytokine stimulation. Both we and others have shown that EA.hy926 cells upregulate IL-8 release and produce nitric oxide in response to TNF- and IL-1 (Villarette and Remick, 1995; Galley et al., 1997) and express inducible cyclo-oxygenase in response to IL-1 (Ristimaki et al., 1994). The present study also demonstrates that IL-8 mRNA from EA.hy926 and EVC304 cells, as well as HUVECs, is increased on exposure to TNF-/IL1, but that the time of maximal expression is later for ECV304 cells compared to the other endothelial cell types. Therefore the lack of E-selectin expression appears to be selective and does not extend to other pro-inflammatory functions. Adherence of polymorphonuclear leucocytes to the endothelial cell lines increased when the endothelial cells were activated with TNF- and IL-1, but the number of adherent PMN was much lower than the adherence to activated primary endothelial cells. In addition we have found that the level of adherence of PMN to activated HUVECs can be reduced to that of the cell lines using an anti-E-selectin blocking antibody (data not shown). These results suggest that lack of E-selectin expression has profound effects on the adherence of PMN, despite normal ICAM-1 expression and IL-8 production, indicating the functional significance of the reduced E-selectin expression. Adhesion of neutrophils and T cells after 4 h and 18 h stimulation with TNF respectively was similar in both the EA.hy926 cell line and HUVECs (Thornhill et al., 1993), but adhesion of T cells to EA.hy926 cells was not increased in response to IL-4 or IFN- as shown in HUVEC’s (Thornhill and Haskard, 1990; Thornhill et al., 1990). Cartright et al. (1995) have shown that the series of transformed endothelial cell lines, termed SGHEC-1 to SGHEC-13, express cell surface E-selectin in response to TNF- or IL-1 as measured by flow cytometry, but do not release immunoreactive sE-selectin. In the present study cell surface and soluble E-selectin was barely detectable in ECV304 or EA.hy296 cells, and was at least 100-fold lower than HUVECs. Another spontaneously transformed endothelial cell line, C11STH, which expresses von Willebrand factor and ICAM-1, is able to upregulate cell surface
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E-selectin expression in response to TNF-, but soluble E-selectin was not measured (Cockerill et al., 1994). Although the endothelial cell-specific phenotypic marker, von Willebrand factor, does not alter with the passage number of primary umbilical cord endothelial cells, the constitutive and LPS- or cytokine-induced expression of ICAM-1 decreased by about 65% and 40% respectively after six passages (Klein et al., 1995). After passage 3, the production of E-selectin in response to LPS or TNF-/IL-1 stimulation was shown to be negligible (Klein et al., 1995). The HUVECs used in the present study were all used before passage 3 and were all cultured using the same media, serum concentration etc., since these may affect cell function (Mutin et al., 1996). Endothelial cells from different cord donors may also have varying responses (Heukens et al., 1991) and it has been reported that detachment conditions, which were identical in the present study for all cells, may also affect cell viability and responses (Beekhuizen, 1994; Mutin et al., 1996). This inconsistency in cells has led to the expanding use of transformed cell lines, which offer a continuous supply of phenotypically constant endothelial cells with a high proliferative potential, stable antigenicity and no growth factor requirement (Balconi and Dejana, 1986). However, we have shown that the cell lines ECV304 and EA.hy926 have limited ability to express E-selectin. Other studies have also shown decreased E-selectin expression in the EA.hy926 cell line and an inability to respond to IL-4 and IFN- stimulation, whilst ECV304 cells do not express PECAM or VE Cadherin. These cell lines should therefore be used with caution for studies of adhesion, regardless of their ability to express other adhesion molecules. REFERENCES A O, K TK, MI LV, A DC, S CW, 1993. E-selectin supports neutrophil rolling in vitro under conditions of flow. J Clin Invest 92: 2719–2730. A K, P S, B K, H D, 1995. A permanent human cell line (EA.hy926) reserves the characteristics of endothelin converting enzyme from primary human umbilical vein endothelial cells. Life Sci 56: 2331–2341. A K, M Y, 1991. Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells. Biochim Biophys Acta 1092: 336–340. B G, D E, 1986. Cultivation of endothelial cells: limitations and perspectives. Med Biol 64: 231–245.
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cells and is elevated in the serum of patients with septic shock. J Immunol 150: 644–654. N P, S R, T T, P M, G CG, 1991. A monoclonal antibody to the human leukocyte adhesion molecule intercellular adhesion molecule-2. Cellular distribution and molecular characterisation of the antigen. J Immunol 146: 2530–2535. P P, S J, S U, L A, 1995. Role of N-linked glycosylation in expression of E-selectin on human endothelial cells. Eur J Immunol 25: 2452– 2459. P JS, B MP, M DL, L LA, F W, G MA J, 1986. Two distinct monokines, interleukin-1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J Immunol 136: 1680–1687. R A, G S, W J, M T, H T, 1994. Induction of cyclo-oxygenase-2 by interleukin-1 alpha. Evidence for post transcriptional regulation. J Biol Chem 269: 11,769–11,775. S NT, G J, L LJ, C W, L TJ, S RA, 1994. A factor in human plasma permits persistent expression of E-selectin by human endothelial cells. J Invest Dermatol 102: 445–450. S C, W MD, 1997. Potential significance of circulating E-selectin. Circulation 94: 1986–1988. T K, S Y, 1992. Rare spontaneously transformed human endothelial cell line provides useful research tool. In Vitro Cell Devel Biol 28A: 380–382.
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T MH, H DO, 1990. IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor and IFN-gamma. J Immunol 145: 865–872. T MH, K-A U, H DO, 1990. IL-4 increases human endothelial cell adhesiveness for T cells but not for neutrophils. J Immunol 144: 3060–3065. T MH, L J, H DO, 1993. Leucocyte endothelial cell adhesion: a study comparing human umbilical vein endothelial cells and the endothelial cell line EA-hy-926. Scand J Immunol 38: 279–286. T MG, 1989. Neutrophil-endothelial cell interactions: mechanisms of neutrophil adherence to vascular endothelium. J Immunol 142: 2257–2263. O BA, E CJ, H CW, M RT, 1986. Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926. Biochem Cell Biol 64: 699–705. V LH, R DG, 1995. Nitric oxide regulation of IL-8 expression in human endothelial cells. Biochem Biophys Res Commun 211: 671–676. W BJ, T AJ, 1995. Expression of ECE and related membrane peptidases in the EA.hy926 cell line. J Cardiovasc Pharmacol 26: S59–S60. W SK, P M, 1997. Dexamethasone inhibits cytokine induced intercellular adhesion molcule-1 upregulation on endothelial cell lines. Eur J Pharmacol 331: 65–71. W B, H P, L A, L F, R M, B D, I A, S G, 1992. Unique expression of von Willebrand factor by type IIA von Willebrand’s disease endothelial cells. Br J Haematol 81: 401–406.
VARIABILITY IN E-SELECTIN EXPRESSION, mRNA LEVELS AND sE-SELECTIN RELEASE BETWEEN ENDOTHELIAL CELL LINES AND PRIMARY ENDOTHELIAL CELLS H. F. GALLEY*, M. G. BLAYLOCK, A. M. DUBBELS and N. R. WEBSTER Academic Unit of Anaesthesia and Intensive Care, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, U.K. Received 11 June 1999; accepted 30 August 1999
Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor (TNF-) and interleukin-1 (IL-1), median (range) percentage of E-selectinpositive HUVECs increased from 1.6(0.9–6.2)% to 91.4(83.0–96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-/IL-1. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of 2000 Academic Press adhesion. K: endothelial cells; E-selectin; adhesion molecule; leucocyte.
INTRODUCTION E-selectin is an inducible endothelial cell surface glycoprotein of the selectin family of adhesion molecules (Bevilacqua et al., 1989). It mediates the binding of neutrophils, eosinophils, monocytes, T lymphocytes and tumour cells to endothelium. E-selectin can be induced by tumour necrosis factor (TNF-), interleukin-1 (IL-1), lipopolysaccharide (LPS), interleukin-3 (IL-3), and interferon- (IFN-) (Pober et al., 1986). Although human umbilical vein endothelial cells (HUVECs) are widely used as a model of normal endothelium, the isolation procedure is cumbersome and the success rate can be low. In addition, cell activity and phenotype may vary between donor cords and at differing passages (Heurkens *To whom correspondence [email protected] 1065–6995/00/020091+09 $35.00/0
should
be
addressed.
E-mail:
et al., 1991) and primary cell cultures may require exogenous growth factors. Transformed cell lines, in contrast, can provide non-varying endothelial cells with a high proliferative potential, stable antigenicity and no growth factor requirement. Such cell lines express common endothelial cell markers such as von Willebrand factor (Edgell et al., 1983; Takahashi and Sawasaki, 1992) and are thus often assumed to exhibit other physiological responses identical to endothelial cells in vivo. Although some cell lines are anecdotally preferred over others for certain studies, there is little evidence in the literature relating specifically to functional E-selectin expression in endothelial cell lines. We investigated various measures of E-selectin expression and neutrophil adherence in three potential sources of endothelial cells as models of human endothelium: primary endothelial cells from umbilical cords, the spontaneously transformed 2000 Academic Press
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ECV304 endothelial cell line and the hybrid line EA.hy926. MATERIALS AND METHODS Cell lines The cell line ECV304 was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, U.K.). The cell line EA.hy926 was obtained as a gift from Zeneca Pharmaceuticals (Cheshire, U.K.), with permission from Dr Cora-Jean Edgell of the University of South Carolina at Chapel Hill. EA.hy926 cells were maintained in DMEM-glutamax-1 (Life Technologies, Paisley, U.K.), while ECV304 cells were maintained in M199-glutamax-1 (Life Technologies), both media were supplemented with 10% fetal calf serum (Seralab, Crawley, Sussex, U.K.) and 100 U/ml penicillin/100 g/ml streptomycin (Life Technologies). Cell lines were maintained at 37C in a humidified atmosphere of 5% CO2/95% air in 25 cm2 or 75 cm2 filter top tissue culture flasks. Primary endothelial cells Endothelial cells (HUVECs) were isolated from human umbilical cords. A minimum 20 cm length of cord was placed in sterile ice-cold buffer (HBSS without phenol red, containing 4.6 g/l HEPES and 100 U/ml penicillin) and stored at 4C for up to 3 days. Isolation of the cells was carried out in a Class II laminar flow hood. Cords were removed from buffer and sprayed with 70% v/v ethanol. Damaged areas of the cords were discarded and blood clots were removed by gentle squeezing. The vein was then cannulated and flushed with phosphate buffered saline (PBS, Dulbecco’s A, pH 7.4; Sigma Chemical Co Ltd, Poole, Dorset, U.K.) to remove all traces of blood. The vein was refilled with warm 20 ml M199-glutamax-1 (Life Technologies) containing 0.5 g/ml collagenase (type H; Boehringer-Mannheim, Lewes, East Sussex, U.K.) and incubated for 13 min at 37C. The cord was massaged, washed twice with PBS, the washes pooled and centrifuged at 200g for 5 min. The cell pellet was resuspended in 10 ml 20% M199glutamax-1 medium containing 20% FCS, 20 g/ml endothelial cell growth factor (Sigma), 5 U/ml heparin (Leo Laboratories Ltd, Princes Risborough, Kent, U.K.), 100 U/ml penicillin/100 g/ml streptomycin and seeded onto 25 cm2 flasks which had been previously coated with 0.1% w/v gelatin (type B from bovine skin; Sigma). After incubation
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at 37C in a humidified atmosphere of 5% CO2/95% air for 24 h, cells were washed in PBS and the medium was then replaced every 2–3 days. Expression of the endothelial cell antigen, von Willebrand factor, demonstrated by immunofluorescence, was also used to confirm the presence of a pure endothelial cell population. Flow cytometry Both cell lines and HUVECs were maintained in appropriate media containing supplements as described, except that HUVEC medium contained only 10% FCS to ensure standardized experimental conditions. Confluent cells were seeded onto sixwell tissue culture plates at 1106/well in a volume of 3 ml and incubated overnight at 37C. Cells were then exposed to 100 U/ml tumour necrosis factor (TNF-, human recombinant; R&D Systems Europe, Cowley, Oxon, U.K.) and 25 U/ml interleukin-1 (IL-1, human recombinant; R&D Systems) for up to 24 h. For flow cytometry cells were suspended in PBS containing 0.1% bovine serum albumin (BSA) and stained with 10 g/ml of either mouse anti-E-selectin (R&D Systems), mouse anti-ICAM-1 (R&D Systems) or mouse isotype control antibody (Sigma) for 30 min at 4C. Cells were then washed twice in PBS/BSA at 500g for 5 min and incubated with fluoresceinlabelled goat anti-mouse antibody for 30 min at 4C in the dark. Cells were analysed using a FACScan flow cytometer with a 488 nm argon laser (Becton Dickinson, Cowley, Oxon, U.K.), with filter settings for fluorescein isothiocyanate at 530 nm. Ten thousand cells were analysed from each tube and analysis gates were set according to both forward and side scatter profiles. Immunofluorescence Cell were grown to confluence on gelatin-coated chamber slides and incubated with 100 U/ml TNF- and 25 U/ml IL-1 for 6 h, prior to fixing in 0.03 citrate buffer pH 4.5/60% (v/v) acetone for 5 min at room temperature. Cells were then incubated with mouse anti-E-selectin (R&D Systems) or mouse isotype control antibody (Sigma) for 30 min at room temperature, washed in PBS-BSA and stained with fluorescein-conjugated goat anti-mouse antibody for a further 30 min at room temperature, protected from light. For von Willebrand factor, unstimulated fixed cells were incubated with rabbit-anti-Von Willebrand factor antibody for 30 min at room temperature prior to staining with rhodamine-conjugated anti-rabbit
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antibody for a further 30 min at room temperature, protected from light. Stained cells were mounted in Vectashield mountant (Vector Laboratories, Peterborough, U.K.) and viewed under a fluorescence microscope.
Non-adherent cells were gently washed off and discarded and adherent cells were lysed with 500 l 10% Triton X-100 (Sigma) prior to counting. Results were expressed as counts/min in the adherent cell population.
mRNA
Statistical analysis
Total RNA was isolated using guanidinium thiocyanate and phenol extraction (Totally RNA kit, Ambion Inc., Austin, TX, U.S.A.). Quantity and quality of RNA was determined by differential UV absorbance. The ratio of absorbance at 260 nm:280 nm was typically between 1.7–2.0 for pure RNA samples. Total RNA (15 g/lane) was electrophoresed for 4 h at 60 V under denaturing conditions on a 1% agarose gel containing formaldehyde, and blotted onto hybond N + hybridization membrane (Amersham International) overnight. RNA was then bound to the membrane by UV crosslinking and hybridized at 42C overnight to a digoxigenin-labelled E-selectin, IL-8 or -actin probe (R&D Systems). Following stringency washes, chemiluminescent detection was performed using CDP Star (Boehringer Mannheim) according to manufacturer’s instructions. mRNA was visualized by autoradiography and quantified by phosphorimaging.
Data are expressed as median (range) and represent results from a minimum of six separate experiments. Differences were assessed using Kruskal Wallis analysis of variance or Mann–Whitney U test as appropriate. A P value of c0.05 was taken as statistically significant.
Soluble E-selectin Confluent cells were seeded onto 24-well tissue culture plates at 0.4106/well in a volume of 0.5 ml and incubated overnight at 37C. To each well was then added 100 U/ml TNF- and 25 U/ml IL-1, and cells were incubated for a further 24 h. Soluble E-selectin was measured in culture medium using an enzyme immunoassay kit (R&D Systems). Neutrophil adherence HUVECs, EAhy.926 and ECV 304 were seeded into gelatin-coated six-well plates to a density of 5104 cells/cm2 and incubated overnight, prior to activation with TNF- (100 U/ml) and IL-1 (25 U/ml) for 6 h. Human polymorphonuclear leucocytes (PMN) were isolated from healthy donors using a single density gradient procedure as previously described (Goode et al., 1994). Isolated PMN (1107) were incubated with 250 Ci 51Cr (Amersham) for 90 min at 37C. Following incubation, cells were washed and resuspended at 3106 cells/ml and 500 l of the labelled PMN were added to washed and dried endothelial monolayers and incubated for 60 min at 37C.
RESULTS Flow cytometry E-selectin expression increased significantly in all endothelial cells after exposure to TNF-/IL-1 for 24 h (P=0.0001) but expression was approximately 100-fold higher in HUVEC’s by 3 h (Fig. 1). The median percentage of E-selectin -positive HUVECs increased from 1.6(0.9–6.2)% to 91.4(83.0–96.1)% following 3 h incubation with TNF-/IL-1 (P=0.001) and was still elevated at 24 h (57.3(40.9– 73.5)%, P=0.001, Fig. 1). The levels of TNF-/ IL-1 -induced ICAM-1 expression was similar in all three cell types, but basal expression in the absence of cytokine stimulation was significantly higher in ECV304 cells than either HUVECs (P=0.004) or EA.hy926 cells (P=0.004) and the kinetics of the expression was markedly different (Fig. 1B). Immunofluorescence Fluorescence microscopy revealed that staining for von Willebrand factor was present in all cell types but was less intense in the cell lines than in primary endothelial cells (Fig. 2A). None of the endothelial cells showed basal E-selectin expression in the absence of activation (not shown). However, after 6 h exposure of HUVECs to TNF-/IL-1, cell surface expression of E-selectin was seen as intense green staining (Fig. 2B). Minimal staining could be seen in the cell lines (Fig. 2B). mRNA Northern blotting showed that mRNA for E-selectin was not detectable in ECV304 and EA.hy926 cell lines, even after exposure to TNF-/ IL-1 for up to 8 h (Fig. 3). However in HUVECs,
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Fig. 1. The effect of exposure of human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for up to 24 h, on E-selectin (A) and intercellular adhesion molecule-1 (ICAM-1) (B) expression by flow cytometry. Bar and whisker graphs show median, 25th and 75th percentiles with ranges as vertical lines. Data are results obtained from six separate experiments. P value refers to Kruskal Wallis analysis of variance. *Significantly higher than in HUVECs and EA.hy926 cells (P=0.05), Mann–Whitney U test.
mRNA for E-selectin increased after 2 h, and was maximal after 6 h exposure to TNF-/IL-1 (Fig. 3). The ‘housekeeping’ mRNA probe for actin showed equal RNA loading (Fig. 3). All cell types showed TNF-/IL-1-mediated IL-8 mRNA expression, but maximal expression was later for ECV304 cells than both HUVECs and EA.hy926 cells (Fig. 3).
both primary endothelial cells and cell lines, following exposure to TNF-/IL-1 for 6 h, compared to adherence without TNF-/IL-1 (Fig. 5). However, TNF-/IL-1 stimulated adherence of PMN was markedly higher in HUVECs compared to ECV304 (P=0.004) and EA.hy926 (P=0.004) cell lines (Fig. 5).
sE-selectin
DISCUSSION
Basal unstimulated 24 h soluble E-selectin accumulation was significantly higher from HUVECs compared to both of the other cell lines. In medium from HUVECs, median (range) sE-selectin concentration was 131.7(95.7–155.7) pg/ml compared to 47.7(17.1–95.7) pg/ml from EA.hy926 cells (P=0.05) and 17.7(6.1–83.7) pg/ml from ECV304 cells (P=0.01, Fig. 4). When cells were exposed to TNF-/IL-1, sE-selectin accumulation from HUVECs increased significantly to 347(281.8– 395.8) pg/ml, (P=0.004, Fig. 4), but failed to increase in the cell lines (Fig. 4). Adherence The number of adherent PMN, as represented by the counts/min of 51Cr, increased significantly in
We have shown that E-selectin expression is negligible by flow cytometry, immunofluorescence, mRNA analysis or as soluble E-selectin in the human vascular endothelial cell lines ECV304 and EA.hy926, and is considerably less than in primary cell cultures. In addition, the expression of von Willebrand factor, ICAM-1 and IL-8 by these cell lines is different to primary vascular endothelial cells from human umbilical cords. E-selectin is an endothelial cell-specific 110 KDa glycoprotein receptor for sialyl Lewisx and sialyl Lewisa carbohydrate ligands on circulating neutrophils, eosinophils and monocytes and tumour cells. It also targets memory T lymphocytes to sites of cutaneous inflammation. E-selectin is expressed on the surface of endothelial cells in response to
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Fig. 2. (A) Photomicrograph of von Willebrand expression in (i) primary human umbilical cord endothelial cells (ii) ECV304 cells and (iii) EA.hy926 cells. (B) Photomicrograph of E-selectin expression on human umbilical cord endothelial cells following exposure to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for 24 h in (i) primary human umbilical cord endothelial cells (ii) ECV304 cells and (iii) EA.hy926 cells.
Fig. 3. Representative northern blot of mRNA for E-selectin, interleukin-8 (IL-8) and actin in human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, following 0–8 h exposure to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1). Numbers are arbitrary phosphorimager counts/mm2.
inflammatory stimuli and video microscopy has shown the importance of E-selectin in the support of neutrophil rolling under conditions of flow (Abbassi et al., 1993). Adhesion is an important part of the host response to infection and inflammation, enabling the margination of circulating cells. E-selectin also has a role in the adherence of monocytes and the formation of atherosclerotic
plaques. Induction of E-selectin expression is stimulated by endotoxin (LPS), TNF-, IL-1, IL-4, oxidized low-density lipoproteins, hyperthermia, chemotactic peptides, phorbol ester and lipid mediators (reviewed by Tonnesen, 1989). N-glycosylation of the E-selectin molecule is obligatory for endothelial cell surface expression (Pahlsson et al., 1995) and a lower molecular
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400 8 6 4 2 ** 0 – + – + – + TNF-α and IL-1β TNF-α and IL-1β TNF-α and IL-1β HUVECs ECV304 EA.hy926
0 – – + – + + TNF-α and IL-1β TNF-α and IL-1β TNF-α and IL-1β HUVECs ECV304 EA.hy926
Fig. 4. The effect of exposure of human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for 24 h, on soluble E-selectin accumulation in culture medium. Bar and whisker graphs show median, 25th and 75th percentiles with ranges as vertical lines. Data are results obtained from six separate experiments. P value refers to Mann–Whitney U test in HUVECs in the absence and presence of TNF-/IL-1. *Significantly lower than in HUVECs (P=0.05) Mann– Whitney U test; **significantly lower than in HUVECs (P=0.01) Mann–Whitney U test.
Fig. 5. The effect of exposure of human umbilical cord endothelial cells (HUVECs) and the human endothelial cell lines, ECV304 and EA.hy926, to 100 U/ml tumour necrosis factor (TNF-) and 25 U/ml interleukin-1 (IL-1) for 24 h, on adherence of human polymorphonuclear leucocytes to endothelial monolayers. Bar and whisker graphs show median, 25th and 75th percentiles with ranges as vertical lines. Data are results obtained from six separate experiments. P value refers to Mann–Whitney U test in the absence and presence of TNF-/IL-1. *Significantly lower than in HUVECs (P=0.004) Mann–Whitney U test.
weight form of the molecule, termed soluble E-selectin (sE-selectin), which is thought to be a biologically active shed form of E-selectin (Newman et al., 1993) is released from the cell surface. Elevated circulating sE-selectin concentrations have been linked to several inflammatory conditions and atherosclerosis (Kaneko et al., 1996; Smith and Wayne, 1997). ECV304 is a spontaneously transformed cell line which originated from the umbilical cord of a Japanese infant. Expression of the endotheliumspecific marker, von Willebrand factor, has been shown previously (Takahashi and Sawasaki, 1992), which we have confirmed in the present study, although we found that expression was less than that in primary endothelial cells. Factor VIII expression was negative, however (Takahashi and Sawasaki, 1992), and hyaluronic acid metabolism was elevated compared to primary cell cultures (Amanuma and Mitsui, 1991). ECV304 cells also express colony stimulating factor receptor chains, in contrast to HUVECs (Colotta et al., 1993). Quantitative analysis of cell surface molecules in a comprehensive study of HUVECs and two cell lines (Mutin et al., 1997) revealed that ECV304 express tissue factor antigen (CD142) but not
platelet-endothelial cell adhesion molecule (PECAM,CD31) or vascular endothelial cadherin (VE cadherin, CD144), in addition to no detectable expression of E selectin under resting conditions. However, stimulated antigen expression in these cells was not studied. EA.hy926 is a hybrid cell line resulting from fusion of primary human umbilical vein endothelial cells with cells selected from the continuous human cell line A549, originally derived from a human lung carcinoma (Edgell et al., 1990). We found positive staining for von Willebrand factor in this cell line, as previously described (Edgell et al., 1983; van Oost et al., 1986; Wilbourn et al., 1992), but again staining was less than in primary cells. The cell line has been shown to be positive for factor VIII (Edgell et al., 1983), endothelin-converting enzyme (Walkden and Turner, 1995; Ahn et al., 1995), PECAM and VE cadherin (Mutin et al., 1997) but not E-selectin, under resting conditions. We found that E-selectin expression as shown by flow cytometry, immunofluorescence, mRNA levels and detection of sE-selectin, was upregulated in primary umbilical vein endothelial cells in response to TNF- and IL-1 after 2 h and was maximal at 6–8 h, declining by 24 h, as previously
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described. However, E-selectin cell surface expression was much lower under these conditions using the endothelial cell lines ECV304 and EA.hy926. Analysis of cell surface expression of E-selectin in HUVECs in response to TNF- and IL-1 has previously been shown to be maximal at 4–8 h (Sepp et al., 1994; Mutin et al., 1997). Northern analysis of mRNA for E-selectin in HUVECs in previous studies was maximal at 3–6 h (Kaneko et al., 1996; Moers et al., 1997) in accordance with the present study. Increased sE-selectin accumulation from HUVECs after 4–24 h exposure to LPS or cytokines have also been previously described (Sepp et al., 1994; Kaneko et al., 1996; Moers et al., 1997). Despite the relevance to the use of cell lines in studies of regulation of antigen expression in pathophysiological conditions, particularly regulation of the response to inflammation or infection, where the use of activated cells is required, there is little previous work on stimulated E selectin expression by the commonly used cell lines, EA.hy926 and ECV304. Thornhill et al. (1993) reported similar upregulation of cell surface E selectin expression and adhesion in EA.hy926 cells to that found in HUVECs. We also showed that E selectin expression by flow cytometry was upregulated in both EA.hy926 and ECV304 cells, but the magnitude of the increase was much less than in primary endothelial cells. However, closer examination of Thornhill’s data reveals some differences between primary endothelial cells and EA.hy926 cells. E selectin expression, measured using an ELISA technique on paraformaldehyde-fixed cell monolayers, rather than by flow cytometry as in the present study, was noticeably lower in EA.hy926 cells than HUVEC’s at both 6 and 20 h after stimulation with TNF (Thornhill et al., 1993), but, these authors did not perform statistical comparisons between the cell types. Both VCAM-1 and ICAM-1 expression in EA.hy926 were similar to that in HUVECs in response to TNF (Thornhill et al., 1993) but EA.hy926 cells did not respond to IL-4 or IFN- in terms of upregulation of ICAM-1 or VCAM-1, unlike HUVECs (Thornhill and Haskard, 1990; Thornhill et al., 1990). There have been no previous studies describing soluble E selectin, mRNA for E selectin or histochemical studies of endothelial antigens in cell lines compared to HUVECs. The mechanism of the reduced E-selectin expression in the cell lines ECV304 and EH.hy926 is unclear. We have shown that both cell lines constitutively express ICAM-1, confirming previous work on ICAM-1 (Burke-Gaffney and Hellewell 1996a,b; Wheller and Perretti, 1997) and ICAM-2
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(Nortamo et al., 1991). However, basal unstimulated expression was higher in ECV304 cells than either HUVECs or EA.hy926, in the present study, although the level of the response to cytokine stimulation with upregulation of ICAM-1 was similar, despite different kinetics. The cell lines also exhibit other inflammatory responses upon cytokine stimulation. Both we and others have shown that EA.hy926 cells upregulate IL-8 release and produce nitric oxide in response to TNF- and IL-1 (Villarette and Remick, 1995; Galley et al., 1997) and express inducible cyclo-oxygenase in response to IL-1 (Ristimaki et al., 1994). The present study also demonstrates that IL-8 mRNA from EA.hy926 and EVC304 cells, as well as HUVECs, is increased on exposure to TNF-/IL1, but that the time of maximal expression is later for ECV304 cells compared to the other endothelial cell types. Therefore the lack of E-selectin expression appears to be selective and does not extend to other pro-inflammatory functions. Adherence of polymorphonuclear leucocytes to the endothelial cell lines increased when the endothelial cells were activated with TNF- and IL-1, but the number of adherent PMN was much lower than the adherence to activated primary endothelial cells. In addition we have found that the level of adherence of PMN to activated HUVECs can be reduced to that of the cell lines using an anti-E-selectin blocking antibody (data not shown). These results suggest that lack of E-selectin expression has profound effects on the adherence of PMN, despite normal ICAM-1 expression and IL-8 production, indicating the functional significance of the reduced E-selectin expression. Adhesion of neutrophils and T cells after 4 h and 18 h stimulation with TNF respectively was similar in both the EA.hy926 cell line and HUVECs (Thornhill et al., 1993), but adhesion of T cells to EA.hy926 cells was not increased in response to IL-4 or IFN- as shown in HUVEC’s (Thornhill and Haskard, 1990; Thornhill et al., 1990). Cartright et al. (1995) have shown that the series of transformed endothelial cell lines, termed SGHEC-1 to SGHEC-13, express cell surface E-selectin in response to TNF- or IL-1 as measured by flow cytometry, but do not release immunoreactive sE-selectin. In the present study cell surface and soluble E-selectin was barely detectable in ECV304 or EA.hy296 cells, and was at least 100-fold lower than HUVECs. Another spontaneously transformed endothelial cell line, C11STH, which expresses von Willebrand factor and ICAM-1, is able to upregulate cell surface
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E-selectin expression in response to TNF-, but soluble E-selectin was not measured (Cockerill et al., 1994). Although the endothelial cell-specific phenotypic marker, von Willebrand factor, does not alter with the passage number of primary umbilical cord endothelial cells, the constitutive and LPS- or cytokine-induced expression of ICAM-1 decreased by about 65% and 40% respectively after six passages (Klein et al., 1995). After passage 3, the production of E-selectin in response to LPS or TNF-/IL-1 stimulation was shown to be negligible (Klein et al., 1995). The HUVECs used in the present study were all used before passage 3 and were all cultured using the same media, serum concentration etc., since these may affect cell function (Mutin et al., 1996). Endothelial cells from different cord donors may also have varying responses (Heukens et al., 1991) and it has been reported that detachment conditions, which were identical in the present study for all cells, may also affect cell viability and responses (Beekhuizen, 1994; Mutin et al., 1996). This inconsistency in cells has led to the expanding use of transformed cell lines, which offer a continuous supply of phenotypically constant endothelial cells with a high proliferative potential, stable antigenicity and no growth factor requirement (Balconi and Dejana, 1986). However, we have shown that the cell lines ECV304 and EA.hy926 have limited ability to express E-selectin. Other studies have also shown decreased E-selectin expression in the EA.hy926 cell line and an inability to respond to IL-4 and IFN- stimulation, whilst ECV304 cells do not express PECAM or VE Cadherin. These cell lines should therefore be used with caution for studies of adhesion, regardless of their ability to express other adhesion molecules. REFERENCES A O, K TK, MI LV, A DC, S CW, 1993. E-selectin supports neutrophil rolling in vitro under conditions of flow. J Clin Invest 92: 2719–2730. A K, P S, B K, H D, 1995. A permanent human cell line (EA.hy926) reserves the characteristics of endothelin converting enzyme from primary human umbilical vein endothelial cells. Life Sci 56: 2331–2341. A K, M Y, 1991. Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells. Biochim Biophys Acta 1092: 336–340. B G, D E, 1986. Cultivation of endothelial cells: limitations and perspectives. Med Biol 64: 231–245.
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