CXC CHEMOKINE RECEPTOR EXPRESSION ON HUMAN ENDOTHELIAL CELLS

Article No. cyto.1998.0465, available online at http://www.idealibrary.com on

CXC CHEMOKINE RECEPTOR EXPRESSION ON HUMAN ENDOTHELIAL CELLS Craig Murdoch,1 Peter N. Monk,2 Adam Finn1 CXC Chemokines play a important role in the process of leukocyte recruitment and activation at sites of inflammation. However, recent evidence suggests that these molecules can also regulate endothelial cell functions such as migration, angiogenesis and proliferation. In this study we have investigated CXC chemokine receptor expression in both primary cultures of human umbilical vein endothelial cells (HUVEC) and the spontaneously transformed HUVEC cell line, ECV304. We found that both cell types express mRNA for chemokine receptors CXCR1, CXCR2 and CXCR4, but not CXCR3. Flow cytometric analysis revealed low levels of CXCR1 but higher levels of CXCR4 cell surface expression. HUVECs responded to SDF-1á with a rapid and robust calcium flux, however no calcium flux was seen with either IL-8 or Gro-á. HUVECs and ECV304 cells did not proliferate in response to CXC chemokines, although ECV304 cells did migrate towards SDF-1á and IL-8. These data demonstrate that HUVECs and the endothelial cell line, ECV304 express functional CXC chemokine receptors.  1999 Academic Press

It is well established that chemokines play a important role in the process of leukocyte recruitment and activation at sites of inflammation.1,2 Chemokines are a family of small molecular weight proteins (8 to 13 kDa) that are classified into four distinct groups depending on the positioning of the cysteine motif at the N-terminus. The family members include CXC, CC, C and CX3C chemokines of which CXC and CC are the largest and most characterized.3–6 The specific effects of chemokines on their target cells are mediated by seven-transmembrane spanning, G-protein coupled receptors.7 To date a total of fifteen chemokine receptors have been identified. Five receptors selectively bind certain CXC chemokines (CXCR1– 5),8–13 whilst the CC receptor family currently consists of nine receptors, CCR1–8 and CCR10.14–22 A further chemokine receptor known as the Duffy antigen receptor for chemokines (DARC) has been shown to bind promiscuously to both CXC and CC chemokines.23 Until recently, the actions of chemokines and the expression of their receptors have only been described From the 1Division of Child Health and 2Krebs Institute for Biomolecular Research, Western Bank, University of Sheffield, Sheffield, UK Correspondence to: Craig Murdoch, Division of Child Health, Sheffield Children’s Hospital, Western Bank, University of Sheffield, Sheffield S10 2TH, UK; E-mail: mdp94cm@Sheffield. ac.uk Received 30 July 1998; accepted for publication 29 October 1998  1999 Academic Press 1043–4666/99/090704+09 $30.00/0 KEY WORDS: chemokine receptors/endothelial/CXCR4 704

on leukocytes. However, new evidence has shown that non-haematopoietic cell types are capable of binding and responding to a number of chemokines. CXC chemokines, such as IL-8, Gro-á, Gro-â, PF-4 and IP-10 and have been implicated in the regulation of keratinocyte and endothelial cell functions including the stimulation and inhibition of proliferation, angiogenesis and cell migration.24–26 IL-8 has also been shown to increase directly the permeability of endothelial cell monolayers.27 It has therefore been suggested that these receptors, along with endothelial proteoglycans, may serve as facilitators of endothelial cell inflammatory responses.28 The expression of other ‘‘classical’’ seven transmembrane chemoattractant receptors on endothelial cells has recently been reported. Both the complement C5a receptor (C5aR) and the Platelet-activating factor receptor (PAFR) have been shown to be expressed on endothelial cells.29,30 The complement fragment C5a has been found to induce endothelial cells to rapidly express P-selectin, secrete von Willebrand factor, and to increase their adhesiveness for human neutrophils.31 The expression of endothelial chemokine receptors which participate either in the inflammatory response or wound healing is therefore a distinct possibility. We have used reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry and functional assays to look for the presence of CXC chemokine receptors in both primary cultures of human umbilical vein endothelial cells (HUVEC) and the spontaneously transformed HUVEC cell line, ECV304. CYTOKINE, Vol. 11, No. 9 (September), 1999: pp 704–712

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Figure 1.

Expression of chemokine receptor mRNA in HUVEC and ECV304 cells by RT-PCR analysis.

Lane 1, HindIII & EcoRI cut ë DNA; lane 2, CXCR1; lane 3, CXCR2; lane 4, CXCR3; lane 5, CXCR4; lane 6; negative control; lane 7, â-2-microglobulin fragment.

RESULTS RT-PCR analysis of CXC chemokine receptor expression In order to assess the gene expression of the CXC chemokine receptor in endothelial cells, RT-PCR was conducted on RNA extracted from primary cultures of HUVECs and from the HUVEC derived cell line ECV304. RT-PCR analysis and subsequent gel electrophoresis showed bands of equivalent size to that expected for seven transmembrane receptors for both endothelial cell types (Fig. 1). DNA sequencing of these bands confirmed that mRNA for CXCR1, CXCR2 and CXCR4 is present in both HUVECs and their transformed counterpart, the ECV304 cell line. CXCR3 mRNA was found to be absent from both endothelial cell types. The band for CXCR4 on the agarose gel was larger than for the other chemokine receptors and comparable to that of the constitutively expressed â-2-microglobulin gene, suggesting that the gene for this receptor may be relatively abundant in endothelial cells.

Cell surface expression of CXC chemokine receptors The cell surface expression of CXC chemokine receptors on HUVEC and ECV304 cells was evaluated both by flow cytometry and immunocytochemistry using receptor-specific monoclonal antibodies and polyclonal antisera. The anti-CXCR4 antibody used recognises an epitope on the second extracellular loop of the receptor whilst the anti-CXCR2 and antiCXCR1 antisera used both bind specifically to the N-terminus of the receptors. A significant shift is seen in the fluorescence histograms of anti-CXCR1 and anti-CXCR4 stained cells when compared to the preimmune serum or isotype matched IgG control (Fig. 2) suggesting the presence of cell surface protein on both HUVEC and ECV304 cells. The results suggest that CXCR1 is expressed at low levels, whilst CXCR4 is abundantly expressed on the cell surface of both

endothelial cell types. CXCR2 cell surface expression could not be detected by this method. We found that the strength of immunofluorescence staining for CXCR4 was similar to that seen for the HLA class I ABC antigen (data not shown) which is consistent with the RT-PCR data (Fig. 1). CXCR3 expression was not examined as no antiserum was available. The localization of CXCR4 to the cell surface of HUVEC cells was confirmed using immunocytochemical staining (Fig. 3). No observable staining was seen with an isotype matched IgG control (data not shown).

Intracellular calcium mobilization Calcium mobilization studies were carried out to determine whether CXCR1 and CXCR4 expressed by endothelial cells are functionally active. Adherent fura2/AM loaded HUVECs cells were challenged with the CXCR1 ligands IL-8, Gro-á and the CXCR4 ligand, SDF-1á. Stimulation of HUVECs with SDF-1á elicited a rapid and robust increase in intracellular calcium in a concentration-dependent manner (Fig. 4). However, the magnitude of the calcium response was lower than that evoked by stimulation with 4 U/ml thrombin in the same cells. HUVECs challenged with either IL-8 (Fig. 4), Gro-á (1 ìg/ml) or buffer alone failed to produce a calcium response in the same assay system (data not shown).

Endothelial cell proliferation A fluorescence-based assay was used to examine the effects of SDF-1á and IL-8 on endothelial cell proliferation over a 72-h incubation period. Endothelial cells stimulated with varying concentrations of SDF-1á or IL-8 produced equivalent fluorescence signals to unstimulated cells (Fig. 5), suggesting that SDF-1á and IL-8 do not have any detectable proliferation effects on endothelial cells under the conditions tested. Similarly, no detectable effects on proliferation was observed with these chemokines after 24 or 48 h (data not shown).

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Figure 2.

CYTOKINE, Vol. 11, No. 9 (September, 1999: 704–712)

Flow cytometry histograms of CXCR1, CXCR2 and CXCR4 immnofluorescence on HUVEC and ECV304 cells.

All cell types were stained with rabbit anti-human CXCR1 or mouse anti-human CXCR2/CXCR4 followed by FITC-conjugated anti-IgG (solid lines). Controls received equivalent concentrations of preimmune primary antiserum or isotype-matched IgG respectively (broken lines). For all panels data are shown as cell number versus the relative fluorescence. Each histogram shows data from a single representative experiment although each analysis was repeated at least three times.

Figure 3.

Immunocytochemical staining of confluent HUVECs showing CXCR4 cell surface localization.

Units of fluorescence

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IL-8 ng/ml Figure 5.

Effects of IL-8 and SDF-1á on endothelial proliferation.

HUVECs were plated into 96-well plates and stimulated for 72 h with varying concentrations of appropriate chemokine. Fluorescence from three replicate wells were measured and data are represented as mean units of fluorescenceSEM (one of three similar experiments). IL-8 and SDF-1á had no effect on HUVEC proliferation, whilst cells stimulated with VEGF (10 ng/ml) showed marked increases in cell number.

DISCUSSION

Figure 4.

Calcium mobilization in HUVECs.

SDF-1á (2 ìg/ml) produced a robust calcium flux (A), whereas IL-8 (1 ìg/ml) did not (B). Thrombin (4 U/ml) was used as a positive control.

Endothelial cell migration The chemotactic responses of ECV304 cells to both IL-8 and SDF-1á was assessed using a modified Boyden chamber. IL-8 and SDF-1á induced directional migration of endothelial cells (Fig. 6) in a concentration-dependant manner. Both chemokines appeared to cause maximal migration at approximately 100 ng/ml. However, SDF-1á caused more endothelial cells to adhere to the underside of the membrane than IL-8, suggesting that SDF-1á is a more powerful endothelial chemoattractant.

The vascular endothelium plays an active and complex role in the maintenance of blood fluidity, tissue homeostasis, growth factor production and angiogenesis. Furthermore, the endothelium has important roles in antigen presentation and in the adhesion and extravasation of leukocytes to sites of inflammation. Recent evidence has demonstrated that, as well as being producers of chemokines, endothelial cells can undergo chemotaxis, proliferation and angiogenesis in response to certain CXC chemokines such as IL-8, GRO-á and ENA-78 which have the N-terminal Glu-Leu-Arg (ELR) motif.32 Furthermore, the ELR motif within CXC chemokines has been shown to be important in receptor-ligand interactions and cell activation.33,34 These observations suggest that endothelial cells possess receptors which can bind and respond to CXC chemokines specifically. Evidence concerning the presence of endothelialexpressed chemokine receptors has been conflicting. Scho¨nbeck et al.35 reported the presence of mRNA for CXCR1 but not CXCR2 in HUVECs, whilst Petzelbauer et al.36 could not detect the expression of any chemokine receptors in endothelial cells. Recently Gupta et al.37 have shown that endothelial cells express

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Figure 6. SDF-1á (A) and IL-8 (B) induced directional migration of ECV304 cells in a typical ‘‘bell-shaped’’ manner. Peak migration was seen at approximately 100 ng/ml for both SDF-1á and IL-8. Results shown are background corrected with a control of 0.1% BSA and are expressed as the mean number of cells migrating in five microscopic fields in each of three wellsSEM (n=3).

several chemokine receptors. Using an RT-PCR-based strategy utilizing receptor-specific primers, we demonstrate that both HUVECs and the spontaneouslytransformed HUVEC cell line, ECV304 express mRNA for CXCR1, CXCR2, and CXCR4 but not CXCR3. One possible explanation for these conflicting results is heterogeneity amongst HUVEC primary cultures, although we found the same profile of chemokine receptor mRNA expression in the ECV304 cell line as in HUVECs. Flow cytometric analysis of CXC chemokine receptors on both endothelial cell types suggested that CXCR1 is expressed at low levels on the cell surface whilst CXCR4 is abundantly expressed. CXC chemokines that do not contain the ELR motif, such as PF-4 and IP-10 appear to inhibit endothelial cell proliferation, migration and angiogenesis.26,32 We found that mRNA for CXCR3, the receptor that binds IP-10, was absent from endothelial cells, suggesting that the inhibitory effects of this chemokine are not mediated by this receptor. However, it is possible that IP-10 may act through an as yet unidentified receptor. Furthermore, a receptor for PF-4 has yet to be identified.

Calcium mobilization experiments suggest that CXCR4 expressed by endothelium can activate G-protein coupled signalling mechanisms upon ligand binding in a way similar to that described for leukocytes, and confirm that endothelial CXCR4 is functionally active. However, IL-8 and Gro-á failed to produce a calcium flux using the same assay system, a finding also observed by Petzelbauer et al.36 This may be explained by the low level of CXCR1 expression. It is also possible that IL-8/CXCR1 interactions may be signalling via a calcium-independent pathway in these cells. Endothelial cells exhibited directional migration towards both SDF-1á and IL-8 in a concentrationdependent manner, suggesting that CXCR1 receptors may be functionally active on these cells even in the absence of demonstrable calcium mobilization. These observations are similar to previous reports by other investigators24,32,37 who also showed directional migration of endothelial cells toward SDF-1á, IL-8 and other CXC chemokines. The existence of effects of CXC chemokines, in particular IL-8, on endothelial cell proliferation, has been contentious. Koch et al.24 reported a dosedependent growth enhancing effect of IL-8 on HUVEC cultures, whilst Petzelbauer et al.36 did not. Using a fluorescence-based proliferation assay we showed that neither IL-8 or SDF-1á affected endothelial cell growth in our culture system. One possible explanation for these differing results is the presence of contaminating subpopulations of IL-8 responsive cells within primary cultures, such as mast or T cells, that could release endothelial growth factors upon chemokine stimulation and therefore enhance endothelial cell growth. To date there is no evidence to suggest that endothelial cell functions are affected by CC chemokines. However, RT-PCR results obtained in our laboratory show that both HUVECs and ECV304 cells also express low levels of mRNA for several CC chemokine receptors including CCR1, CCR2B, CCR3, CCR4 and CCR5.38 These results suggest that CC chemokines could also induce endothelial cell responses. Furthermore, expression of the promiscuous chemokine receptor DARC was detected on ECV304 cells but not on HUVECs (unpublished observations). DARC has been reported to be expressed on renal and spleen capillary endothelium39 as well as on endothelial cells of venules.40 Our findings suggest that the spontaneously transformed ECV304 cell line may have different characteristics from primary endothelial cells isolated from large veins. It has been suggested that during the course of an inflammatory response chemokines bind to endothelial proteoglycans, producing an immobilized haptotactic chemokine gradient on the endothelial cell surface. Such a chemokine gradient would ensure directional

CXC chemokine receptors on endothelial cells / 709

leukocyte transmigration through the endothelium to the site of inflammation.41,42 It has also been proposed that endothelial cell chemokine receptors along with proteoglycans could participate in the presentation of chemokines to leukocytes.41 However, if chemokine receptors expressed on leukocytes and endothelium are identical they might be expected to bind their ligands in the same manner and compete for chemokines rather than contribute to presentation. If endothelial proteoglycans alone bind and present chemokines they may do so both to leukocyte and to endothelial-expressed chemokine receptors. Such a model has recently been proposed by Gupta et al.37 This model is analogous to the way in which fibroblast growth factor is thought to bind to endothelial proteoglycans, facilitating its interaction with high affinity FGF receptors on the endothelial cell surface.43 A low level of expression and responsiveness of chemokine receptors on endothelial cells may be sufficient to permit cell activation in the presence of high levels of proteoglycan-bound chemokine on the adjacent endothelial cell surface. IL-8 and other CXC chemokines are secreted by a variety of cell types, including endothelial cells, upon activation with other pro-inflammatory mediators and cytokines.3 However, SDF-1á is constitutively expressed by a broad range of tissues, including bone marrow, thymus, spleen and liver. Its expression does not appear to be regulated by pro-inflammatory stimulants.44 It is therefore possible that CXCR4/SDF-1á is not involved in the inflammatory response but may have other cellular functions. Recent studies show that mice devoid of either the SDF-1 or CXCR4 genes die perinatally and present multiple defects of development, including a severe reduction of B cell and myeloid progenitors in the bone marrow.45 Furthermore, mice lacking CXCR4 have defective formation of the large vessels supplying the gastrointestinal tract, and are defective in vascular development, haematopoiesis and cardiogenesis. Consequently, it appears that both CXCR4 and SDF-1 are important in organ vascularization and embryogenesis.46 Recent studies have shown that CXCR447 CCR2b, CCR3, CCR548 and CCR849 serve as co-factors in association with CD4 to permit infection of either T cell tropic or macrophage tropic HIV-1 strains into permissive cells. Furthermore, some HIV isolates, particularly HIV-2, have been shown to gain entry to cells that do not express CD4, suggesting that some HIV strains can enter cells by utilizing CXCR4 alone.50 The expression of CXCR4 on endothelial cells suggests that HIV could infect endothelial cells by a CD4-independent pathway. Corbeil et al.51 have shown that some HIV-1 isolates can infect HUVECs in vitro. Therefore HIV may have more widespread cellular targets that first envisaged.

It is possible that the expression of CXC chemokine receptors on endothelial cells may facilitate the recruitment of phagocytic cells to sites of inflammation or have some other functional role within the immune response or neo-vascularization processes. If this is so, it is also possible that these receptors may be involved in the pathophysiology of several acute or chronic disease states.

MATERIALS AND METHODS Cell culture Endothelial cells were isolated from human umbilical cord veins by 0.1% collagenase (Sigma) digestion and cultured in flasks coated with 2% gelatin as described previously.52 Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ìg/ml streptomycin was used to maintain the cells. HUVECs were used at passage 2 and were verified as endothelial by a cobblestone appearance and specific immunofluorescence staining for lectin Ulex europaeus I and von Willebrand factor (data not shown). The endothelial cell line ECV30453 was obtained from the European Collection Animal Cell Cultures (Aylesbury, UK) and cultured as above, except that 5% FCS was used.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from cultured HUVECs and ECV304 cells using RNAzol B (Biogenesis) according to the manufacturer’s instructions. RNA from these cells was reverse transcribed using Superscript II reverse transcriptase (Life Technologies) and receptor-specific oligonucleotide primers for CXCR1, CXCR2, CXCR3 and CXCR4 based on the published nucleotide sequences.8–11 PCR was performed using 2.5 ìl cDNA added to a reaction mixture containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, 200 ìM deoxynucleotide triphosphates, 1 ìM of each receptorspecific primer and 2.5 units Taq polymerase (Life Technologies) in a reaction volume of 50 ìl. The enzyme was added after heating the probe to 94C (hot start). Samples were incubated in a Techne PHC-3 thermal cycler for a total of 35 cycles. Each cycle consisted of 1 min at 95C, 1.5 min at 59C and 1.5 min at 72C. The amplification of a segment of the constitutively expressed â-2-microglobulin gene was used as an endogenous control. PCR products were visualized on a 1.5% TAE agarose gel containing ethidium bromide under UV illumination. DNA fragments whose sizes corresponded to that of seven-transmembrane receptors were gel-purified and subjected to DNA sequence analysis.

Flow cytometry Endothelial cells were removed from flasks nonenzymatically and resuspended at 2105 cells in ice cold wash buffer (0.2% BSA-PBS+0.1% sodium azide). Cells were incubated with either 1 in 100 dilutions of rabbit polyclonal anti-CXCR1 antiserum or 10 ìg/ml mouse monoclonal anti-human CXCR2 (Serotec, clone HC2) or mouse

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monoclonal anti-human CXCR4 (R & D Systems, clone 12G5) antibody for 40 min at 4C. Cells were kept on ice to minimize down-modulation and internalization of receptors. After washing twice with cold wash buffer, cells were incubated with FITC-conjugated anti-mouse or anti-rabbit IgG (Serotec) in the dark for 30 min at 4C. Finally, cells were washed twice more and resuspended in wash buffer. FACS analysis was done with a FACScan flow cytometer (Becton Dickinson). Controls received preimmune primary antiserum or isotype-matched irrelevant IgG at the same concentration.

Immunocytochemistry Cultured HUVECs were washed with PBS, fixed with 2% paraformaldehyde and stained with either 10 ìg/ml monoclonal anti-CXCR4 or 10 ìg/ml isotype matched IgG control antibody, for 1 h at room temperature. Cells were washed, stained with FITC-conjugated rabbit anti-mouse IgG for 30 min at room temperature, washed again and mounted with Citifluor (Agar Scientific). Photographs were taken using a Nikon SLR camera attached to a Leica DM IRB inverted fluorescence microscope.

Ca2+ mobilization assay Microfluorimetry was used for measurements of intra cellular calcium [Ca2+ ]i. HUVECs were grown on poly-lysine-coated glass coverslips and loaded with 2 ìM fura-2/AM added to the culture medium for 45 min at 37C. Coverslips were transferred to assay buffer (in mM: NaCl 137, KCl 5.36, MgSO4 0.81, Na2PO4 0.34, KH2PO4 0.44, NaHCO3 4.17, HEPES 10, CaCl2 1.26 and glucose 2.02) and fluorescence of fura-2 in individual cells was measured using a Nikon diaphot microscope with an epifluorescence 40 objective. Chemokines were added from concentrated stocks in water. To establish the integrity of endothelial cells, [Ca2+ ]i was also measured using thrombin as a control stimulant; buffer alone was used as a negative control. CXC chemokine activity was checked using a flow cytometric calcium mobilization-based assay on isolated neutrophils.

Proliferation assay Increases in HUVEC cell number were detected by labelling cellular DNA with the fluorescent dye propidium iodide. Endothelial cells were plated at a density of 1104 cells/well in a 96-well plate and incubated in culture medium for 24 h to allow adherence. After washing, DMEM with 10% FCS, supplemented with or without chemokine, was added and the cells incubated for 72 h at 37C, 5% CO2. At the end of the culture period endothelial cells were fixed with 3.7% formaldehyde, stained with 5 ìg/ml propidium iodide for 5 min at room temperature in the dark and then air dried. Fluorescence was measured at 530 nm excitation and 620 nm emission using an automated fluorescent microplate reader (Denly Wellfluor) . The amount of fluorescence detected was assumed to be directionally proportional to the number of cells within a well.54 Results are expressed as mean fluorescence values from triplicate wells SEM.

Migration assay Migration of ECV304 cells was performed using a 48-well chemotaxis chamber (Neuroprobe). The chemokine

CYTOKINE, Vol. 11, No. 9 (September, 1999: 704–712)

in DMEM+0.1% BSA was added to the lower wells and overlaid with a fibronectin (4.0 ìg/ml) coated 8 ìM pore polyvinylpyrrolidone-coated polycarbonate filter. Fifty microlitres of a cell suspension containing 3104 cells were added to the upper wells and the chamber incubated for 5 h at 37C in a humidified atmosphere of 5% CO2. Membranes were fixed with methanol and stained with Harris’ haematoxylin. Cells that had migrated and were attached to the lower side of the membrane were counted per high power field (HPF) using a light microscope. Results are expressed as the mean number of cells migrating in five microscopic fields in each of three wells SEM.

Acknowledgements The authors would like to thank staff of the Jessops Hospital for Women Maternity Unit for umbilical cord collection, Ruth Shepherd and Molly Hashmi for help with calcium assays and Arthur Moir and Paul Brown for synthesis of oligonucleotide primers and DNA sequencing. The funding of this work included financial support from the Arthritis and Rheumatism Council (P. N. Monk, M0543) and the Children’s Appeal, Sheffield Children’s Hospital.

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