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IFN- Induced Adenosine Production on the Endothelium: A Mechanism Mediated by CD73 (Ecto-5'-Nucleotidase) Up-Regulation¹

Jussi Niemelä^*, Tiina Henttinen^*, Gennady G. Yegutkin^*, Laura Airas^*, Anna-Maija Kujari^*, Pertti Rajala and Sirpa Jalkanen^2,*

^* MediCity Research Laboratory and Department of Medical Microbiology, Turku University, Turku and National Public Health Institute, Turku, Finland; and Department of Surgery, Turku University Central Hospital, Turku, Finland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD73 (ecto-5'-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN- produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN- treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1, TNF-, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN- is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The interaction between lymphocytes and endothelial cells is a multistep process. To be able to penetrate the vessel wall and to reach the target site, circulating cells use a very finely regulated set of adhesion molecules. Enhanced adhesion to endothelium and subsequent transmigration of recirculating leukocytes through the endothelial lining of vessel wall into the tissue are characteristic for inflammation. Moreover, the release of pro- and anti-inflammatory cytokines in a high extent takes place at sites of inflammation. Those cytokines are potent regulators of the expression of adhesion molecules (1, 2, 3).

Ecto-5'-nucleotidase (CD73) is a 70-kDa GPI-anchored cell surface molecule with ecto-enzymatic activity. It is abundantly expressed on the vascular endothelium and at a low level on certain subpopulations of human lymphocytes. It is part of the purine salvage pathway by degrading nucleoside-5'-monophosphates (AMP and IMP) into nucleosides such as adenosine and inosine (4, 5). CD73 has also been suggested to mediate homing of skin-infiltrating lymphocytes in vivo (6). Triggering of CD73 on the surface of lymphocytes, but not on endothelial cells, results in the shedding of the CD73 and increased adhesion of lymphocytes to endothelium via LFA-1 clustering (7, 8). This phenomenon is especially interesting in light of the fact that the cDNA and protein structure are very similar on both cell types (7).

Adenosine, a purine nucleoside product of CD73 enzyme activity, has a role in many physiological and pathological events. It binds to specific receptors on the cell surface. To date, four different subtypes of G protein-coupled adenosine receptors, A1R, A2aR, A2bR, and A3R, have been cloned (9). Ecto-5'-nucleotidase activity is shown to be an important mediator of the anti-inflammatory effect of methotrexate and sulfasalazine in vitro and in vivo in the murine air pouch inflammation model through increasing extracellular adenosine levels (10). Adenosine, by binding to A1 and A2 receptors, regulates pathological consequences of inflammation by controlling leukocyte binding to endothelium (11) and acts as an anti-inflammatory agent by binding to A2 and A3 receptors, through the inhibition of neutrophil degranulation (12). Adenosine also decreases eosinophil migration through activation of A3 receptor (13). Adenosine, converted from neutrophil-derived AMP, leads to increased endothelial barrier function by endothelial A2bR activation. This promoting effect of AMP is CD73 mediated and is followed by an increase in intracellular cAMP (14). Recently, a critical role for A2a receptor has been shown in decreasing systemic and tissue-specific inflammatory responses in vivo (15).

To date, practically nothing is known about the regulation of endothelial CD73 expression and function. However, in inflammation there may be some inducers secreted that in vivo specifically control endothelial CD73 expression. This hypothesis was suggested by a previous finding, that CD73 is up-regulated in inflamed skin (6).

As adenosine, having an anti-inflammatory and cell-protective effect, plays an important role in controlling the extent and consequences of inflammation, this work was designed to identify the factors responsible for the regulation of CD73 expression as well as ecto-5'-nucleotidase-mediated adenosine production.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells, Abs, and reagents

HUVEC (3) were isolated using a method modified from Jaffe et al. (16), and were cultured on gelatin-coated cell culture flasks in complete medium, as described earlier (7). Human PBL from healthy volunteers were isolated using Ficoll-Hypaque (Histopague-1077; Pharmacia, Uppsala, Sweden). PBL and U266B1 cell line, a gift from J.-Y. Bonnefoy (Glaxo Institute for Molecular Biology, Geneva, Switzerland), was cultured in RPMI 1640 medium containing 10% FCS, 4 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Anti-CD73 mAb 4G4 (mouse IgG1), anti-ICAM-1 mAb 5C3 (IgG1) (17), anti-CD31 mAb 2C8 (IgG1), and mAb 3G6 (mouse IgG1) against chicken T cells as a negative control Ab were used. , Methyleneadenosine 5'-diphosphate (AMPCP)³ and AMP were from Sigma-Aldrich (St. Louis, MO).

Inductions and immunofluorescence stainings

IL-1, IL-4, TNF-, and IFN- (Genzyme, Cambridge, MA); IFN- (Wellferon; The Wellcome Foundation, London, U.K.); LPS from Escherichia coli serotype O:55 (Difco Laboratories, Detroit, MI); and dibutyryl cAMP, PMA, thrombin from human plasma, histamine, and FMLP from Sigma-Aldrich were used for inductions, as indicated in Table I. For every time point, a control flask was incubated without inducers.

1) To study the effect of a panel of different inducers on surface expression of CD73 immunofluorescence analyses were performed, as reported before (17). In brief, HUVEC were treated with or without inducers and detached with 5 mM of EDTA-trypsin. A total of 5 x 10⁵ cells for each staining was incubated with saturating concentrations of mAb 3G6 negative control (neg co), 4G4 (anti-CD73), and 5C3 (anti-ICAM-1) as hybridoma supernatants or purified Ab (final concentration, 10 µg/ml) for 20 min at 4°C and washed twice. Then the cells were incubated for 20 min at 4°C with 1/100 diluted FITC-conjugated sheep anti-mouse-IgG mAb (Sigma-Aldrich) containing 5% AB serum. Finally, the cells were washed twice and fixed with 1% paraformaldehyde. All incubations and washes were performed with PBS containing 2% FCS and 1 mM of NaN₃. Fluorescence was then detected using FACS (BD Biosciences, San Jose, CA). The difference between control and treated cells was calculated from: fold difference = treated cells (MFI, anti-CD73 - MFI, neg co)/nontreated cells (MFI, anti-CD73 - MFI, neg co).

2) For the detection of intracellular CD73, lymphocytes were permeabilized before immunofluorescence stainings by incubating them for 2 min in acetone at -20°C. Then the cells were washed with RPMI 1640 medium containing 5% FCS and stained and analyzed by FACS, as described above.

3) To study the distribution of CD73 on HUVEC, cells were seeded on 10-mm-diameter gelatin-coated coverslips and cultured in the presence or absence of IFN- (100 U/ml) for 72 h. The cells were washed twice with PBS and then incubated for 30 min on ice with saturating concentration of mAb 4G4 (anti-CD73). After rinsing three times with PBS, coverslips were incubated further with FITC-conjugated sheep anti-mouse IgG mAb diluted 1/100 for 20 min on ice, washed three times in PBS, and mounted on glass slides in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The cells were examined on a Zeiss (Oberkochen, Germany) LSM 510 laser-scanning confocal microscope.

CD73 RNA analysis

For RNA isolation, 2 x 10⁵ HUVEC were seeded onto six-well plates. Typically, RNA was isolated after 4–5 days postseeding. IFN- (100 U/ml) was added to wells 72, 24, or 12 h before RNA isolation. Total RNA was isolated using RNase Easy Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. Before real-time RT-PCR measurement, 1–2 µg of total RNA was treated with DNase I, and cDNA was made by using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD), according to the manufacturer’s instructions. Finally, cDNA was treated with RNase H (Life Technologies). CD73 primers 5'-CTG GGA GCT TAC GAT TTT GCA-3' and 5'-CCT CGC TGG TCT GCT CCA-3' and probe 5'-CCA ACG ACG TGC ACA GCC GG-3' were designed using Primer Express computer software (PE Biosystems, Foster City, CA). Primers and probe for GAPDH housekeeping gene were used as internal controls. The real-time RT-PCR measurements were performed using TaqMan Universal PCR Master Mix and ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA). The expression of the housekeeping gene GAPDH was used as a reference for normalization, and the relative increase of CD73 mRNA expression between control and IFN--treated cells was calculated.

Ecto-5'-nucleotidase assay

Ecto-5'-nucleotidase activity was assayed by TLC, as described previously (18). Briefly, the standard enzyme assay contained in a final volume of 120 µl of RPMI 1640, 4–6 x 10⁴ detached HUVEC (or 1 x 10⁵ lymphoid cells), 5 mmol/L -glycerophosphate, and the indicated concentrations of AMP with tracer [2-³H]AMP (sp. act., 18.6 Ci/mmol; Amersham, Little Chalfont, U.K.). Incubation times were chosen to ensure the linearity of the reaction with time, so that the amount of the converted AMP did not exceed 7–10% of the initially introduced substrate. Aliquots of the mixture were applied to Alugram SIL G/UV₂₅₄ TLC sheets (Macherey-Nagel, Düren, Germany) and separated with isobutanol/isoamyl alcohol/2-ethoxyethanol/ammonia/H₂0 (9:6:18:9:15) as solvent. ³H-labeled AMP and its dephosphorylated nucleoside derivatives were visualized in UV light and quantified using a Wallac-1409 -spectrometer.

Permeability assay

To evaluate barrier function of confluent monolayers, HUVEC were seeded (50,000 cells/insert) on Transwell insert polycarbonate filters (6.5 mm in diameter, 0.4 in µm pore size; Costar, Cambridge, MA). The filters were treated for 1–2 h with fibronectin and air dried before seeding endothelial cells. Typically, monolayers were studied 4–5 days postseeding. HUVEC were either induced with IFN- (100 U/ml) for 72 h before the studies of monolayer permeability or grown in medium without IFN-. Transport across endothelial monolayers was assessed by measuring the flux of FITC-labeled dextran (500 µg/ml, M_r 70,000). Endothelial monolayers were pretreated with AMP (50 µM) for 15 min before the FITC-dextran transport was initiated. To evaluate the role of CD73 enzymatic activity on the endothelial cell permeability, the flux of FITC-dextran was measured in the presence or absence of a specific inhibitor of ecto-5'-nucleotidase, AMPCP (100 µM). In certain experiments, AMPCP was added to the upper and lower chambers 30 min before the transport was initiated by adding FITC-labeled dextran. The inserts were removed from the bottom chamber (Visiplate; PerkinElmer Life Sciences, Turku, Finland) at the time points 10, 20, 30, 40, and 100 min, and FITC-labeled dextran was measured directly from the bottom chambers in a fluorometer (TECAN Ultra fluorescence reader; Tecan Group, Maennedorf, Switzerland) using 485 and 535 nm as the excitation and emission wavelengths, respectively.

Patient characteristics

Twelve patients having superficial epithelial bladder cancer were evaluated for operation from 1 to 3 wk before the actual operation. In connection to the evaluation visit, biopsies were taken from normal area of the bladder and from the tumor. Fifty million units of IFN-2b (IntronA; Schering-Plough, Kenilworth, NJ) were instilled to the bladder 1 day before the operation. The cystectomy was performed and the patients underwent conventional ureteroenterocutaneostomy, enterocystoplasty, or ureteroenteoumbilicostomy as the reconstructive operation. Three patients did not receive IFN- before operation. Two of them received 100 mg epirubicin (a standard treatment in the Turku University Hospital; Pharmorubicin; Pharmacia) instilled to the bladder 1 day before the operation, and one did not receive anything. Their tumors were analyzed before (biopsy) and after the operation and used as controls. All patients were Caucasian males. Patient characteristics appear in Table II. The study protocol was approved by the Ethical Board of the Turku University Hospital, and informed consent was obtained from each patient.

View this table: [in this window] [in a new window]   Table II. Patient characteristics of IFN--treated patients

The bladder sample specimens were snap frozen in liquid nitrogen and cut into 5-µm sections. Subsequently, serial sections were stained with anti-CD73 mAb 4G4, anti-CD31 mAb 2C8, or 3G6 (negative control) as primary Abs, and peroxidase-conjugated rabbit anti-mouse IgG (DAKO A/S, Glostrup, Denmark) was used as a second-stage Ab. The reaction was developed by adding 3,3'-diaminobenzidine tetrahydrochloride (Polysciences, Warrington, PA) in PBS. All incubations were 20 min, with saturating mAb concentrations, followed by two washes with PBS. The number of positive vessels/microscopic field (x200) was counted, and intensity of the staining was semiquantitatively evaluated. A combined score from 0 to 3 was given to each sample. Score 0 was assigned to samples with no positive blood vessels, and score 3 to samples with staining equal to inflamed tonsil. Scores 1 and 2 were adjusted to cover the staining patterns in between. All samples were read blindly by two independent readers, and the mean of their scores is presented for each sample.

Statistical analysis

Data are presented as mean ± SEM of individual experiments. Statistical comparisons were made using Student's t test, and p values <0.05 were taken as significant. Data from kinetic experiments were subjected to computer analyses using the Michaelis-Menten equation to determine the K_m and V_max values (GraphPad Prism version 3.0, San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD73 expression is up-regulated on endothelial cells with IFN-

This work was designed to find potent regulators of CD73 expression or CD73-based ecto-5'-nucleotidase enzyme activity after cells were exposed to a wide range of well-known inducers. First inductions were done for 4 min or 2 h for rapidly acting inducers (i.e., inducers known to release preformed molecules from intracellular storage granules) PMA, FMLP, dibutyryl cAMP, thrombin, and histamine and for 4 or 20–24 h for slowly acting inducers (i.e., inducers leading to de novo synthesis of new molecules) IL-1, LPS, IL-4, TNF-, IFN-, and IFN- (see Table I). The only inducers leading to a marked change in the CD73 expression on HUVEC were IFN- and IFN-, with doses of >200 U/ml after induction for 20–24 h. As IFN- is rather widely used in the clinical medicine, its effects were evaluated in more detail.

IFN- increases endothelial CD73 expression in a time- and dose-dependent manner

Practically all nonactivated HUVEC bear CD73 on their surface when analyzed by FACS (7). Therefore, to measure the increase in expression of CD73 molecules on cell surface, we analyzed the MFI of HUVEC, as described in Materials and Methods. To further study the kinetics of IFN- up-regulation, confluent monolayers of HUVEC were incubated using different doses of IFN- for the indicated periods of time. CD73 expression was increased time dependently almost 2-fold (92.4 ± 11.5%; n = 9) after 72 h with 1000 U/ml IFN- (Fig. 1a). Longer exposure of the HUVEC to IFN- did not cause further significant increase in CD73 expression (data not shown). A similar pattern of CD73 up-regulation was seen after induction with IFN- (data not shown). Up-regulation of CD73 expression was also dose dependent, as in concentrations ranging from 10 to 1000 U/ml the highest increase in intensity was observed at 1000 U/ml (Fig. 1b). Comparable increase subsequent to IFN- treatment took place in HUVEC grown in Transwell chambers (data not shown). Immunofluorescence stainings followed by confocal laser microscopy revealed that IFN- treatment does not induce any significant changes in the distribution or polarization of CD73 on HUVEC surface. Instead, CD73 is more intensely, but similarly distributed on the cell surface (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Induction of CD73 surface expression on HUVEC by IFN- is both time and dose dependent. a, HUVEC were exposed to 1000 U/ml IFN- for indicated time periods. b, HUVEC were cultured with different concentrations of IFN- for 72 h. Relative means of MFI ± SEM of three to nine experiments are shown. Control expression is the expression of CD73 without IFN- at each time point. Background (the negative control staining) is subtracted. *, p < 0.05; **, p < 0.01.

View larger version (65K): [in this window] [in a new window]   FIGURE 2. Induction of CD73 with IFN- leads to increased expression rather than changes in its distribution. HUVEC were either grown in medium or induced with IFN- for 72 h, and the cell surface expression of CD73 was detected with mAb 4G4 against CD73 and FITC-conjugated anti-mouse IgG Ab. The characteristic distribution of CD73 on control (a and b) and on IFN--treated (c and d) endothelial cells. On control HUVEC CD73 is expressed on the cell surface in a punctate-like pattern. After IFN- induction, expression of CD73 is more intense, but surface distribution is similar as on control HUVEC. Images (a and c) are projections generated from confocal serial sections of fluorescently labeled cells, and images (b and d) are confocal XZ profiles (Z-scans). Bar = 10 µm.

Induction of CD73 RNA expression by IFN-

Next, we wanted to determine whether increase in surface CD73 expression is mediated by increase in CD73 RNA expression. Quantitative real-time PCR revealed prominent induction of CD73 mRNA expression after 12- and 24-h exposure to IFN- (100 U/ml) (2.0 ± 0.5-fold increase and 2.1 ± 0.2-fold increase, respectively, compared with untreated cells after normalization to GAPDH, p < 0.05). Treatment of HUVEC with IFN- for 72 h resulted in maximal increase of CD73 mRNA (2.5 ± 1.1-fold increase, p < 0.05) (Fig. 3).

View larger version (8K): [in this window] [in a new window]   FIGURE 3. IFN- increases the relative CD73 mRNA expression in HUVEC. HUVEC were incubated with IFN- (100 U/ml) for 12, 24, and 72 h or left untreated (0 h). Total RNA was isolated, and real-time PCR was used to determine CD73 mRNA levels. Data were calculated relative to internal control gene (GAPDH) and are expressed as fold increase over untreated cells (0 h) ± SEM at each indicated time. The data present the means of three experiments; *, p < 0.05.

Despite the structural similarity of endothelial and lymphoid CD73, IFN- promotes different effects on these two cell types

After finding out that CD73 expression on endothelial cells is IFN- inducible, we wanted to determine whether CD73 on lymphocytes would also behave similarly in the same conditions. A total of 1000 U/ml IFN- did not increase CD73 expression on PBL significantly (data not shown). Even with longer induction time up to 48 h, we observed only minor changes in CD73 expression on lymphocyte surface. To exclude the possibility that freshly isolated lymphocytes do not survive well in culture conditions and, therefore, fail to up-regulate CD73, we also treated CD73-expressing lymphoid cell line U266B1 with IFN-. They also were incapable of up-regulating their CD73 expression even after 48 or 72 h of induction. Instead, there was a decrease at 48-h time point after IFN- treatment as compared with control cells (relative MFI 82.1 ± 5.6% vs 100%; n = 3).

To elucidate whether there is also up-regulation in intracellular CD73 protein level, PBL and HUVEC were permeabilized with acetone before immunofluorescence staining after IFN- induction. No up-regulation of intracellular expression of CD73 could be observed in PBL. Similar results were obtained when analyses were done with FACS and fluorescence microscopy. In HUVEC, a slightly increased intracellular staining with anti-CD73 mAb was seen after IFN- induction (data not shown).

CD73 is up-regulated in vessels of healthy and tumor areas of bladder after treatment with IFN- in vivo

To investigate whether IFN- would also regulate the expression of CD73 in vivo, tissue specimens from superficial epithelial bladder cancers were collected before and after IFN-2b treatment, stained, and analyzed (Table II). In two tumors, the malignant cells were CD73 positive, reflecting the fact that also some epithelial cells are CD73 positive. IFN- produced a clear up-regulation of CD73 in vascular endothelium both in normal and cancer tissue in vivo when compared with expression levels before and after treatment in specimens of the control patients treated with epirubicin (Fig. 4). However, no CD73 up-regulation was detected among the few normal lymphocytes present within the tumors. Similarly, the expression level of tumor cells remained constant during IFN-2b treatment in those tumors that were positive for CD73 (Fig. 5). Three patients, who did not receive IFN- and were used to control the possible up-regulation of CD73 caused by the biopsy and operation itself, did not show any significant increase in their CD73 expression (one patient did not show any change and two patients had an increase of 0.5 in endothelial CD73 expression within the tumor tissue). Thus, the mean change of the control patients was 0.3, and that of treated patients 1.3 (p = 0.02).

View larger version (30K): [in this window] [in a new window]   FIGURE 4. IFN- induces CD73 expression in vivo. A summary of semiquantitative analysis of immunohistochemical stainings of urine bladder samples from healthy (a) and tumor (b) areas before and after IFN- treatment is presented.

View larger version (115K): [in this window] [in a new window]   FIGURE 5. IFN- induces expression of endothelial CD73 also in vivo in patients suffering from bladder carcinoma. a, A specimen stained with anti-CD73 mAb before IFN- treatment. The vessels are practically negative for CD73 (score 0). b, A serial section of the same sample stained with anti-CD31 mAb demonstrating the vessels. c and d, Serial sections from a sample of the same patient taken after IFN- treatment stained with anti-CD73 mAb (c) or with anti-CD31 mAb (d). CD31-positive vessels express CD73 abundantly (score 3). e–h, An example of a tumor (t) expressing CD73 before (e) and after (g) IFN- treatment. Also in this case, IFN- increased CD73 expression on endothelial cells (e, score 1 and g, score 2). f and h, Anti-CD31 stainings. a--d and g--h, The arrows point to individual vessels. e and f, The arrows point toward a neovascular network of vessels. Insets in d and h show stainings with a negative control Ab 3G6. Scale bar 100 µm in all figures.

IFN- increases ecto-5'-nucleotidase activity on endothelial cells

To determine whether IFN--induced increase of CD73 expression on HUVEC is accompanied by concomitant induction in ecto-5'-nucleotidase activity, we applied a radiochemical assay for direct measurement of [³H]AMP conversion into [³H]adenosine. Pretreatment of HUVEC monolayers with IFN- (1000 U/ml for 48 h) caused significant increase in the rate of [³H]AMP hydrolysis (Fig. 6a), whereas no significant activation of the enzyme activity was detected after PBL treatment with IFN- (Fig. 6b). To further elucidate the mechanism of ecto-5'-nucleotidase activation, kinetic analysis of [³H]AMP hydrolysis by control and IFN--treated HUVEC was conducted, and these saturating curves can be seen in Fig. 6c. Statistical analysis revealed that IFN- significantly increased the maximum hydrolysis rate (V_max) of 5'-nucleotidase as compared with nontreated cells (525 ± 30 vs 350 ± 29 nmol/10⁶ cells/h) without any modification of the enzyme affinity (K_m 50–60 µmol/L). These data suggest that IFN- increases the number of enzymatically active 5'-nucleotidase molecules on the endothelial surface rather than induces conformational changes of the enzyme catalytic site. Interestingly, use of the same approach with other nucleotide [³H]ATP did not reveal significant changes of ATP-hydrolyzing activities after HUVEC treatment with IFN- (data not shown), confirming the specificity of ecto-5'-nucleotidase induction. To ensure that CD73 is not continuously secreted from lymphocytes into cell culture supernatant producing increased enzyme activity, we analyzed [³H]AMP conversion into [³H]adenosine in cell culture medium from IFN--induced lymphocytes and nontreated control cells. No significant change in enzymatic activity of cell culture medium was found between control and IFN- treatment (data not shown).

View larger version (17K): [in this window] [in a new window]   FIGURE 6. IFN- increases cell surface ecto-5'-nucleotidase activity. HUVEC (a) and PBL (b) were pretreated for 48 h without () or with 1000 U/ml IFN- (). Ecto-5'-nucleotidase activity was assayed by using 300 µmol/L [3H]AMP and expressed on ordinate as nmoles of substrate dephosphorylated by 106 cells per hour (mean ± SEM; n = 4–5). *, p < 0.05 as compared with control cells. c, Rate of [3H]AMP hydrolysis by control (•) and IFN--treated () HUVEC vs substrate concentration plot. Values are expressed as mean ± SEM for two independent experiments. The kinetic parameters (Vmax and Km) were calculated from the presented curves and summarized in the text.

IFN- increases HUVEC membrane function

To study whether IFN--up-regulated CD73 expression and CD73-mediated increase in adenosine production are able to regulate HUVEC membrane function, we measured the flux of FITC-labeled dextran through confluent endothelial monolayers growing on permeable insert wells. At all time points examined, there was a significant difference (p < 0.05) in the permeabilities of HUVEC treated with IFN- (100 U/ml) for 3 days compared with untreated HUVEC, as indicated by decreased flux of FITC-dextran (Fig. 7a). Pretreatment of HUVEC monolayers with a specific CD73 enzyme inhibitor, AMPCP, reversed the permeability decrease associated with IFN- treatment as demonstrated by increased flux of FITC-dextran (Fig. 7b).

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Effect of IFN- on permeability of HUVEC monolayers. a, HUVEC were plated on porous polystyrene membrane (pore size of 0.4 µm) and grown to confluency. HUVEC were grown in medium or treated with 100 U/ml IFN- for 72 h. Fifteen minutes after adding AMP, the membrane function was analyzed by measuring the flux of 70-kDa FITC-dextran through HUVEC monolayer to the lower chamber with a fluorometer. The FITC-dextran flux was measured up to 100 min. Values are means ± SEM, n = 3. *, p < 0.05 as compared with IFN--treated cells. b, Confluent monolayers were exposed to AMPCP (100 µM), a specific ecto-5'-nucleotidase inhibitor, 30 min before addition of FITC-dextran. Data shown are mean values ± SEM, n = 3. *, p < 0.05 as compared with IFN--treated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

During normal cell maturation, CD73 expression and its enzymatic activity increase (22, 23). One of the known activators is the end product of the enzymatic activity of CD73 itself: adenosine increases CD73 expression and enzymatic activity in a paracrine fashion (24). The human CD73 promoter sequence contains possible binding sites for many transcription factors such as Sp1, Sp1/AP-2, cAMP response element, and Sp1/NF-AT (25), and among them is agtttcgtatcac sequence, which rather closely resembles IFN--stimulated response elements in certain known genes. Although this sequence does not fully match the consensus site (agtttcnntttcnc) of those known genes (26), it remained still possible that this sequence in CD73 gene acts as an IFN- response element. Therefore, we tested whether nuclear proteins isolated from HUVEC can bind to this sequence by using EMSA (data not shown). Based on these results, IFN- seems not to use this element, but instead exerts its effect indirectly inducing other mediators, which then cause up-regulation of CD73.

None of the rapidly acting factors tested in this work was able to up-regulate CD73. This is interesting in light that expression of lymphocyte CD73 can be increased by triggering of CD38 in 20 min. In this case, the additional CD73 is translocated to the cell surface from a pre-existing intracellular pool (27). Based on the fact that a relatively long time was needed for IFN- to up-regulate CD73 in vitro, we may argue that its up-regulation is not an early event at sites of inflammation in vivo either. It may further indicate that up-regulation of CD73 and increased production of adenosine are the body’s own defense mechanisms to decrease and limit inflammation. This is well in line with the findings that adenosine prevents cell damage during heart and central nervous ischemia (9, 28, 29). After hypoxia, ecto-5'-nucleotidase activity increases due to phenomenon known as preconditioning (30, 31, 32). This results in release of large amounts of adenosine, leading to increased resistance of cells to infarction, for example, in cardiac hypoxia.

The prerequisite for adenosine formation is availability of AMP. AMP is an important intermediate metabolite of ecto-enzymatic ATP metabolism that can be either irreversibly hydrolyzed into adenosine by ecto-5'-nucleotidase or regenerated into ATP via ecto-nucleotidase kinase reactions. Concentration of AMP is not a limiting factor, because CD73 functions as a master switch, determining the shift from the ATP-consuming/adenosine-producing pathway to the ATP-generating pathway, and whenever more AMP is needed for adenosine production less ATP is generated (18, 33). Moreover, pathological conditions such as hypoxia and tissue injury trigger release of high amounts of AMP (34, 35).

IFNs are very potent immunomodulatory substances. They produce antiproliferative effects, induce antiviral resistance, and regulate immune responses (36). Levels of IFN- in healthy individuals are usually low, but in inflammatory conditions IFN- is secreted already at early stages of inflammation from APCs. The lower concentrations used in our experiments that cause up-regulation of CD73 are comparable to those found endogeneously in patients suffering from inflammations (21, 37), and thus expected to up-regulate CD73 also in vivo. Interestingly, IFN- seems to maintain the integrity of vascular wall, because it has been shown to enhance endothelial barrier function of bovine retinal microvascular endothelial cells (38). The mechanisms were not elucidated in this earlier work by Gillies and Su, but our present results strongly suggest that up-regulation of CD73 contributes to this phenomenon via increased adenosine production.

IFN- has also been used to treat various cancers. In this work, we found that bladder carcinoma patients treated with IFN- up-regulate their CD73 expression specifically on endothelial cells. This type of up-regulation was not detectable in normal lymphocytes always present in variable numbers within the tumor tissues or in malignant cells of two tumors positive for CD73. CD73 positivity is thought to provide survival advantage as a salvage pathway for CD73-positive tumor cells when the patients are treated by antimetabolites that block the de novo synthesis of purines (39, 40). In contrast, in the hypoxic conditions often present in tumors, increased adenosine production might give the normal cells, especially endothelial cells and CD73-positive tumor-infiltrating lymphocytes, an advantage to survive better than CD73-negative cancer cells, which have a higher proliferation rate and thus are more vulnerable to cytotoxic drugs. Trophic actions of adenosine on endothelial cells may be advantageous to cancer growth as such, because angiogenesis is a prerequisite for tumor growth (41).

Our present results of the distinct effects of IFN- on endothelial and lymphocyte CD73 further demonstrate the difference between the cell types to regulate the expression of CD73. This difference is not explained by lack of IFN- receptors on lymphocytes, because both B and T cells express high affinity receptor for IFN- (42). In our earlier work, we discovered that triggering of CD73 with anti-CD73 mAb (that mimics the ligand binding) results in shedding of lymphocyte, but not endothelial cell CD73, although the cDNA sequences of CD73 in these cell types are practically identical (7, 8). In this context, it is worth mentioning that the amount of CD73 varies markedly between lymphocytes and endothelial cells. Only 10–15% of lymphocytes express CD73, and the expression level is low in comparison with, for example, HUVEC, which all are positive for CD73. This type of cell-specific differences in amount and regulation of CD73 may be fundamental for appropriate behavior of lymphocytes, the role of which is to actively deaminate the existing adenosine and extravasate to lymphoid tissues or to sites of inflammation. In contrast, adenosine is necessary for endothelial cells to maintain their barrier function.

In conclusion, we have demonstrated in this work how CD73 can be up-regulated both in vitro and in vivo by IFN-. As adenosine is highly anti-inflammatory in its nature, manipulation of its endogenous production via up-regulation of CD73 may be a potential way to treat harmful inflammatory conditions such as reperfusion injuries in connection to myocardial infarction and stroke.

	Acknowledgments

 
We thank Maritta Pohjansalo, Laila Reunanen, Sari Mäki, and Pirjo Heinilä for excellent technical assistance; Anne Sovikoski-Georgieva for secretarial help; and Erkki Nieminen for help with the figures.

	Footnotes

 
¹ This work was supported by grants from the Finnish Academy, the Sigrid Juselius Foundation, the Finnish Cancer Union, the Ida Montin Foundation, the Paulo Foundation, the Research and Science Foundation of Farmos, the Finnish Cultural Foundation, and the Turku Graduate School of Biomedical Sciences.

² Address correspondence and reprint requests to Dr. Sirpa Jalkanen, MediCity Research Laboratory, Turku University, Tykistökatu 6A, FIN-20520 Turku, Finland. E-mail address: sirpa.jalkanen{at}utu.fi

³ Abbreviations used in this paper: AMPCP, , methyleneadenosine 5'-diphosphate; MFI, mean fluorescence intensity; neg co, negative control.

Received for publication May 28, 2003. Accepted for publication November 20, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Butcher, E. C.. 1991. Leukocyte-Endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67:1033.[Medline]
2. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
3. Carlos, T. M., J. M. Harlan. 1994. Leukocyte-Endothelial adhesion molecules. Blood 84:2068.[Abstract/Free Full Text]
4. Thompson, L. F., J. M. Ruedi, A. Glass, G. Moldenhauer, P. Moller, M. G. Low, M. R. Klemens, M. Massaia, A. H. Lucas. 1990. Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (CD73). Tissue Antigens 35:9.[Medline]
5. Zimmermann, H.. 1992. 5'-Nucleotidase: molecular structure and functional aspects. Biochem. J. 285:345.
6. Arvilommi, A.-M., M. Salmi, L. Airas, K. Kalimo, S. Jalkanen. 1997. CD73 mediates lymphocyte binding to vascular endothelium in inflamed human skin. Eur. J. Immunol. 27:248.[Medline]
7. Airas, L., J. Niemelä, M. Salmi, T. Puurunen, D. J. Smith, S. Jalkanen. 1997. Differential regulation and function of CD73, a glycosyl-phosphatidylinositol-linked 70-kD adhesion molecule, on lymphocytes and endothelial cells. J. Cell Biol. 136:421.[Abstract/Free Full Text]
8. Airas, L., J. Niemelä, S. Jalkanen. 2000. CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism. J. Immunol. 165:5411.[Abstract/Free Full Text]
9. Olah, M. E., G. L. Stiles. 1995. Adenosine receptor subtypes: characterization and therapeutic regulation. Annu. Rev. Pharmacol. Toxicol. 35:581.[Medline]
10. Morabito, L., M. C. Montesinos, D. M. Schreibman, L. Balter, L. F. Thompson, R. Resta, G. Carlin, M. A. Huie, B. N. Cronstein. 1998. Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5'-nucleotidase-mediated conversion of adenine nucleotides. J. Clin. Invest. 101:295.[Medline]
11. Cronstein, B. N., R. I. Levin, M. Philips, R. Hirschhorn, S. B. Abramson, G. Weissmann. 1992. Neutrophil adherence to endothelium is enhanced via adenosine A₁ receptors and inhibited via adenosine A₂ receptors. J. Immunol. 148:2201.[Abstract]
12. Bouma, M. G., T. M. M. A. Jeunhomme, D. L. Boyle, M. A. Dentener, N. N. Voitenok, F. A. J. M. van den Wildenberg, W. A. Buurman. 1997. Adenosine inhibits neutrophil degranulation in activated human whole blood: involvement of adenosine A2 and A3 receptors. J. Immunol. 158:5400.[Abstract]
13. Knight, D., X. Zheng, C. Rocchini, M. Jacobson, T. Bai, B. Walker. 1997. Adenosine A3 receptor stimulation inhibits migration of human eosinophils. J. Leukocyte Biol. 62:465.[Abstract]
14. Lennon, P. F., C. T. Taylor, G. L. Stahl, S. P. Colgan. 1998. Neutrophil-derived 5'-adenosine monophosphate promotes endothelial barrier function via CD73-mediated conversion to adenosine and endothelial A_2B receptor activation. J. Exp. Med. 188:1433.[Abstract/Free Full Text]
15. Ohta, A., M. Sitkovsky. 2001. Role of G-protein-coupled adenosine receptors in down-regulation of inflammation and protection from tissue damage. Nature 414:916.[Medline]
16. Jaffe, E. A., R. L. Nachman, C. G. Becker, C. R. Minick. 1973. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J. Clin. Invest. 52:2745.
17. Airas, L., M. Salmi, S. Jalkanen. 1993. Lymphocyte-Vascular adhesion protein-2 is a novel 70-kDa molecule involved in lymphocyte adhesion to vascular endothelium. J. Immunol. 151:4228.[Abstract]
18. Yegutkin, G. G., T. Henttinen, S. Jalkanen. 2001. Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions. FASEB J. 15:251.[Abstract/Free Full Text]
19. Constantin, G., M. Majeed, C. Giagulli, L. Piccio, J. Y. Kim, E. C. Butcher, C. Laudanna. 2000. Chemokines trigger immediate ₂ integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13:759.[Medline]
20. Butcher, E. G., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
21. Pirisi, M., C. Fabris, P. Toniutto, E. Falleti, S. Tisminetzky, M. Gerotto, G. Soardo, D. Vitulli, M. Del Forno, F. Baralle, E. Bartoli. 1997. Endogenous interferon- concentration and outcome of interferon treatment in patients with chronic hepatitis C. Dig. Dis. Sci. 42:767.[Medline]
22. Edwards, N. L., E. W. Gelfand, L. Burk, H. M. Dosch, I. H. Fox. 1979. Distribution of 5'-nucleotidase in human lymphoid tissues. Proc. Natl. Acad. Sci. USA 76:3474.[Abstract/Free Full Text]
23. Resta, R., S. W. Hooker, K. R. Hansen, A. B. Laurent, J. L. Park, M. R. Blackburn, T. B. Knudsen, L. F. Thompson. 1993. Murine ecto-5'-nucleotidase (CD73): cDNA cloning and tissue distribution. Gene 133:171.[Medline]
24. Narravula, S., P. F. Lennon, B. U. Mueller, S. P. Colgan. 2000. Regulation of endothelial CD73 by adenosine: paracrine pathway for enhanced endothelial barrier function. J. Immunol. 165:5262.[Abstract/Free Full Text]
25. Spychala, J., A. G. Zimmermann, B. S. Mitchell. 1999. Tissue-specific regulation of the ecto-5'-nucleotidase promoter: role of the cAMP response element site in mediating repression by the upstream regulatory region. J. Biol. Chem. 274:22705.[Abstract/Free Full Text]
26. Darnell, J. E., Jr, I. M. Kerr, G. R. Stark. 1994. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415.[Abstract/Free Full Text]
27. Peola, S., P. Borrione, L. Matera, F. Malavasi, A. Pileri, M. Massaia. 1996. Selective induction of CD73 expression in human lymphocytes by CD38 ligation: a novel pathway linking signal transducers with ecto-enzyme activities. J. Immunol. 157:4354.[Abstract]
28. Heurteaux, C., I. Lauritzen, C. Widmann, M. Lazdunski. 1995. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K⁺ channels in cerebral ischemic preconditioning. Proc. Natl. Acad. Sci. USA 92:4666.[Abstract/Free Full Text]
29. Linden, J.. 2001. Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection. Annu. Rev. Pharmacol. Toxicol. 41:775.[Medline]
30. Kitakaze, M., M. Hori, T. Morioka, T. Minamino, S. Takashima, Y. Okazaki, K. Node, K. Komamura, K. Iwakura, T. Itoh, et al 1995. 1-Adrenoceptor activation increases ecto-5'-nucleotidase activity and adenosine release in rat cardiomyocytes by activating protein kinase C. Circulation 91:2226.[Abstract/Free Full Text]
31. Kitakaze, M., K. Node, T. Minamino, K. Komamura, H. Funaya, Y. Shinozaki, M. Chujo, H. Mori, M. Inoue, M. Hori, T. Kamada. 1996. Role of activation of protein kinase C in the infarct size-limiting effect of ischemic preconditioning through activation of ecto-5'-nucleotidase. Circulation 93:781.[Abstract/Free Full Text]
32. Minamino, T., M. Kitakaze, T. Morioka, K. Node, K. Komamura, H. Takeda, M. Inoue, M. Hori, T. Kamada. 1996. Cardioprotection due to preconditioning correlates with increased ecto-5'-nucleotidase activity. Am. J. Physiol. 270:H238.
33. Henttinen, T., S. Jalkanen, G. G. Yegutkin. 2003. Adherent leukocytes prevent adenosine formation and impair endothelial barrier function by ecto-5'-nucleotidase/CD73-dependent mechanism. J. Biol. Chem. 278:24888.[Abstract/Free Full Text]
34. Bhatnagar, A.. 2003. Surviving hypoxia: the importance of rafts, anchors, and fluidity. Circ. Res. 92:821.[Free Full Text]
35. Madara, J. L., T. W. Patapoff, B. Gillece-Castro, S. P. Colgan, C. A. Parkos, C. Delp, R. J. Mrsny. 1993. 5'-Adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers. J. Clin. Invest. 91:2320.
36. Schindler, C. W.. 2002. Series introduction: JAK-STAT signaling in human disease. J. Clin. Invest. 109:1133.[Medline]
37. Ronnblom, L., G. V. Alm. 2001. A pivotal role for the natural interferon -producing cells (plasmacytoid dendritic cells) in the pathogenesis of lupus. J. Exp. Med. 194:F59.
38. Gillies, M. C., T. Su. 1995. Interferon-2b enhances barrier function of bovine retinal microvascular endothelium in vitro. Microvasc. Res. 49:277.[Medline]
39. Ujházy, P., M. Klobusická, O. Babusíková, P. Strausbauch, E. Mihich, M. J. Ehrke. 1994. Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin. Int. J. Cancer 59:83.[Medline]
40. Pieters, R., A. J. P. Veerman. 1988. The role of 5'nucleotidase in therapy-resistance of childhood leukemia. Med. Hypotheses 27:77.[Medline]
41. Burnstock, G.. 2002. Purinergic signaling and vascular cell proliferation and death. Arterioscler. Thromb. Vasc. Biol. 22:364.[Abstract/Free Full Text]
42. Paul, W. E.. 1993. Fundamental Immunology, Chapter 21 815. Raven, New York.

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