TGF-Β1 Prevents Up-Regulation of the P2X7 Receptor by IFN-Γ and LPS in Leukemic THP-1 Monocytes

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Biochimica et Biophysica Acta 1798 (2010) 2058–2066

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TGF-β1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes

Saﬁna Gadeock a,1, Jimmy N.S.N. Tran b,c,1, Jennifer G. Georgiou b,1, Iman Jalilian a, Rosanne M. Taylor c, James S. Wiley b,d, Ronald Sluyter a,b,⁎

a School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia b Department of Medicine, Nepean Clinical School, University of Sydney, Penrith, New South Wales, Australia c Faculty of Veterinary Science, University of Sydney, Sydney, New South Wales, Australia d Florey Neuroscience Institutes, University of Melbourne, Melbourne, Victoria, Australia

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Article history: The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and Received 18 March 2010 can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation Received in revised form 20 July 2010 of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell- Accepted 21 July 2010 surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and Available online 27 July 2010 mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP- induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for Keywords: γ + Purinergic receptor 3 days with IFN- and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium uptake into P2X receptor differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced Transforming growth factor ethidium+ uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of Monocyte P2X7 expression and ATP-induced ethidium+ uptake in a concentration-dependent fashion with a maximum Macrophage 2+ effect at 5 ng/ml and with an IC50 of ~0.4 ng/ml. Moreover, ATP-induced YO-PRO-1 uptake and IL-1β fl In ammation release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up- regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity. © 2010 Elsevier B.V. All rights reserved.

1. Introduction maturation and release of interleukin (IL)-1β and IL-18 [5]. Predomi- nately due to this property, P2X7 is considered an important sentinel of The P2X7 receptor is a trimeric ATP-gated cation channel predom- immunological danger and infection [6,7], and a potential target of next inately expressed on lymphoid and myeloid leukocytes [1,2]. Activation generation adjuvants [8]. P2X7 function varies between different of this receptor causes the flux of Ca2+,Na+ and K+,aswellasthe human individuals, which in part, can be explained by numerous uptake of large organic cations and anions. The permeability pathways polymorphisms that alter receptor expression and/or function [9,10]. for these large molecules are unknown, but it appears that more than Relative P2X7 expression and function also differs between various one pathway may exist and that these pathways vary between cell types leukocyte subtypes [11,12], and is up-regulated upon differentiation of [3,4]. P2X7 activation can induce a variety of downstream events monocytes to either macrophages [13] or dendritic cells [14].Moreover, including activation of the NALP3 inflammasome, and the subsequent pro-inflammatory molecules such as interferon-γ,(IFN-γ), tumour necrosis factor-α and lipopolysaccharide (LPS) can up-regulate P2X7 expression and function in monocytes/macrophages [15–17].The effects of pro-inflammatory molecules on P2X7 are not restricted to γ Abbreviations: IL, interleukin; IFN-γ, interferon-γ; LPS, lipopolysaccharide; TGF-β1, leukocytes, as IFN- can also up-regulate P2X7 expression and function transforming growth factor-β; PMA, 12-myristate 13-acetate; mAb, monoclonal in epithelial cells [18]. However the role of other soluble molecules in antibody; 7AAD, 7-aminoactinomycin D; Ab, antibody; MFI, mean fluorescence the modulation of P2X7 expression and function remain unknown. intensity Following on from the pioneering work of Blanchard [19] and Dubyak ⁎ Corresponding author. School of Biological Sciences, University of Wollongong, [20–22] and their colleagues, the human leukemic monocytic cell line Wollongong, Australia. Tel.: +61 2 4221 5508; fax: +61 2 4221 4135. E-mail address: [email protected] (R. Sluyter). THP-1 has become a well-established model to study the expression and 1 These authors contributed equally to this work. function of P2X7. THP-1 cells express no to low amounts of P2X7, however

0005-2736/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2010.07.022 S. Gadeock et al. / Biochimica et Biophysica Acta 1798 (2010) 2058–2066 2059 the expression of this receptor can be induced following differentiation gentamycin and 2 mM GlutaMAX (complete media) at 37 °C/5% with IFN-γ and LPS, or to a lesser extent with 12-myristate 13-acetate CO2. THP-1 cells were differentiated as described [21]. Briefly, cells (PMA) [21]. In addition to cation fluxes [21],activationofP2X7in were plated at 1×106 cells/ml and cultured for 3 days in 5 ml fresh differentiated THP-1 cells results in number of downstream signalling complete media containing 1000 U/ml IFN-γ and 1 μg/ml LPS. TGF-β1 events including: cell lysis [19]; activation of phospholipase D [20], was added as indicated. Untreated THP-1 cells were plated at mitogen-activated protein kinases [23] and caspase-1 [24];nuclear 3×105 cells/ml and cultured as above. translocation of NF-κB [23]; dissociation of nonmuscle myosin from the P2X7 complex [25] and subsequent inhibition of phagocytosis [26]; release of mature IL-1β [27] and IL-18 [28]; shedding of microvesicles 2.3. Cell-surface receptor measurements [29]; and killing of intracellular mycobacteria [30].THP-1monocyteshave also been widely used to screen a number of potential P2X7 antagonists THP-1 cells (5×105) were pre-incubated in 100 μl NaCl medium leading to the development of some highly specific and potent P2X7 (145 mM NaCl, 5 mM KCl, 5 mM D-glucose, 0.1% BSA, 10 mM HEPES, pH + antagonists [31,32]. Finally, this cell line was used to establish the blocking 7.5) containing 10% human AB serum and 0.01% NaN3 for 10 min, then action of the only available human anti-P2X7 monoclonal antibody (mAb) incubated with fluorochrome-conjugated mAb and 7AAD (to exclude [16]. dead cells) for 20 min, washed and the mean channel fluorescence Transforming growth factor-β1(TGF-β1) has pleiotropic effects in a intensity (MFI) determined using a FACSCalibur or LSR II flow cytometer variety of biological processes including inflammation and immunity, (BD) and Cell Quest software (BD) or FlowJo software (Tree Star, where it is involved in homeostasis and tolerance, as well as the Ashland, OR) respectively. initiation and resolution of the immune response [33].AssuchTGF-β1 has many regulatory roles in restricting the damage caused by the immune response but without negating the elimination of pathogens 2.4. Ethidium+ and propidium2+ uptake measurements [33]. The action of TGF-β1 varies between cell types and their state of differentiation, and the combination of cytokines within the extracel- Ethidium+ or propidium2+ uptake into THP-1 cells suspended in lular environment [34].TGF-β1 can down-modulate the expression and KCl medium (150 mM KCl, 5 mM D-glucose, 0.1% BSA, 10 mM HEPES, function of several cell-surface receptors including CD1d on dendritic pH 7.5), as used extensively by our group for over 17 years to assess cells [35],FcεRI on mast cells [36] and Fas on follicular dendritic cells P2X7 pore formation and to avoid the inhibitory effect of Na+ on dye [37]. Whether this important immunomodulatory cytokine affects P2X7 uptake, was measured by time-resolved flow cytometry as described is unknown. Using immunocytochemical and molecular techniques, and [38]. Briefly, THP-1 cells (1×106) were incubated in 1 ml of KCl fluorescent cation dye uptake measurements, we demonstrate that TGF- medium at 37 °C. Ethidium+ or propidium2+ (both 25 μM) was β1 abrogates the up-regulation of P2X7 expression and function by IFN- added, followed 40 s later by ATP (1 mM). In some experiments, cells γ and LPS in THP-1 cells. were pre-incubated with KN-62 or DMSO (diluent control) for 5 min. The linear mean channel of fluorescent cation dye intensity was 2. Materials and methods measured at 5-s intervals using a FACSCalibur flow cytometer and WinMDI 2.8 software (http://www.scripps.edu). Ethidium+ or 2.1. Reagents propidium2+ uptake (P2X7 function) was quantified as the difference in arbitrary units of area under the fluorescent cation dye uptake RPMI-1640 medium (containing 10 mM HEPES), LPS (Escherichia coli curves in the presence and absence of ATP during the first 5 min of 055:B5), ATP, ethidium bromide, propidium iodide, trypan blue, nigericin incubation. and DMSO were from Sigma (St. Louis, MO). FCS (heat-inactivated before use), gentamycin and GlutaMAX were from Invitrogen (Grand Island, NY). Recombinant human IFN-γ and TGF-β1 were from Roche (Mannheim, 2.5. YO-PRO-12+ uptake measurements Germany), and R&D Systems (Minneapolis, MN), respectively. KN-62 and 7-amino actinomycin D (7AAD) were from Alexis Biochemicals (Lausen, YO-PRO-12+ uptake into THP-1 cells suspended in NaCl medium Switzerland). YO-PRO-12+ was from Molecular Probes (Invitrogen, was measured by a fixed-time flow cytometric assay as described [39]. Eugene, OR). Protease inhibitor cocktail tablets (complete, Mini, EDTA- Briefly, THP-1 cells (1×106) in 1 ml of NaCl medium containing 1 μM free) and phenyl-methyl-sulfonyl-fluoride were from Roche Diagnostics YO-PRO-12+ were incubated in the absence or presence of 1 mM ATP (Pensberg, Germany). SuperSignal® West Pico Chemiluminescent Sub- for 15 min at 37 °C. Incubations were stopped by addition of an equal strate was from Pierce (Rockford, IL). Other reagent grade chemicals were volume of ice-cold NaCl medium containing 20 mM MgCl2 and from Sigma or Amresco (Solon, OH). centrifugation. Cells were washed once with NaCl medium. YO-PRO- Murine anti-human P2X7 mAb (clone L4) [16] and isotype control 12+ uptake (expressed as MFI) was measured using a LSR II flow mAb (clone WMD7) were purified from hybridoma supernatant and cytometer and FlowJo software. conjugated to FITC as previously described [11]. FITC-conjugated murine anti-human CD14 (clone TUK4) and isotype control mAb were from Dako (Glostrup, Denmark). PE-conjugated murine anti-human CD39 mAb 2.6. IL-1β release measurements (clone TU66), FITC-conjugated murine anti-human CD86 (clone 2331) and MHC class II (HLA-DR; clone L243) mAb were from BD (San Jose, CA). ATP- and nigericin-induced IL-1β release from THP-1 cells was FITC- and PE-conjugated isotype control mAb were from eBioscience (San measured using a modification of a protocol used previously for Diego, CA) and BD, respectively. Rabbit anti-rat P2X7 (C-termini epitope) human monocytes [40]. THP-1 cells were suspended at 5×105 viable polyclonal antibody (Ab) and blocking peptide were from Alomone Labs (trypan blue exclusion) cells/ml in RPMI-1640 medium containing (Jerusalem, Israel). Rabbit anti-actin Ab was from Sigma. Peroxidase- 0.1% BSA (pH 7.5) and plated into 24-well plates (0.5 ml/well). Cells conjugated goat anti-rabbit IgG Ab was from Rockland (Gilbertsville, PA). were incubated in the absence or presence of 3 mM ATP or 20 μM

nigericin for 15 min at 37 °C/5% CO2.Thesampleswerethen 2.2. Cell culture centrifuged at 11,000g for 10 s and the cell-free supernatants stored at −20 °C for less than 2 weeks before assaying. Amounts of IL-1β The human monocytic leukemic cell line, THP-1, was maintained were measured using a human IL-1β ELISA (eBioscience) according to in RPMI-1640 medium supplemented with 10% FCS, 5 μg/ml the manufacturer's instructions. 2060 S. Gadeock et al. / Biochimica et Biophysica Acta 1798 (2010) 2058–2066

2.7. P2X7 expression by immunoblotting

THP-1 cells were washed three times in ice-cold PBS and then lysed (1×107 cells/ml) in ice-cold 50 mM BisTris (pH 7.0) containing 750 mM 6-aminohexanoic acid, 1% n-dodecyl β-D-maltoside, 1 mM phenyl-methyl-sulfonyl-fluoride and a protease inhibitor cocktail (used according to the manufacturer's instructions) over 60 min, sheared 10 times through a 21 G needle and centrifuged (16,000g for 10 min at 4 °C). Protein concentrations of cell supernatants were determined using the CB-XTM Protein Assay (G-Biosciences, St Louis, MO) according to the manufacturer's instructions. Cell supernatants (8 μg/lane) were then separated under reducing conditions (5% 2- Fig. 1. P2X7 is present on the cell-surface of differentiated but not undifferentiated THP-1 mercaptoethanol) using 4–20% gradient iGels (NuSep, Austell, GA) cells. THP-1 cells, incubated for 3 days in the absence (undifferentiated) or presence and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). (differentiated) of 1000 U/ml IFN-γ and 1 μg/ml LPS, were labelled with FITC-conjugated anti-P2X7 (solid line) or isotype control (shaded) mAb and 7AAD, and the relative cell- Immunoblotting was performed as described [41] using anti-P2X7 or surface expression (MFI) determined by flow cytometry. Representative results from eight anti-actin Ab followed by peroxidase-conjugated anti-rabbit Ig experiments are shown. antibody, and with Ab binding visualized using chemiluminescent substrate and Amersham Hyperfilm ECL (GE Healthcare, Little Chalfont, Buckinghamshire, UK). 3.2. The P2X7 antagonist KN-62 impairs ATP-induced ethidium+ uptake

To confirm that the ATP-induced ethidium+ uptake into differen- 2.8. P2X7 expression by RT-PCR tiated THP-1 cells was mediated by P2X7, cells were pre-incubated in the presence of the P2X7 antagonist KN-62 [44] or DMSO, and the Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, ATP-induced ethidium+ uptake measured. Pre-incubation with 1 μM Hilden, Germany) according to the manufacturer's instructions. Previ- KN-62 inhibited the ATP-induced ethidium+ uptake by 95.1±2.3% ously described primers to P2X7 (forward primer 5'-GGATGGTGAAC- (Fig. 3). Basal ethidium+ uptake in presence of either KN-62 or DMSO CAGCAGCTA-3'; reverse primer 5'-AAGCCACTGTACTGCCCTTC-3') [42] was similar results not shown. and β-actin (forward 5'-TGGTGGGCATGGGTCAGAAG-3'; reverse 5'- GTCCCGGCCAGCCAGGTCCAG-3') [43] were obtained from Sigma- 3.3. ATP induces ethidium+ but not propidium2+ uptake Genosys (Castle Hill, Australia). PCR amplification was performed using SuperscriptTM III One-Step RT-PCR System Platinum Taq DNA The size exclusion cut-off for P2X7 permeants varies between polymerase (Invitrogen) and corresponding primers for 30 cycles different cell types [4]. To partly define the permeability pathway (95 °C, 15 s; 50 °C, 30 s; 72 °C, 60 s) for P2X7 and 30 cycles (95 °C, following P2X7 activation in differentiated THP-1 cells, the ability of ATP 15 s; 61 °C, 30 s; 72 °C, 60 s) for β-actin. PCR products were separated to induce propidium2+ uptake was compared to ethidium+ uptake. As on a 2% agarose gel and visualized using ethidium bromide staining. above, ATP induced ethidium+ uptake into differentiated THP-1 cells (mean arbitrary units of ethidium+ uptake of 7128±2492; Fig. 4). In contrast, propidium2+ uptake in the presence of ATP was negligible 2.9. Statistical analysis (mean arbitrary units of propidium2+ uptake of 31±54; Fig. 4).

Data is presented as the mean±SD. Differences between treat- 3.4. TGF-β1 prevents the up-regulation of P2X7 by IFN-γ and LPS ments were compared using either the unpaired Student's t-test for single comparisons to control samples or ANOVA for multiple Next, the effect of TGF-β1 on the up-regulation of P2X7 function was comparisons (using Tukey's post test) using Prism 5 for Mac OS X assessed. THP-1 cells were incubated for 3 days with both IFN-γ and LPS in Version 5.0a (GraphPad Software, San Diego, CA) with P b0.05 considered signiﬁcant.

3. Results

3.1. IFN-γ and LPS up-regulate P2X7 expression and function

Differentiation of THP-1 cells with IFN-γ and LPS over 3 days up- regulates P2X7 expression and function [21,22].Therefore,toconfirm these results, THP-1 cells were incubated in the absence or presence of IFN-γ and LPS as previously described [21], and the amount of cell- surface P2X7 and ATP-induced ethidium+ uptake (P2X7 function) measured by flow cytometry. Undifferentiated cells expressed low to negligible amounts of cell-surface P2X7, whereas cell-surface P2X7 was significantly higher on THP-1 cells following differentiation with IFN-γ and LPS (MFI of 0.9±0.8 and 28.7±13.2 respectively, Pb0.01; Fig. 1). This difference in the amount of cell-surface P2X7 corresponded with Fig. 2. ATP induces ethidium+ uptake into differentiated but not undifferentiated THP-1 differences in ATP-induced ethidium+ uptake. ATP induced negligible cells. THP-1 cells, incubated for 3 days in the absence (undifferentiated; ○, ●)or amounts of ethidium+ uptake into undifferentiated THP-1 cells, but presence (differentiated; Δ, ▲) of 1000 U/ml IFN-γ and 1 μg/ml LPS, were suspended in KCl medium at 37 °C. Ethidium+ (25 μM) was added, followed 40 s later by the addition induced significant ethidium+ uptake into differentiated cells (mean of 1 mM ATP (arrow). Mean channel of cell-associated fluorescence intensity was + arbitrary units of ethidium uptake of 28±79 and 9456±5087 measured by time-resolved flow cytometry for cells incubated in the absence (Δ, ○)or respectively, Pb0.01; Fig. 2). presence of ATP (▲, ●). Representative results from eight experiments are shown. S. Gadeock et al. / Biochimica et Biophysica Acta 1798 (2010) 2058–2066 2061

Fig. 3. The P2X7 antagonist, KN-62, impairs ATP induced-ethidium+ uptake into differentiated THP-1 cells. THP-1 cells, incubated for 3 days with 1000 U/ml IFN-γ and 1 μg/ml LPS, were suspended in KCl medium at 37 °C. Cells were pre-incubated for 5 min with either DMSO (○, ●)or1μMKN-62(▲), ethidium+ (25 μM) was then added followed 40 s later by the addition of 1 mM ATP (arrow). Mean channel of cell-associated fluorescence intensity was measured by time-resolved flow cytometry for cells incubated in the absence (Δ)orpresenceofATP(●, ▲). Representative results from three experiments are shown. the presence or absence of increasing concentrations of TGF-β1, and the cell-surface P2X7 expression and the ability of ATP to induce ethidium+ uptake measured. Cell-surface P2X7 expression on differentiated THP-1 was abrogated in a concentration-dependent fashion by TGF- β1 with a maximal inhibitory effect observed at 5 ng/ml (69.5±7.3% inhibition) and with an IC50 of 0.46±0.25 ng/ml (Fig. 5A). This inhibitory effect of TGF-β1oncell-surfaceP2X7expression corresponded with decreased amounts in ATP-induced ethidium+ uptake, with a maximal inhibitory effect observed at 5 ng/ml (71.7±10.6% inhibition) and with Fig. 5. TGF-β1 prevents the up-regulation of P2X7 expression and function of IFN-γ and an IC50 of 0.37±0.25 ng/ml (Fig. 5B). LPS-treated THP-1 cells. THP-1 cells were incubated for 3 days with 1000 U/ml IFN-γ, Next, we examined if delayed addition of TGF-β1 could prevent 1 μg/ml LPS and 0–10 ng/ml TGF-β1 (as indicated). (A) Cells were labelled with FITC- the up-regulation of P2X7 expression and function by IFN-γ and LPS. conjugated anti-P2X7 or isotype control mAb and 7AAD, and the relative cell-surface THP-1 cells were incubated in the presence of IFN-γ and LPS for 3 days expression (MFI) determined by flow cytometry. (B) Cells were suspended in KCl medium at 37 °C, ethidium+ (25 μM) was added, followed 40 s later by the addition of with 10 ng/ml TGF-β1 added on either days 0, 1 or 2. As above, cell- 1 mM ATP, and the mean channel of cell-associated fluorescence intensity was fi surface P2X7 expression was signi cantly lower in THP-1 cells measured by time-resolved flow cytometry. Curves are presented as a percentage of the incubated with TGF-β1 for 3 days compared to cells incubated in maximal (A) cell-surface P2X7 expression or (B) ATP-induced ethidium+ uptake of IFN- the absence of TGF-β1(Fig. 6A). Cell-surface P2X7 expression on THP- γ and LPS-treated THP-1 cells incubated in the absence of TGF-β1. Results expressed as 1 cells incubated with TGF-β1 for the final 2 days was lower, albeit not the mean±SD (n=5).

significantly, compared to cells incubated in the absence of TGF-β1 (Fig. 6A). Addition of TGF-β1 on the last day of incubation had no significant effect on cell-surface P2X7 expression with values similar to those of cells incubated in the absence of TGF-β1(Fig. 6A). A similar pattern was observed regarding P2X7 function. As above, ATP- induced ethidium+ uptake was significantly lower in THP-1 cells incubated with TGF-β1 for 3 days compared to cells incubated in the absence of TGF-β1(Fig. 6B). ATP induced ethidium+ uptake into THP- 1 cells incubated with TGF-β1forthefinal 2 days was also significantly lower compared to cells incubated in the absence of TGF-β1, but to a lesser extent than 3 days incubation with TGF-β1 (Fig. 6B). The addition of TGF-β1 on the last day of incubation had no significant effect on ATP induced ethidium+ uptake with values similar to those of cells incubated in the absence of TGF-β1(Fig. 6B). To demonstrate that that effect of TGF-β1 on P2X7 function was not limited to ethidium+ uptake or to cells suspended in depolarising KCl medium, we first examined ATP-induced YO-PRO-12+ uptake in Fig. 4. ATP induces ethidium+ but not propidium2+ uptake into differentiated THP-1 cells. THP-1 cells suspended in NaCl medium as described [39]. ATP induced THP-1 cells, incubated for 3 days with 1000 U/ml IFN-γ and 1 μg/ml LPS, were suspended significant YO-PRO-12+ uptake into THP-1 cells differentiated with + 2+ in KCl medium at 37 °C. Ethidium (○, ●)orpropdium (Δ, ▲)(both25μM) was added, IFN-γ and LPS, but not undifferentiated cells (Fig. 7A). The presence of followed 40 s later by the addition of 1 mM ATP (arrow). Mean channel of cell-associated TGF-β1 during the incubation of THP-1 cells with IFN-γ and LPS fluorescence intensity was measured by time-resolved flow cytometry for cells incubated fi 2+ in the absence (○, Δ)orpresenceofATP(●, ▲). Representative results from three signi cantly decreased YO-PRO-1 uptake by 53±1% (Fig. 7A). Next, experiments are shown. we examined ATP-induced IL-1β release from THP-1 cells as described 2062 S. Gadeock et al. / Biochimica et Biophysica Acta 1798 (2010) 2058–2066

Fig. 6. Delayed addition of TGF-β1reducesitsefficacy in preventing the up-regulation of P2X7 expression and function of IFN-γ and LPS-treated THP-1 cells. THP-1 cells were incubated for 3 days with 1000 U/ml IFN-γ and 1 μg/mlLPSintheabsenceof TGF-β1 (0 h), or in the presence of 10 ng/ml TGF-β1forall3days(72h),thefinal 2 days (48 h) or the final day (24 h) of incubation. (A) Cells were labelled with FITC- conjugated anti-P2X7 or isotype control mAb and 7AAD, and the relative cell-surface expression (MFI) determined by flow cytometry. (B) Cells were suspended in KCl medium at 37 °C, ethidium+ (25 μM)wasadded,followed40 slaterbythe additionof 1 mM ATP, and the mean channel of cell-associated fluorescence intensity was measured by time-resolved flow cytometry. Ethidium+ uptake data are expressed as arbitrary units of area under uptake curve. Results are expressed as the mean±SD (n =5);**Pb0.01 compared to 0 h. Fig. 7. TGF-β1 prevents the up-regulation of ATP-induced YO-PRO-12+ uptake and IL-1β γ β release of IFN- and LPS-treated THP-1 cells. THP-1 cells were incubated for 3 days with [40]. Nigericin, which induces IL-1 release independently of P2X7 1000 U/ml IFN-γ and 1 μg/ml LPS in the absence or presence of 10 ng/ml TGF-β1, or in the activation, was used as a positive control. ATP induced a small but absence of IFN-γ and LPS. (A) Cells were suspended in NaCl medium containing 1 μMYO- significant release of IL-1β from THP-1 cells differentiated with IFN-γ PRO-12+, and incubated in the absence or presence of 1 mM ATP at 37 °C for 15 min. and LPS, but not from undifferentiated cells (Fig. 7B). The presence of Incubations were stopped by addition of NaCl medium containing 20 mM MgCl2 and 2+ fl β γ centrifugation, and the YO-PRO-1 uptake (MFI) determined by ow cytometry. (B) Cells TGF- 1 during the incubation of THP-1 cells with IFN- and LPS were suspended in RPMI-1640 medium containing 0.1% BSA, and incubated in the absence β decreased IL-1 release by 79±26%, although this failed to reach or presence of 3 mM ATP or 20 μM nigericin for 15 min at 37 °C/5% CO2. The samples were statistical significance (Fig. 7B). This decrease in ATP-induced IL-1β centrifuged and the amount of IL-1β in cell-free supernatants measured by ELISA. Results release was not due to differences in IL-1β synthesis or release are expressed as the mean±SD (n=3); *Pb0.05 and **Pb0.01 to corresponding basal; †† b γ mechanisms between the two groups as nigericin caused a similar IL- P 0.01 compared to THP-1 cells differentiated with IFN- and LPS, and incubated with ATP. 1β release from differentiated cells incubated in the absence or presence of TGF-β1(Fig. 7B). despite the presence of actin (Fig. 8A), although a faint band at 75 kDa, 3.5. TGF-β1 prevents the up-regulation of total P2X7 by IFN-γ and LPS the predicted size of P2X7, was observed in one of the three experiments performed (results not shown). In contrast, this proce- Human monocytes contain a large pool of intracellular P2X7 [11], dure revealed the presence of a major band at 75 kDa in IFN-γ and which traffics to the cell surface during differentiation to macro- LPS-treated THP-1 cells incubated in the presence or absence of TGF- phages [17]. Therefore, to determine if the effect of TGF-β1in β1(Fig. 8A). A minor band at 55 kDa was also detected in these cells in differentiated THP-1 cells is due to altered P2X7 trafficking or to a two of the three experiments performed. Of note, the intensity of the decrease in total P2X7 protein, whole lysates of THP-1 cells were 75 and 55 kDa bands were consistently reduced in the IFN-γ and LPS- prepared and the amount of P2X7 examined by immunoblotting. treated THP-1 cells incubated in the presence of TGF-β1 compared to Actin was included as a loading control. Immunoblotting with an anti- IFN-γ and LPS-treated cells incubated in the absence of TGF-β1, P2X7 polyclonal Ab failed to detect any bands in untreated THP-1 cells despite the presence of equal amounts of actin (Fig. 8A). Pre- S. Gadeock et al. / Biochimica et Biophysica Acta 1798 (2010) 2058–2066 2063

(Fig. 8B). In contrast, P2X7 mRNA expression in IFN-γ and LPS-treated cells incubated in the presence of TGF-β1 was lower than IFN-γ and LPS-treated cells incubated in the absence of TGF-β1, despite similar expression of β-actin mRNA (Fig. 8B).

3.7. The inhibitory effect TGF-β1 on P2X7 is not due to up-regulation of CD39

The function of purinergic receptors on myeloid leukocytes can be modulated by presence of cell-surface ecto-nucleotidases such as the ecto-ATPDase CD39 [45]. Moreover, P2X7 function is lower in monocyte-derived Langerhans compared to monocyte-derived dendritic cells, which corresponds to differences in CD39 but not P2X7 cell-surface expression [14]. Therefore, the possibility remains that the reduced amount of P2X7 function caused by TGF-β1 in THP-1 cells is partly due to the up-regulation of CD39. Therefore, the expression of cell-surface CD39 was assessed. CD39 however was not detected on either undifferentiated THP-1 cells (MFI of 0.0±0.0; n=3), or on cells incubated with IFN-γ and LPS in the presence or absence of TGF-β1 (MFI of 0.0±0.0 and 0.0±0.0 respectively; n=3).

3.8. TGF-β1 prevents the up-regulation of CD86 but not CD14 or MHC class II by IFN-γ and LPS

Differentiation of THP-1 cells with IFN-γ and LPS up-regulates CD14 [46]. Therefore, it was determined if the TGF-β1 effect on THP-1 cells incubated with IFN-γ and LPS was specific to P2X7 or could also affect other cell-surface receptors. THP-1 cells incubated with IFN-γ and LPS in the presence or absence of TGF-β1, as well as undifferentiated cells were labelled with a panel of mAb and the expression of cell-surface markers examined by flow cytometry. Undifferentiated THP-1 cells expressed negligible to low levels of cell- surface CD14, CD86 and MHC class II, as well as P2X7 (Fig. 9). Differentiation of THP-1 cells with IFN-γ and LPS increased the cell- surface expression of P2X7, CD14, CD86 and MHC class II (Fig. 9). As above (Figs. 5A and 6A), the presence of TGF-β1 during the incubation of THP-1 cells with IFN-γ and LPS significantly decreased P2X7 expression (Fig. 9). Similarly, TGF-β1 significantly decreased CD86 expression on cells incubated with IFN-γ and LPS (Fig. 9). In contrast, CD14 and MHC class II expression was similar on IFN-γ and LPS- treated THP-1 cells incubated in the presence or absence of TGF-β1 (Fig. 9).

4. Discussion

Fig. 8. TGF-β1 prevents the up-regulation of total P2X7 protein and mRNA of IFN-γ and P2X7 expression and function can be up-regulated in the human LPS-treated THP-1 cells. (A, B) THP-1 cells were incubated for 3 days with 1000 U/ml IFN-γ leukemic monocytic cell line THP-1 upon incubation with IFN-γ and μ β and 1 g/ml LPS in the absence (Dx) or presence (TGF) of 10 ng/ml TGF- 1, or in the LPS [21,22]. Using this model of monocyte/macrophage differentia- absence of IFN-γ and LPS (Nil). (A) Whole cell lysates were separated by electrophoresis, tion, we demonstrated that TGF-β1 prevents the up-regulation of transferred to nitrocellulose membrane and probed with either (upper panel)anti-P2X7Ab or (lower panel) anti-actin Ab. (B) (Upper panel)P2X7and(lower panel) β-actin mRNA was P2X7 by IFN-γ and LPS. The effect of TGF-β1 was maximal at 5 ng/ml, ampliﬁed by RT-PCR, separated on a 2% agarose gel and visualised using ethidium bromide where it reduced the up-regulation of P2X7 cell-surface expression staining. (A, B) Results are representative of three experiments. and P2X7-induced ethidium+ uptake by ~70%. The effect of TGF-β1

was concentration-dependent with an IC50 of ~0.40 ng/ml. This incubation of the anti-P2X7 polyclonal Ab with blocking peptide inhibitory effect was most prominent when TGF-β1 was present abolished immunoreactivity of both the 75 and 55 kDa bands results during the entire 3-day incubation with IFN-γ and LPS, however TGF- not shown. β1 could partially impair P2X7 up-regulation when added 1 day after the commencement of differentiation with IFN-γ and LPS. The parallel 3.6. TGF-β1 prevents the up-regulation of P2X7 mRNA by IFN-γ and LPS decrease in cell-surface P2X7 expression and P2X7 function in THP-1 cells parallels observations in leukocytes, where P2X7 function Incubation of THP-1 cells with IFN-γ and LPS up-regulates P2X7 correlates with cell-surface P2X7 expression [11,17]. Finally, this mRNA [21]. Therefore, to determine if the effect of TGF-β1in effect of TGF-β1 was not limited to ethidium+ uptake, as ATP-induced differentiated THP-1 cells is due to a decrease in P2X7 mRNA, RNA YO-PRO-12+ uptake and IL-1β release was also impaired in differen- was isolated from THP-1 cells and the amount of P2X7 mRNA tiated THP-1 cells incubated with TGF-β1 compared to differentiated examined by RT-PCR. β-actin was included as a house keeping control. cells incubated without TGF-β1. Although, it should be noted that the P2X7 mRNA was present in undifferentiated THP-1 cells, and amount of ATP-induced IL-1β released from THP-1 cells incubated for incubation with IFN-γ and LPS up-regulated P2X7 mRNA expression 3 days with IFN-γ and LPS is lower than the amount of ATP-induced 2064 S. Gadeock et al. / Biochimica et Biophysica Acta 1798 (2010) 2058–2066

increased amounts of a P2X7 variant, which impairs P2X7 expression and function as observed in some epithelial cell lines [49],as immunoblotting revealed a decrease in both full-length P2X7 (75 kDa) and a shorter P2X7 variant (55 kDa). The reduced P2X7 function following TGF-β1 incubation was also not due to increased levels of the ecto-ATPDase CD39. THP-1 cells, regardless of treatment, failed to express cell-surface CD39, however we cannot exclude the possibility that other ecto-nucleotidases [50] play an ancillary role in the reduced P2X7 function in TGF-β1-treated THP-1 cells. Finally, the inhibitory effect of TGF-β1 is not due to a general block in THP-1 cell differentiation per se as this cytokine failed to impair the up- regulation of CD14 and MHC class II. The THP-1 cell line is a well-established model used to study the P2X7 and its downstream effects (see Introduction). Moreover, THP-1 cells differentiated with IFN-γ and LPS demonstrate characteristics of primary macrophages including plastic adherence [21], phagocytosis of latex beads [26] and killing of intracellular mycobacteria [30].Ourstudy confirms the presence of functional P2X7 in THP-1 cells following differentiation with IFN-γ and LPS. Originally, it was observed that incubation of THP-1 cells with IFN-γ and LPS up-regulated ATP-induced cation fluxes [21], and then subsequently shown that these cation fluxes were impaired by the P2X7 antagonist KN-62 [22]. Similarly, we found that KN-62 impaired ATP-induced ethidium+ uptake in differentiated THP-1 cells confirming that the uptake of this organic cation is also mediated by P2X7. Our study also confirms the absence of functional P2X7 in undifferentiated THP-1 cells as observed by others [20,21].In contrast another group [23] has demonstrated the presence of functional P2X7 in undifferentiated THP-1 cells whereby the most potent P2X7 agonist, 2'- and 3'-0(4-benzoylbenzoyl) ATP, induced mitogen-activated protein kinase activation, NF-κB translocation and IL- 1β release. The reasons for these differences are unknown but may reflect differences in cell lines between laboratories, differences in the relative sensitivities of the different assays, or differences in the relative activation thresholds including amounts of cell-surface P2X7 expression required for different P2X7-mediated events. The detection of P2X7 Fig. 9. TGF-β1 prevents the up-regulation of CD86 but not CD14 or MHC class II of IFN-γ and mRNA and low to negligible amounts of cell-surface P2X7, as well as the LPS-treated THP-1 cells. Untreated THP-1 cells,aswellasTHP-1cellsincubatedfor3dayswith detection of P2X7 protein (in one of three experiments) in undifferen- 1000 U/ml IFN-γ and 1 μg/ml LPS in the absence or presence of 10 ng/ml TGF-β1, were tiated THP-1 cells in our study suggests these cells may contain low fl labelled with uorochrome-conjugated mAb and 7AAD, and the relative cell-surface amounts of functional P2X7 but which were undetectable by our cation expression (MFI) determined by flow cytometry. Results are expressed as the mean±SD †† β (n=5).*Pb0.05 and **Pb0.01 to untreated THP-1 cells; Pb0.01 compared to THP-1 cells uptake and IL-1 release assays. differentiated with IFN-γ and LPS. The permeability pathways that mediate the passage of large organic ions following P2X7 activation have not been fully elucidated, although recent data indicates that the dissociation of non-muscle IL-1β release from THP-1 cells incubated for 3 h with PMA and then myosin from the P2X7 complex may be involved in P2X7 pore for 6–24 h with LPS [27,29] or for 2 days with IFN-γ and then for 4 h formation [25]. In this study, ATP induced the uptake of ethidium+ with LPS [47]. The reason for this difference is unknown, but (314 Da) and YO-PRO-12+ (375 Da), but not propidium2+ (415 Da) prolonged (3 days) incubation of THP-1 cells with LPS and/or possibly into differentiated THP-1 cells. This result is similar to those observed IFN-γ may result in the loss of IL-1β prior to ATP or nigericin in human lymphocytes [51] and dendritic cells [52], but contrasts incubation. Alternatively, 3-day incubation of THP-1 cells with IFN-γ ATP-induced uptake of propidium2+ uptake in osteoblasts [53] and and LPS may not induce large amounts IL-1β synthesis. P2X7-transfected HEK-293 cells [54]. The opening of pannexin-1 has The mechanism by which TGF-β1 abrogates the up-regulation of been implicated in the P2X7-mediated uptake of ethidium+ in cell-surface P2X7 by IFN-γ and LPS in THP-1 cells has been partially macrophages and P2X7-transfected HEK-293 cells [55], but the elucidated. Immunoblotting of whole cell lysates with anti-P2X7 Ab presence of pannexin-1 in THP-1 cells and whether this hemichannel demonstrated that TGF-β1 prevents the synthesis of the total amount is permeable to propidium2+ is unknown. Nevertheless our data of P2X7 protein in differentiated THP-1 cells. Moreover, this reduction suggests that propidium2+ may enter via a different permeability in P2X7 protein paralleled a decrease in P2X7 mRNA expression pathway to that of ethidium+, or that the pore size of the P2X7 suggesting that transcription rather than translation is abrogated by permeability pathway may differ between cell types. TGF-β1 in THP-1 cells incubated with IFN-γ and LPS. However, we In addition to P2X7, IFN-γ and LPS up-regulated the expression of cannot exclude the possibility that TGF-β1 may be involved in some CD14, CD86 and MHC class II in THP-1 cells. Although various post-transcription event such as increased P2X7 mRNA decay, as TGF- inflammatory stimuli can up-regulate each of these receptors in THP- β1 can increase the rate of decay of chemokine mRNA induced by LPS 1 cells [46,56,57], in relation to incubation with both IFN-γ and LPS in murine macrophages [48]. It appears unlikely that the inhibitory this has only been previously demonstrated for CD14 [46]. 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