STAT1-Dependent Pathways Macrophages

Modulation of TNF-α Gene Expression by IFN- γ and Pamidronate in Murine Macrophages: Regulation by STAT1-Dependent Pathways This information is current as of September 26, 2021. Kae Takagi, Masatoshi Takagi, Siva Kanangat, Kenneth J. Warrington, Hidenobu Shigemitsu and Arnold E. Postlethwaite J Immunol 2005; 174:1801-1810; ; doi: 10.4049/jimmunol.174.4.1801 Downloaded from http://www.jimmunol.org/content/174/4/1801

References This article cites 60 articles, 24 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/174/4/1801.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 26, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Modulation of TNF-␣ Gene Expression by IFN-␥ and Pamidronate in Murine Macrophages: Regulation by STAT1-Dependent Pathways1

Kae Takagi,† Masatoshi Takagi,‡ Siva Kanangat,*† Kenneth J. Warrington,† Hidenobu Shigemitsu,*† and Arnold E. Postlethwaite2*†

Aminobisphosphonates are drugs used in the treatment of hypercalcemia, Paget’s disease, osteoporosis, and malignancy. Some patients treated with aminobisphosphonates have a transient febrile reaction that may be caused by an increased serum concentration of proinflammatory cytokines. Aminobisphosphonates induce the production of certain proinflammatory cytokines in vitro, especially in cells of monocytic lineage. A unique feature of aminobisphosphonates is that they bind the V␥2V␦2 class of T cells, which are found only in primates, and stimulate cytokine production. The effects of aminobisphosphonates on other cells, Downloaded from including macrophages, are incompletely understood. We show in this study that treatment of murine macrophages with pamidronate, a second generation aminobisphosphonate, induces TNF-␣ production. Furthermore, pretreatment of murine macrophages with pamidronate before stimulation with IFN-␥ significantly augments IFN-␥-dependent production of TNF-␣. This pamidronate-mediated augmentation of TNF-␣ production results in sustained phosphorylation of the tyrosine residue at position 701 of STAT1 after IFN-␥ treatment. Our data suggest that this sustained phosphorylation results from inhibition of protein tyrosine phosphatase activity. We also show that pamidronate treatment increases TNF-␣ production in vivo in mice. Pami- http://www.jimmunol.org/ dronate-augmented TNF-␣ production by macrophages might be a useful strategy for cytokine-based anticancer therapy. The Journal of Immunology, 2005, 174: 1801–1810.

isphosphonates are compounds with a chemical structure munopathogenesis of some diseases (12). Most effects of IFN-␥ similar to that of inorganic pyrophosphate. Those that can be attributed to several gene products that are regulated by the B have two amino chains covalently bound to the central JAK-STAT pathway (13–15), a signaling pathway that has been carbon atom are called aminobisphosphonates (e.g., risedronate, implicated in the mediation of biologic responses induced by many pamidronate, ibandronate, olpadronate, and alendronate) (1). Ami- cytokines (16, 17). A recent report showed that the diverse gene by guest on September 26, 2021 nobisphosphonates are strikingly effective against clinical disor- expression patterns mediated by STAT1-dependent pathways, ders associated with bone resorption, including tumor-associated STAT1-independent mechanisms, and the balance between these osteolysis and hypercalcemia, Paget’s disease, and osteoporosis two pathways play an important role in the biological response to (2). However, treatment with aminobisphosphonate results in a IFN-␥ (18). transient febrile reaction in some patients (3, 4). The mechanism TNF-␣ plays important roles in host defense against infections underlying this reaction is not fully understood, but increased pro- and tumors and in mediating inflammatory and immune responses. duction of the proinflammatory cytokines IL-1, IL-6, IFN-␥, and It triggers the local expression of chemokines and cytokines and TNF-␣ is thought to play a role (3–8), and bisphosphonate treat- promotes the adhesion, extravasation, attraction, and activation of ment of peripheral mononuclear cells or macrophages in vitro has ␣ been reported to increase the production of the inflammatory cy- leukocytes at the site of infection (19, 20). Most TNF- is pro- tokines IL-1, IL-6, TNF-␣, and IFN-␥ (7, 9–11). duced by activated macrophages after completion of a series of IFN-␥, the main cytokine through which T cells activate mac- reactions: precursor differentiation (recruitment and differentiation rophages, is produced by activated T and NK cells. The complex of immature blood-derived mononuclear phagocytes into compe- genetic programs elicited by IFN-␥ in the immune system account tent, cytokine-responsive macrophages by activators at sites of in- for the diverse activities of IFN-␥ in host defense and in the im- flammation), priming (induction, by certain cytokine signals, of a receptive or primed state in which the inflammatory macrophages are not yet cytotoxic), and the trigger reaction (the ability of † *Department of Veterans Affairs Medical Center, Memphis, TN 38104; Divisions of primed macrophages to develop full functional activity in response Connective Tissue Diseases and Pulmonary Medicine, Department of Medicine, Uni- versity of Tennessee Health Science Center, Memphis, TN 38163; and ‡Department to trigger signals). The cytokine priming and trigger signals form of Hematology/Oncology, St. Jude Children’s Research Hospital, Memphis, TN the basis of a regulatory system that sets the threshold and deter- 38105 mines the onset of macrophage effector activity (21). IFN-␥ acti- Received for publication July 20, 2004. Accepted for publication November 8, 2004. vates macrophages and induces them to produce a variety of cy- The costs of publication of this article were defrayed in part by the payment of page ␣ charges. This article must therefore be hereby marked advertisement in accordance tokines, including TNF- (22–28). This signal pathway has been with 18 U.S.C. Section 1734 solely to indicate this fact. believed to use the JAK-STAT pathway (25). Production of 1 This work was supported by a Merit Review Grant funded by the U.S. Department TNF-␣ is also induced by stimulation of macrophages with LPS, of Veterans Affairs. which induces the activation of multiple forms of NF-␬B (29–31). 2 Address correspondence and reprint requests to Dr. Arnold E. Postlethwaite, De- However, LPS also activates the JAK-STAT pathway (32). It is partment of Medicine, University of Tennessee College of Medicine, 956 Court Av- enue, Room G326, Memphis, TN 38163. E-mail address: [email protected] not completely understood how the JAK-STAT pathway and

NF-␬B family members interact to regulate TNF-␣ gene expres- Technologies) to obtain cDNA. The cDNA was amplified by 18 and 22 sion. Recent reports suggest that bisphosphonates have important cycles of PCR using the following primer pairs: TNF-␣: sense, 5Ј- GGGCAGGTCTACTTTGGAGTCATT-3Ј; and antisense, 5Ј-ATTCTG effects on macrophages (11, 33). One of these effects is thought to Ј Ј ␬ AGACAGAGGCAACCTGAC-3 ; and GADPH: sense, 5 -AATCCCATC be activation of the NF- B pathway to increase production of ACCATCTTCCAGGAG-3Ј; and antisense, 5Ј-CACCCTGTTGCTGTAGC TNF-␣ by macrophages (33). However, the molecular mechanism CAAATTC-3Ј. Amplified PCR products were electrophoresed in a 2% by which bisphosphonates modulate the cytokine network is not agarose gel, stained with ethidium bromide, and photographed under UV light. fully understood. Preparation of cell lysates The purpose of the present study was to investigate the mech- 6 anism by which pamidronate acts on murine macrophages and Cell lysates were prepared from 10 cells by washing the cells with PBS ␥ ␣ and incubating them in 150 mM NaCl containing 1.0% Nonidet P-40, 0.1% augments IFN- -dependent TNF- production by these cells. We SDS, 0.1% sodium deoxycholate, 5 mM EDTA, 10 mM Tris-HCl (pH 7.4), show that treatment of murine macrophages with pamidronate in- and protease inhibitors for 30 min on ice. Lysate was cleared by centrif- duces TNF-␣ production. Furthermore, pretreatment of murine ugation at 13,000 ϫ g for 15 min at 4°C. ␥ macrophage with pamidronate before stimulation with IFN- sig- Western blot analysis nificantly augments IFN-␥-dependent production of TNF-␣. This enhancement of IFN-␥-mediated TNF-␣ production by pami- The total protein concentration in the lysates was measured using the DC protein assay kit (Bio-Rad). Samples containing 30 ␮g of protein were dronate is the result of prolonged STAT1 phosphorylation as a 3 boiled in sample buffer, and the denatured proteins were separated by SDS- result of inhibition of protein tyrosine phosphatases (PTPs). PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Certain types of malignant tumors are sensitive to TNF-␣ (34, Blots were probed with the following primary Abs: anti-phospho-STAT1

35), and low dose TNF-␣ enhances the cytotoxic effects of che- Y701, anti-STAT1, anti-phospho-p38 T180/Y182, anti-p38, and anti- Downloaded from motherapeutic agents on tumor cells (36, 37). Activation of mac- phospho-protein kinase RNA dependent (anti-phospho-PKR) T446/451 (Cell Signaling Technology); anti-␤-actin (I19), anti-JAK2 (N17), and anti- rophages by aminobisphosphonates might therefore be useful in PKR (K17; Santa Cruz Biotechnology). Primary Abs were detected using cytokine-mediated cancer therapy. HRP-conjugated anti-rabbit or anti-mouse secondary Abs (Amersham Bio- sciences) by ECL (Amersham Biosciences). Phospho-STAT1 expression Materials and Methods was quantiﬁed using NIH image 1.63.

Cells and cell culture Flow cytometry http://www.jimmunol.org/ Murine peritoneal macrophages were isolated by using a previously de- Macrophages were pretreated with PBS or pamidronate (20 ␮M) for 2 h, scribed protocol (38) after i.p. injection of thioglycolate (BD Biosciences) then incubated with PBS, murine IFN-␥ (10 pg/ml), or pamidronate and into 6- to 12-wk-old C57BL/6J mice. After culture of the macrophages in murine IFN-␥ for 4 h with a Golgi plug (BD Biosciences) to block cytokine plastic flasks for 2 h, floating cells were removed by extensive washing, secretion. After washing with PBS, macrophages were incubated with anti- and attached cells were maintained in RPMI 1640 medium (Invitrogen Life mouse CD11b-allophycocyanin (BD Biosciences) or rat IgG2-allophyco- Technologies) containing 9% FBS, 100 U/ml penicillin, and 100 ␮g/ml cyanin isotype control (BD Biosciences) for 20 min at 4°C. These cells streptomycin. More than 90% of the cultured cells were macrophages as were washed again in PBS, then fixed by incubation in 4% paraformalde- determined by flow cytometric analysis of CD11b-positive cells. Cells of hyde for 30 min at 4°C and washed with PBS containing 0.5% BSA. the 293T and RAW 264.7 lines were obtained from the American Type Aliquots of 2 ϫ 106 cells/100 ␮l were placed in each assay tube to which Culture Collection and grown in DMEM (Invitrogen Life Technologies) we added 100 ␮l of Cytofix/Cytoperm buffer (BD Biosciences). Samples by guest on September 26, 2021 containing 9% FBS, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. All were incubated for 20 min at room temperature, rinsed with Perm/Wash cultures were incubated at 37°C in 5% CO2 and a humidified environment. buffer (BD Biosciences), then incubated in PBS containing 5% BSA for 10 Human monocytes from healthy volunteers were isolated by using a pos- ϩ min at room temperature. Anti-phospho-STAT1 Y701 Ab (Cell Signaling itive selection protocol to enrich for CD14 monocytes according to the Technology) or rabbit IgG isotype control (Sigma-Aldrich) was added to manufacturer’s instructions (Miltenyi Biotec) and incubated with RPMI the assay tubes, and incubation was continued for 30 min at room temper- 1640 containing 9% FBS. ature. After rinsing with 0.5% BSA in PBS, secondary Ab (FITC-conjugated anti-rabbit IgG; Jackson ImmunoResearch Laboratories) was added, Reagents and samples were incubated for 30 min at room temperature. After rinsing We used commercially available pamidronate (Novartis Pharmaceuticals), in 0.5% BSA in PBS, we added PE-conjugated anti-TNF-␣ Ab or PE- murine IFN-␥ (R&D Systems), and human IFN-␥ (BD Biosciences). Clo- conjugated mouse IgG isotype control (BD Biosciences) to each tube and dronate was a gift from Dr. G. Palmieri (University of Tennessee Health incubated the samples for 20 min at room temperature. Cells were washed Science Center). The JAK inhibitor 2-(1,1-dimethylethyl)-9-fluoro-3,6-di- with PBS again before being subjected to flow cytometry on a FACSCali- hydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one (39) and the NF-␬B in- bur instrument (BD Biosciences). hibitor SN50 were obtained from Calbiochem (40). Sodium orthovanadate, Immunoprecipitation staurosporine, and anisomycin were obtained from Sigma-Aldrich. Cell lysates were prepared from mouse peritoneal macrophages by incu- ELISA bating the cells in lysis buffer (150 mM NaCl containing 1.0% Nonidet Supernatants from cultured cells were harvested 18 h after culture with P-40, 0.5% Tween 20, 5 mM EDTA, 10 mM Tris-HCl (pH 7.4), and pro- PBS and murine or human IFN-␥, pamidronate alone, pamidronate with tease inhibitors) for 30 min on ice. Lysate was cleared by centrifugation at ϫ murine or human IFN-␥, clodronate alone, or clodronate with murine 13,000 g for 15 min at 4°C, then precleared by incubation with rabbit IFN-␥. The TNF-␣ concentration in supernatants was determined by IgG and protein A/G agarose for 60 min at 4°C with rotation. Precleared ␮ ␮ ELISA (R&D Systems). The detection limits of the assay were between 5 lysates were immunoprecipitated with 5 l of anti-STAT1 Ab and 25 lof and 3000 pg/ml. Each result is expressed as the mean value of three or four protein A/G agarose (Santa Cruz Biotechnology) for 120 min at 4°C with independent experiments performed in duplicate. rotation. After extensive washing with lysis buffer, immunoprecipitants were subjected to SDS-PAGE and transferred to a polyvinylidene difluo- Semiquantitative RT-PCR analysis to measure TNF-␣ mRNA ride membrane. Src homology domain 2-containing phosphatase 1 (SHP2) concentration (PTPN11) was detected with anti-SHP2 Ab (ab9214; Abcam). TC-PTP (PTPN2) was detected with mouse mAb 3E2 (gift from Dr. M. L. Trem- Total RNA was isolated from 106 murine peritoneal macrophages using blay, McGill University, Montreál, Canada). TRIzol (Invitrogen Life Technologies), and 1 ␮g of total RNA was subjected to RT using poly(dT) oligomer and SuperScript II (Invitrogen Life Phosphatase assay Cell lysates were prepared from RAW 264.7 cells by incubation in lysis 3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; ChIP, chro- buffer (25 mM Tris-HCl (pH 7.4), 10 mM 2-ME, 2 mM EDTA, 1.0% matin immunoprecipitation; PKR, protein kinase RNA dependent; pNPP, p-nitrophe- Triton X-100, and protease inhibitors) with sonication. Lysate was cleared nyl phosphate; SHP2, Src homology domain 2-containing phosphatase 1; TC-PTP, T by centrifugation at 13,000 ϫ g for 15 min at 4°C, then subjected to cell PTP. phosphatase assay, for which 50 ␮g of cell lysate was mixed with 100 ␮l The Journal of Immunology 1803

of p-nitrophenyl phosphate (pNPP) liquid substrate system (Sigma- cantly augmented TNF-␣ production compared with that obtained Aldrich) with pamidronate or sodium orthovanadate and incubated at room by IFN-␥ treatment alone ( p Ͻ 0.001; Fig. 1A). Increased secre- temperature. Absorbance at 405 nm was measured using a Benchmark Plus tion of TNF-␣ was detected even at 1 pg/ml IFN-␥ when macro- Microplate Spectrophotometer (Bio-Rad). phages were pretreated with pamidronate (Fig. 1A). Similar aug- Luciferase assay mentation was observed when cells were pretreated with up to 20 ␮ ␥ The luciferase assay was conducted as previously described (41). Briefly, M pamidronate before stimulation with 10 pg/ml IFN- . How- 293T cells were seeded in 96-well plates at 50% confluent density. Cells ever, 100 ␮M pamidronate, with or without IFN-␥, induced mas- were transfected with a firefly luciferase reporter plasmid (2ϫM67SJE Luc sive cell death, and we were unable to detect any residual TNF-␣. plasmid, gift from Dr. Y. Eugene Chin, Brown University School of Med- Pamidronate itself (20 ␮M) weakly induced TNF-␣ production ␬ icine, Providence, RI) or NF- B luciferase reporter plasmid (gift from Dr. (70 Ϯ 49.9 pg/ml) in the absence of IFN-␥ (Fig. 1B). Cells treated K. Tago, St. Jude Children’s Research Hospital, Memphis, TN) using Li- pofectamine 2000 transfection reagent (Invitrogen Life Technologies). The with clodronate, which is not an aminobisphosphonate, showed no 2ϫM67SJE Luc plasmid was used to measure STAT1 activation. The Re- increase in TNF-␣ production; indeed, clodronate seemed to sup- nilla luciferase reporter plasmid pRL-SV40 (Promega) was used as an press IFN-␥-mediated TNF-␣ production in a dose-dependent internal control. After 24 h, cells were treated with 20 ␮M pamidronate or ␥ ␮ manner (Fig. 1C). Pamidronate also induced augmentation of PBS for 4 h, then human IFN- (50 g/ml) or PBS was added, and incu- ␣ bation was continued for 6 h. Firefly and Renilla luciferase activities were TNF- production by human macrophages cultured in the pres- measured using the dual luciferase reporter assay system (Promega) and a ence of low concentrations of IFN-␥ ( p Ͻ 0.001; Fig. 1D). MicroLumat Plus 96V luminometer (Berthold Technologies). ␥ ␣ Chromatin immunoprecipitation (ChIP) assay Modulation of IFN- -mediated expression of TNF- mRNA by pamidronate Downloaded from Cells were washed once with PBS after removal of culture medium, then incubated in 6 ml of 1% formaldehyde in PBS for 10 min at room tem- TNF-␣ production is regulated at transcriptional and translational perature to cross-link proteins to DNA. The cross-linking reaction was levels (19). To determine whether increased production of TNF-␣ quenched by adding 2 ml of 1 M glycine in PBS and continuing the in- is associated with an increase in TNF-␣ mRNA expression, we cubation at room temperature for 10 min. Cells were washed twice with evaluated TNF-␣ mRNA expression by semiquantitative RT-PCR. cold PBS, collected into tubes, and lysed with 50 mM Tris-HCl (pH 7.5), ␣ 1% Nonidet P-40, 0.5% Tween 20, and 150 mM NaCl. After centrifugation TNF- mRNA was not detected in macrophages treated with PBS http://www.jimmunol.org/ for 15 min at 4°C, the cell pellet was resuspended in PIPA buffer (50 mM (Fig. 2, lane 1, 18- and 22-cycle products). TNF-␣ mRNA was Tris-HCl (pH 7.5), containing 1% Nonidet P-40, 0.05% SDS, 0.5% sodium barely detected in macrophages stimulated with pamidronate alone deoxycholate, 1 mM EDTA, 150 mM NaCl, and protease inhibitors), then (lane 2, 18- and 22-cycle products). As expected, IFN-␥ treatment sonicated (three times, 20 s each time) on ice. Cell lysates were precleared ␣ by incubation with rabbit IgG (Sigma-Aldrich) and protein A/G-agarose induced an increase in the expression of TNF- mRNA (lane 3, (Santa Cruz Biotechnology) coated with salmon sperm DNA (Invitrogen 18- and 22-cycle products). However, greater induction of TNF-␣ Life Technologies) for1hat4°Cwith rotation. Cross-linked DNA-protein mRNA occurred in macrophages treated with IFN-␥ and pamidr- complexes were immunoprecipitated by anti-phospho-STAT1 Y701 Ab onate (lane 4, 18- and 22-cycle products). Pamidronate treatment and protein A/G-agarose coated with salmon sperm DNA. After extensive did not affect the expression of GAPDH mRNA. These results washing in RIPA buffer, immunoprecipitated phospho-STAT1-DNA com- ␣ plexes were dissociated by heating at 65°C overnight with decross-link indicate that enhanced production of TNF- protein by pami- by guest on September 26, 2021 buffer (50 mM Tris-HCl (pH 7.0), 5 mM EDTA, 10 mM DTT, and 1% dronate is associated with a concomitant increase in the amount SDS). DNA was extracted by phenol/chloroform purification, followed by of TNF-␣ mRNA transcribed. ethanol precipitation with glycogen. Precipitated DNA was dissolved in 20 ␮l of Tris-EDTA buffer and subjected to PCR. Pamidronate affects mainly the TNF-␣ pathway that is mediated Measurement of serum TNF-␣ concentration by the JAK-STAT pathway Sixteen C57BL/6J mice, 6–12 wk of age, were given a single i.p. injection Because the murine TNF-␣ promoter contains response elements of 0.1 ml of PBS alone, PBS containing pamidronate (0.1 ml of 200 ␮g/ ␥ ␥ ␬ ␥ for IFN- (the -activated sequence or GAS element) and NF- B, ml/mouse), PBS containing murine IFN- (0.1 ml of 1000 pg/ml/mouse), ␣ or PBS containing pamidronate and murine IFN-␥ at the same concentra- which plays a key role in LPS-mediated TNF- production (31), tions as given individually. Serum concentrations of TNF-␣ were deter- IFN-␥ signaling may involve the NF-␬B pathway as well as the mined by ELISA (kit from R&D Systems) 2 h later. JAK-STAT pathway. To determine which pathway is preferentially modulated by pamidronate, we treated cells with a JAK Statistical analysis inhibitor or NF-␬B inhibitor (SN50) before pretreatment with We used the Mann-Whitney U test for statistical analysis. pamidronate and stimulation with IFN-␥. JAK-mediated phosphorylation of STAT1 at Y701 was partly inhibited at an Results inhibitor concentration of 100 nM and was completely inhibited ␥ ␣ Effect of pamidronate and IFN- on release of TNF- by mouse at 500 nM (Fig. 3A). When cells were pretreated with the JAK macrophages in vitro inhibitor, TNF-␣ production by IFN-␥ was reduced by 82%, To determine whether IFN-␥ induction of TNF-␣ production is and pamidronate-augmented production of TNF-␣ by IFN-␥ modulated by pamidronate, we measured, by ELISA, TNF-␣ concen- was reduced by 89% ( p Ͻ 0.05; Fig. 3B). However, pretreat- trations in supernatants from cultures of mouse peritoneal macro- ment with the NF-␬B inhibitor SN50 did not suppress TNF-␣ phages stimulated with IFN-␥ after pretreatment with pamidronate or production resulting from treatment with IFN-␥ and pami- without pamidronate pretreatment. As previously reported, IFN-␥ dronate or IFN-␥ alone (Fig. 3B), but this pretreatment did activated mouse peritoneal macrophages in vitro and induced suppress LPS-mediated TNF-␣ secretion by 71% ( p Ͻ 0.05; TNF-␣ production in a dose-dependent manner (Fig. 1A). TNF-␣ Fig. 3C). When we assessed the effects of the inhibitors on was not abundantly secreted at a low (10 pg/ml) IFN-␥ concen- TNF-␣ production by macrophages stimulated with pami- tration (mean TNF-␣ concentration, 31 Ϯ 42 pg/ml), but incuba- dronate, but not IFN-␥, the JAK inhibitor reduced TNF-␣ tion of macrophages with 100 pg/ml IFN-␥ markedly stimulated production in a dose-dependent manner ( p Ͻ 0.05; Fig. 3D). secretion of TNF-␣ (mean TNF-␣ concentration, 1053 Ϯ 389 pg/ The NF-␬B inhibitor had no effect on pamidronate-induced ml; p Ͻ 0.001). Pretreatment of macrophages with 20 ␮M pam- TNF-␣ production (Fig. 3D). These results strongly suggest that idronate before exposure to low concentrations of IFN-␥ signifi- pamidronate preferentially modulates the JAK-STAT pathway. 1804 PAMIDRONATE-DEPENDENT TNF-␣ PRODUCTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Effects of IFN-␥ and pamidronate on TNF-␣ secretion by murine (A–C) and human (D) macrophages. Mean concentrations of TNF-␣ in culture supernatants were measured by ELISA in three or four independent experiments. A, Dose dependency of TNF-␣ production on IFN-␥. The TNF-␣ concentration was measured 16 h after IFN-␥ stimulation following pretreatment with pamidronate at a final concentration of 20 ␮M(f) or without pamidronate (PBS only; Ⅺ) for 2 h. *, Statistically significant difference (p Ͻ 0.05) in TNF-␣ levels between samples treated with IFN-␥ alone and those treated with IFN-␥ and pamidronate. B, Dose dependency of TNF-␣ production on pamidronate. The TNF-␣ concentration was measured 16 h after stimulation with IFN-␥ at a final concentration of 10 pg/ml (f) or without IFN-␥ stimulation (PBS only; Ⅺ). *, Statistically significant difference (p Ͻ 0.05) in TNF-␣ levels between samples treated with IFN-␥ alone and those treated with IFN-␥ and pamidronate. C, Dose dependency of TNF-␣ production on clodronate. The TNF-␣ concentration was measured 16 h after treatment with IFN-␥ at a final concentration of 10 pg/ml. D, TNF-␣ production by human macrophages treated with PBS, pamidronate (Pam; final concentration, 20 ␮M), human IFN-␥ (hIFN␥; final concentration, 10 pg/ml), or 20 ␮M pamidronate 2 h before stimulation with 10 pg/ml human IFN-␥ (Pam ϩ hIFN␥).

STAT1 phosphorylation status and STAT1 expression mented the phosphorylation of STAT1. The expression of JAK2 Previous reports have suggested that increased transcription of was unchanged by these stimulations (Fig. 4A). Next we examined TNF-␣ mRNA by IFN-␥ uses the JAK-STAT1 pathway (25, 42). STAT1 expression and Y701 phosphorylation in macrophages at IFN-␥ stimulation increases the expression of STAT1, and phosphorylation of Y701 in STAT1 by JAK2 leads to rapid dissociation of the receptor-STAT1 complex and translocation of STAT1 into the nucleus as a homodimer (14, 16, 17). This process may be necessary for an IFN-␥-mediated increase in TNF-␣ mRNA transcription. To determine whether pamidronate induces an IFN-␥- mediated increase in the expression or phosphorylation of STAT1, we compared the amounts of STAT1 and STAT1 phosphorylated FIGURE 2. Expression of TNF-␣ mRNA by cultured murine macro- ␥ ␥ at Y701 in pamidronate-treated and IFN-␥-stimulated murine mac- phages treated with pamidronate, IFN- , or pamidronate and IFN- . The amount of TNF-␣ mRNA was measured by semiquantitative RT-PCR as- rophages using anti-STAT1 and anti-phospho-Y701-STAT1 Abs say (18 and 22 cycles) in macrophages treated with PBS (lane 1), pam- in Western blot analyses. As expected, STAT1 expression was idronate (ﬁnal concentration, 20 ␮M; lane 2), murine IFN-␥ (ﬁnal concen- ␥ increased by IFN- stimulation (Fig. 4A). Pamidronate treatment tration, 10 pg/ml; lane 3), or pamidronate and murine IFN-␥ (lane 4). had no effect on STAT1 expression or STAT1-Y701 phosphory- Pamidronate treatment lasted 2 h; IFN-␥ treatment lasted 16 h. GAPDH lation, but IFN-␥ treatment resulted in an increase in STAT1-Y701 was used as a control. Representative data from three independent exper- phosphorylation, and treatment with pamidronate and IFN-␥ aug- iments are shown. The Journal of Immunology 1805 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Inhibitors of JAK and NF-␬B modulate TNF-␣ production resulting from treatment with pamidronate and IFN-␥. A, Inhibition of STAT1 phosphorylation by JAK inhibitor. Murine macrophages in culture were treated with JAK inhibitor 60 min before stimulation with 10 pg/ml IFN-␥, harvested, and subjected to Western blot analysis 30 min after IFN-␥ treatment. Blots were probed with Abs to phospho-STAT1 and STAT1. Representative data from two independent experiments are shown. B, The TNF-␣ concentration in culture supernatants was measured by ELISA; mean values from three independent experiments are shown. Murine macrophages in culture were treated with PBS, 500 nM JAK inhibitor, or the NF-␬B inhibitor SN50 at 50 ␮g/ml for 1 h, then with PBS (lanes 1 and 3) or pamidronate (final concentration, 20 ␮M)for2h(lanes 2 and 4), and finally stimulated with IFN-␥ (final concentration, 10 pg/ml) for 16 h (lanes 3 and 4). C, TNF-␣ production by LPS stimulation was markedly reduced by the NF-␬B inhibitor SN50. Murine macrophages in culture were treated with 500 nM JAK inhibitor or 50 ␮g/ml SN50 60 min before stimulation with 5 ng/ml LPS; culture supernatants were subjected to ELISA 16 h after LPS treatment. D, TNF-␣ production resulting from treatment with pamidronate (final concentration, 20 ␮M) was dose dependently reduced by pretreatment with JAK inhibitor, but was unaffected by pretreatment with the NF-␬B inhibitor SN50 at a final concentration of 50 ␮g/ml. different times after IFN-␥ stimulation alone or with pamidronate the cells were positive for STAT1 phosphorylation (Fig. 4C). We also treatment (Fig. 4B). Increased STAT1 expression was detected evaluated intracellular TNF-␣ by double-labeling the cells with anti- 30 min after IFN-␥ stimulation, and this increase was sustained TNF-␣ and anti-phospho-STAT1 Abs. Before treatment with IFN-␥ for up to 3 h after stimulation. This result was unaffected by or pamidronate, no cells were positive for TNF-␣; 2% of the cells pretreatment with pamidronate. Phosphorylation of STAT1 was were positive for TNF-␣ and phospho-STAT1 after treatment with detected 30 min after addition of IFN-␥ and reached a peak at pamidronate alone, and 14% were positive for TNF-␣ and phospho- 1 h, after which it gradually declined. However, pretreatment STAT1 after treatment with IFN-␥ alone. In contrast, 21% of the cells with pamidronate prolonged STAT1 phosphorylation for at were positive for TNF-␣ and phospho-STAT1 after treatment with least 3 h after stimulation (Fig. 4B). IFN-␥ and pamidronate (Fig. 4D). These Western blot and flow cy- To confirm the Western blot findings and to evaluate TNF-␣ pro- tometry findings suggest that prolonged phosphorylation of STAT1 duction by phospho-STAT1-positive cells, we labeled macrophages may be related to increased TNF-␣ production. with anti-phospho-Y701-STAT1 and anti-TNF-␣ Abs and subjected them to flow cytometry 4 h after IFN-␥ treatment. Before treatment with IFN-␥ or pamidronate, 0.5% of the cells were positive for Prolonged phosphorylation of STAT1 results from inhibition of STAT1 phosphorylation. After treatment with pamidronate alone, 9% tyrosine phosphatase activity of the cells were positive for STAT1 phosphorylation. After treatment To determine whether prolonged phosphorylation of STAT1 re- with IFN-␥ alone, 48% of the cells were positive for STAT1 phos- sults from pamidronate-mediated increases in JAK2 kinase activ- phorylation, and after treatment with pamidronate and IFN-␥, 83% of ity, inhibition of tyrosine phosphatase activity, or both of these 1806 PAMIDRONATE-DEPENDENT TNF-␣ PRODUCTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. STAT1 phosphorylation status after treatment of murine macrophages with IFN-␥ or IFN-␥ and pamidronate. A, Cells were treated without (Ϫ) or with (ϩ) pamidronate (final concentration, 20 ␮M) for 2 h before stimulation with IFN-␥ (ϩ; final concentration, 10 pg/ml) for 4 h. Whole cell lysates were analyzed by Western blot with anti-JAK2 and anti-phospho-Y701-STAT1 Abs; stripped blots were reprobed with a generic anti-STAT1 Ab to determine the amount of STAT1 present. Blots were also probed for ␤-actin as a control for protein loading. Similar results were obtained in two repeat studies. B, Time course of phospho-STAT1 expression after treatment for up to 3 h with IFN-␥ (final concentration, 10 pg/ml), no pretreatment (PBS), or pretreatment with pamidronate (final concentration, 20 ␮M) for 2 h. Blots were probed with Abs to phospho-STAT1, STAT1, and ␤-actin, as described above. The bar graph in the lower panel shows the relative densitometric values of the phospho-STAT1 bands. Similar results were obtained in two repeat studies. C, Flow cytometric analysis of phospho-STAT1 expression. Macrophages incubated without (PBS) or with pamidronate (final concentration, 20 ␮M) for 2 h were fixed 4 h after stimulation with or without IFN-␥ (final concentration, 10 pg/ml). D, Percentages of cells determined by flow cytometry to be positive for phospho-STAT1 or phospho-STAT1 and TNF-␣ (1) after treatment with PBS, pamidronate (Pam), IFN-␥, or pamidronate and IFN-␥ (Pam ϩ IFN-␥). Representative data from two independent experiments are shown. mechanisms, we performed pulse-chase experiments using the pro- amounts of phosphorylated STAT1 were detected in PBS-treated tein kinase inhibitor staurosporine, which also inhibits JAK2 (43), and pamidronate-treated cells 30 min after IFN-␥ stimulation, we and measured the residual amount of phosphorylated STAT1 at speculated that pamidronate inhibits tyrosine phosphatase. To de- several time points (30, 35, 40, and 50 min) after treatment of termine whether pamidronate does inhibit protein phosphatase ac- murine macrophages with IFN-␥ alone or IFN-␥ and pamidronate tivity, we conducted an in vitro phosphatase assay using pNPP as (Fig. 5A). We detected a similar amount of phosphorylated STAT1 a substrate. Sodium orthovanadate, a known PTP inhibitor, inhib- in macrophages treated with IFN-␥ alone or IFN-␥ and pami- ited protein phosphatase activity in the cell lysate. However, pamidronate 30 min after IFN-␥ stimulation. However, in macrophages dronate also inhibited protein phosphatase activity in a dose-de- stimulated with IFN-␥ alone, phospho-STAT1 was dephosphory- pendent manner (Fig. 5B). These results suggest that pamidronate lated within 20 min after addition of staurosporine (50 min after prolongs the phosphorylation status of STAT1 Y701 after IFN-␥ IFN-␥ treatment). In contrast, dephosphorylation of STAT1 oc- treatment by inhibiting phosphatase activity. Another possibility is curred at a much slower rate after addition of staurosporine to cells that impaired dephosphorylation of STAT1 results from pami- treated with pamidronate. Because the expression of JAK2 was dronate-mediated interference with the ability of the phosphatase unchanged by addition of pamidronate (Fig. 4A), and similar to bind to phosphorylated STAT1. To investigate this possibility, The Journal of Immunology 1807 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. A, Inhibition of phosphatase activity by pamidronate. Murine macrophages were incubated with or without pamidronate for 2 h before IFN-␥ stimulation. Stimulation with IFN-␥ for 30 min was followed by a staurosporine chase (0.25 ␮M) for another 5–20 min. Cell extracts were analyzed by Western blot with anti-phospho-STAT1, generic anti-STAT1, and anti-␤-actin Abs. The bar graph in the lower panel shows the relative densitometric values of the phospho-STAT1 bands. Similar results were obtained in two repeat studies. B, In vitro phosphatase assay. Purified cell lysates were mixed with the substrate pNPP, and their absorbance at 405 nm was measured at various time points as indicated in the graph. The reaction was performed without pamidronate (control), in the presence of pamidronate at several concentrations (6.5, 12.5, and 25 ␮M), or in the presence of 12.5 ␮M sodium orthovanadate. C, Western blots showing PTPN2- and PTPN11-bound STAT1. Cells were treated with PBS or pamidronate (Pam; final concentration, 20 ␮M) for 2 h before stimulation with IFN-␥ (final concentration, 10 pg/ml) for 4 h. Whole cell lysates were immunoprecipitated with anti-STAT1 Ab, and STAT1 bound to PTPN2 or PTPN11 was detected by Western blot with anti-PTPN2 and anti-PTPN11 Abs. The total amount of immunoprecipitated STAT1 in the lysate was detected by anti-STAT1 Ab (lower panel). D, Western blots showing activation of p38 MAPK and PKR. Cells were treated with PBS or pamidronate (Pam; final concentration, 20 ␮M) for 2 h before stimulation with IFN-␥ (final concentration, 10 pg/ml) for 4 h. Cells were also treated with anisomycin (5 ␮g/ml) for 30 min as a positive control for p38 MAPK phosphorylation. Whole cell lysates were analyzed by Western blot with anti- phospho-p38 MAPK T180/Y182 and anti-phospho-PKR T446/451 Abs; stripped blots were reprobed with a generic anti-p38 MAPK or anti-PKR Ab. we measured the amounts of SHP2 (PTPN11) and TC-PTP with pamidronate, IFN-␥, or pamidronate and IFN-␥. We found no (PTPN2), which is known to dephosphorylate STAT1 Y701, p38 MAPK phosphorylation after any of these treatments. Phos- bound to STAT1 by immunoprecipitating STAT1 from cell ly- phorylated PKR levels were elevated after IFN-␥ stimulation, as sates. There was no significant difference between the amounts of expected, but treatment with pamidronate did not augment phos- STAT1 bound to SHP2 (PTPN11) and to TC-PTP (PTPN2), even phorylated PKR levels (Fig. 5D). These experiments excluded the after treatment with pamidronate or IFN-␥ (Fig. 5C). IFN-␥ also involvement of p38 MAPK and PKR pathways. activates RNA-dependent PKR, and it has a crucial role in the translational regulation of TNF-␣ production (44). TNF-␣ mRNA STAT1 reporter assay and ChIP assay stabilization is regulated by p38 MAPK, which also controls the To determine whether sustained phosphorylation of STAT1 con- stability of several proinflammatory mRNAs (45). We therefore tributes to an increase in transcription of TNF-␣ mRNA, we used investigated activation of p38 MAPK or PKR after stimulation the luciferase assay to assess STAT1-dependent transcription after 1808 PAMIDRONATE-DEPENDENT TNF-␣ PRODUCTION

FIGURE 7. Elevation of serum TNF-␣ concentration in mice by injection of pamidronate and IFN-␥. The serum TNF-␣ concentration was de- FIGURE 6. STAT1-dependent transcription of TNF-␣. A, STAT1 or termined by ELISA. Mice were injected with PBS, pamidronate (Pam), NF-␬〉-dependent activation of transcription as measured by luciferase as- IFN-␥, or pamidronate and IFN-␥ (Pam ϩ IFN-␥). Mean values from three Downloaded from say. We transiently transfected 293T cells with 2ϫM67SJE Luc or NF-␬〉 independent experiment are shown. reporter plasmid and a pRL-SV40 control plasmid. Twenty-four hours after transfection, cultures were incubated with PBS or pamidronate (Pam; final concentration, 20 ␮M) for 4 h, then with or without human IFN-␥ (hIFN␥; final concentration, 50 pg/ml) for 6 h. We prepared whole cell extracts and measured luciferase activity in them according to the manufacturer’s in- TNF-␣ concentration in mice treated with pamidronate and IFN-␥ structions. The graph shows mean values from three independent experi- Ͻ was statistically significantly higher ( p 0.001) than that in mice http://www.jimmunol.org/ ments. The firefly luciferase activity was calculated relative to Renilla lu- treated with pamidronate or IFN-␥ alone. ciferase activity, and the relative increases in firefly luciferase activity were standardized to that observed after PBS treatment alone (ϭ1). f, STAT1 Ⅺ ␬〉 Discussion activation; , NF- activation. B, Murine macrophages were incubated ␥ with PBS or pamidronate (Pam; final concentration, 20 ␮M) for 2 h, then LPS and IL-2 act synergistically with IFN- , activate macro- ␣ treated with or without IFN-␥ (final concentration, 10 pg/ml) for 4 h. phages, and cause enhanced production of TNF- (24, 46–49). Genomic TNF-␣ promoter DNA sites to which phospho-STAT1 was We have shown that treatment of human or murine macrophages bound were identified by ChIP assay. We included a positive control of with pamidronate augments the IFN-␥-stimulated production of 0.1% input of genomic DNA PCR product (Posi). Representative data from TNF-␣, and that in murine macrophages, this pamidronate- two independent experiments are shown. modulated augmentation of TNF-␣ production is regulated at the by guest on September 26, 2021 transcriptional level. Additional findings strongly suggested that pamidronate modulates the JAK-STAT pathway, but not the transfecting 293T cells with a STAT1 reporter plasmid. Pami- NF-␬B pathway, because the NF-␬B inhibitor SN50 was unable to dronate treatment alone activated the STAT1 reporter plasmid suppress pamidronate-mediated augmentation of TNF-␣ produc- (mean, 2.8-fold relative increase in activation; p Ͻ 0.1), as did tion by IFN-␥ and pamidronate-dependent TNF-␣ production, but treatment with IFN-␥ alone (mean, 3.0-fold increase; p Ͻ 0.1); the a JAK inhibitor preferentially suppressed IFN-␥-dependent TNF-␣ combination of IFN-␥ and pamidronate increased STAT1 reporter production, pamidronate-mediated augmentation of TNF-␣ pro- activity (mean, 5.0-fold increase; p Ͻ 0.05). However, no NF-␬〉 duction by IFN-␥, and pamidronate-dependent TNF-␣ production reporter activity was detected under these conditions (Fig. 6A). To (Fig. 3). In addition, we observed prolonged phosphorylation of determine whether this increased STAT1 reporter activity contrib- STAT1 at Y701 after pamidronate pretreatment and IFN-␥ stim- utes to TNF-␣ promoter activity in murine macrophages in culture, ulation, and we showed that this prolonged phosphorylation of we used the ChIP assay. Incubation of cells with IFN-␥ alone for STAT1 results from inhibition of tyrosine phosphatase activity. 4 h induced an increase in the binding of STAT1 to the TNF-␣ We then demonstrated STAT1-dependent transcription of TNF-␣, promoter, whereas pretreatment with pamidronate alone did not; and finally, we confirmed in vivo our in vitro findings by showing the combination of pamidronate with IFN-␥ enhanced the binding an elevation of the serum TNF-␣ concentration in mice injected of phospho-STAT1 to the TNF-␣ promoter (Fig. 6B). with pamidronate and IFN-␥. Treatment of macrophages with pamidronate alone weakly in- ␣ Elevation of serum TNF- concentration after injection of mice duced TNF-␣ production. This induction also involved an increase ␥ with pamidronate and IFN- in vivo in TNF-␣ mRNA expression and increased binding of STAT1 to Our in vitro experiments prompted us to determine whether pami- the TNF-␣ promoter, as shown by the luciferase assay. However, dronate enhances IFN-␥-induced TNF-␣ production in vivo. we did not detect phosphorylation of STAT1 at Y701 by Western Groups of four mice were given i.p. PBS alone, pamidronate (20 blot analysis (Fig. 7). It is difficult to explain the increased TNF-␣ ␮g) alone, IFN-␥ (100 pg) alone, or pamidronate (20 ␮g) and production resulting from treatment with pamidronate alone using IFN-␥ (100 pg). Two hours later, we measured the serum TNF-␣ our hypothesis that sustained phosphorylation of STAT1 results in concentration by ELISA. In PBS-injected mice, the TNF-␣ con- augmentation of TNF-␣ production, but this discrepancy may sim- centration was below the detection limit of the assay (i.e., Ͻ5 ply be explained by different sensitivities of Western blot detection pg/ml). The mean serum TNF-␣ concentration was 77.2 Ϯ 19.3 and flow cytometric detection, given that we observed weak pg/ml in mice injected with pamidronate alone, 55.7 Ϯ 24.3 pg/ml STAT1 phosphorylation by flow cytometry. Furthermore, because in mice injected with IFN-␥ alone, and 433.8 Ϯ 294.1 pg/ml in a JAK inhibitor suppressed TNF-␣ secretion induced by treatment mice injected with pamidronate and IFN-␥ (Fig. 7). The serum with pamidronate alone, it appears that pamidronate itself modifies The Journal of Immunology 1809

pends on these tyrosine phosphatase activities. Our data show that treatment of macrophages with pamidronate before IFN-␥ stimulation elevates phosphorylation of STAT1-Y701. Bisphospho- nates, which are organic pyrophosphonate analogues, may inﬂu- ence biochemical pathways involved in phosphate metabolism, including protein phosphorylation and dephosphorylation. Indeed, several groups have found that alendronate and other bisphosphonates, including pamidronate, can inhibit several PTPs (53–57). The inhibitory effect of these compounds appears to result from oxidation of the active site cysteine residue in PTP. The results of our pulse-chase experiment and in vitro phosphatase assay suggested that pamidronate-treated macrophages have impaired phosphatase activity. These observations strongly suggest that pamidronate acts as a STAT1 tyrosine phosphatase inhibitor. We spec- ulate that if pamidronate inhibits PTPs, it may be a target for PTPN2 or PTPN11; the action of these enzymes on pamidronate would result in sustained phosphorylation of STAT1. Sustainably activated STAT1 may then bind to the TNF-␣ promoter for longer

FIGURE 8. Our proposed model of the interactions among IFN-␥, the than usual, as shown by our ChIP assay. This mechanism (Fig. 8) Downloaded from ␣ JAK-STAT1 pathway, TNF- transcription, and pamidronate in mouse may be invoked in the enhanced production of TNF-␣ we observed ␥ macrophages. Engagement of IFN- with its receptor leads to aggregation after IFN-␥ stimulation after pamidronate treatment. of the receptor complex; sequential phosphorylation of JAK1, JAK2, and ␥ Previous studies have shown increased serum concentrations of IFN- receptor 1 (IFNGR1); and subsequent recruitment of STAT1. ␣ STAT1 is phosphorylated by activated JAK2 kinase and translocated to the TNF- and IL-6 after injection of bisphosphonates in patients with nucleus, where it participates in the regulation of TNF-␣ transcription. increased bone turnover or malignancy (6, 7), but it has not been

Pamidronate acts as a PTP inhibitor and thereby allows sustained phos- clear why bisphosphonates have this effect. In primates, activation http://www.jimmunol.org/ phorylation of STAT1. Previous studies suggested that pamidronate acti- of the V␥2V␦2 class of T cells may be one of the crucial outcomes vates the NF-␬B pathway. This also might result in enhanced TNF-␣ of increased production of proinflammatory cytokines (58, 59). transactivation. The results of our in vitro studies strongly suggest that macrophages are involved in increased production of proinflammatory cytokines. Furthermore, our in vivo study showed that pami- the STAT pathway. However, solely on the basis of our experi- dronate-treated mice exhibit an increased serum concentration of ment with the NF-␬B inhibitor SN50, we cannot exclude involve- TNF-␣ and that combined treatment with pamidronate and IFN-␥ ment of the NF-␬B pathway, as suggested in a previous report enhances TNF-␣ production to a significantly greater extent than (33); examination of NF-␬B knockout mice will provide additional that induced by treatment with IFN-␥ alone. Our in vitro findings by guest on September 26, 2021 insight into this mechanism. Another explanation for the discrep- have allowed us to elucidate one mechanism of pamidronate- ancy is that pamidronate treatment induces IFN-␥ secretion by induced cytokine secretion. As mentioned above, pamidronate has macrophages, the secreted IFN-␥ stimulates the IFN receptor, and been used to treat various diseases, including malignancies. Treatment the macrophages then produce TNF-␣. However, we detected no of malignant tumors with pamidronate commonly results in induction IFN-␥ secretion by pamidronate-stimulated macrophages by of apoptosis, inhibition of tumor cell adhesion and invasion, and an- ELISA (data not shown). Although we used purified macrophages, tiangiogenic effects (60). We now propose that pamidronate-mediated we cannot exclude the possibility that our cell preparations con- augmentation of TNF-␣ production by macrophages could also con- tained residual mononuclear cells. Therefore, pamidronate-medi- tribute to the antitumor effect of aminobisphosphonates. ated TNF-␣ production may result from modulation of the cytokine network by pamidronate. LPS-mediated signaling is also Acknowledgments ␥ known to be involved in the STAT1 pathway (32). IFN- -depen- The 2xM67SJE luciferase plasmid was a gift from Dr. Y. Eugene Chin, the ␣ dent TNF- production probably uses more than one pathway. NF-␬B luciferase plasmid was a gift from Dr. Kenji Tago, and the 3E2 Usually, many cytokine signals are transiently activated by sev- anti-TC-PTP Ab was a gift from Dr. Michael L. Tremblay. We thank eral mechanisms after a ligand interacts with its receptor (e.g., Patricia Wheller and Usha Nair for technical assistance, and internalization of receptor, degradation of the signal-mediating Dr. Kenji Tago for helpful discussions. protein, phosphorylation or dephosphorylation of the signal-mediating protein, and nuclear translocation of the signal-mediating References transcription factor). Indeed, induction of activation of the JAK- 1. Rogers, M. J., S. Gordon, H. L. Benford, F. P. Coxon, S. P. Luckman, STAT pathway by IFN-␥ is regulated in this way (50). Our ex- J. Monkkonen, and J. C. Frith. 2000. Cellular and molecular mechanisms of periments confirmed that phosphorylation of Y701 in STAT1 oc- action of bisphosphonates. Cancer 88:2961. 2. Mundy, G. R., and T. Yoneda. 1998. Bisphosphonates as anticancer drugs. curred transiently after IFN-␥ stimulation, as previously suggested. N. Engl. J. Med. 339:398. PTP plays an important role in the signal transduction pathways 3. Adami, S., A. K. Bhalla, R. Dorizzi, F. Montesanti, S. Rosini, G. Salvagno, and V. Lo Cascio. 1987. The acute-phase response after bisphosphonate administra- that control cellular growth, differentiation, and activity by dephos- tion. Calcif. Tissue Int. 41:326. phorylating phosphorylated tyrosine residues. The extent of ty- 4. Adami, S., and N. Zamberlan. 1996. Adverse effects of bisphosphonates: a com- rosine phosphorylation is controlled by the dynamic equilibrium parative review. Drug Safety 14:158. 5. Schweitzer, D. H., M. Oostendorp-van de Ruit, G. Van der Pluijm, C. W. Lowik, between protein-tyrosine kinases and PTPs (51, 52). The phos- and S. E. Papapoulos. 1995. Interleukin-6 and the acute phase response during phorylation status of STAT1-Y701 also depends on the balance treatment of patients with Paget’s disease with the nitrogen-containing bisphos- between JAK2 activity and tyrosine phosphatase activity. Two phonate dimethylaminohydroxypropylidene bisphosphonate. J. Bone Miner. Res. 10:956. PTPs, PTPN2 and PTPN11, are involved in this regulation (41, 6. Sauty, A., M. Pecherstorfer, I. Zimmer-Roth, P. Fioroni, L. Juillerat, M. Markert, 43). It is speculated that dephosphorylation of STAT1-Y701 de- H. Ludwig, P. Leuenberger, P. Burckhardt, and D. Thiebaud. 1996. Interleukin-6 1810 PAMIDRONATE-DEPENDENT TNF-␣ PRODUCTION

and tumor necrosis factor ␣ levels after bisphosphonates treatment in vitro and in 264 macrophages: the role of a bisphosphonate metabolite. Eur. J. Pharm. Sci. patients with malignancy. Bone 18:133. 8:109. 7. Thiebaud, D., A. Sauty, P. Burckhardt, P. Leuenberger, L. Sitzler, J. R. Green, 34. Senzer, N., S. Mani, A. Rosemurgy, J. Nemunaitis, C. Cunningham, C. Guha, A. Kandra, J. Zieschang, and P. Ibarra de Palacios. 1997. An in vitro and in vivo N. Bayol, M. Gillen, K. Chu, C. Rasmussen, et al. 2004. TNFerade biologic, an study of cytokines in the acute-phase response associated with bisphosphonates. adenovector with a radiation-inducible promoter, carrying the human tumor ne- Calcif. Tissue Int. 61:386. crosis factor ␣ gene: a phase I study in patients with solid tumors. J. Clin. Oncol. 8. Santini, D., B. Vincenzi, G. Avvisati, G. Dicuonzo, F. Battistoni, M. Gavasci, 22:592. A. Salerno, V. Denaro, and G. Tonini. 2002. Pamidronate induces modifications 35. Mueller, H. 1998. Tumor necrosis factor as an antineoplastic agent: pitfalls and of circulating angiogenetic factors in cancer patients. Clin. Cancer Res. 8:1080. promises. Cell. Mol. Life Sci. 54:1291. 9. Pietschmann, P., P. Stohlawetz, S. Brosch, G. Steiner, J. S. Smolen, and 36. Curnis, F., A. Sacchi, and A. Corti. 2002. Improving chemotherapeutic drug M. Peterlik. 1998. The effect of alendronate on cytokine production, adhesion penetration in tumors by vascular targeting and barrier alteration. J. Clin. Invest. molecule expression, and transendothelial migration of human peripheral blood 110:475. mononuclear cells. Calcif. Tissue Int. 63:325. 37. Lejeune, F. J. 2002. Clinical use of TNF revisited: improving penetration of 10. Pennanen, N., S. Lapinjoki, A. Urtti, and J. Monkkonen. 1995. Effect of liposo- anti-cancer agents by increasing vascular permeability. J. Clin. Invest. 110:433. mal and free bisphosphonates on the IL-1␤, IL-6 and TNF␣ secretion from RAW 38. Unkeless, J. C., S. Gordon, and E. Reich. 1974. Secretion of plasminogen acti- 264 cells in vitro. Pharm. Res. 12:916. vator by stimulated macrophages. J. Exp. Med. 139:834. 11. Sugawara, S., M. Shibazaki, H. Takada, H. Kosugi, and Y. Endo. 1998. Con- 39. Thompson, J. E., R. M. Cubbon, R. T. Cummings, L. S. Wicker, R. Frankshun, trasting effects of an aminobisphosphonate, a potent inhibitor of bone resorption, B. R. Cunningham, P. M. Cameron, P. T. Meinke, N. Liverton, Y. Weng, et al. on lipopolysaccharide-induced production of interleukin-1 and tumour necrosis 2002. Photochemical preparation of a pyridone containing tetracycle: a Jak pro- factor ␣ in mice. Br. J. Pharmacol. 125:735. tein kinase inhibitor. Bioorg. Med. Chem. Lett. 12:1219. 12. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to 40. Lin, Y. Z., S. Y. Yao, R. A. Veach, T. R. Torgerson, and J. Hawiger. 1995. interferon-␥. Annu. Rev. Immunol. 15:749. Inhibition of nuclear translocation of transcription factor NF-␬B by a synthetic 13. Darnell, J. E., Jr., I. M. Kerr, and G. R. Stark. 1994. Jak-STAT pathways and peptide containing a cell membrane-permeable motif and nuclear localization transcriptional activation in response to IFNs and other extracellular signaling sequence. J. Biol. Chem. 270:14255. proteins. Science 264:1415. 41. Wu, T. R., Y. K. Hong, X. D. Wang, M. Y. Ling, A. M. Dragoi, A. S. Chung, 14. Darnell, J. E., Jr. 1997. STATs and gene regulation. Science 277:1630. A. G. Campbell, Z. Y. Han, G. S. Feng, and Y. E. Chin. 2002. SHP-2 is a Downloaded from 15. Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al. 1996. Targeted and serine residues in nuclei. J. Biol. Chem. 277:47572. disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in 42. Ramana, C. V., M. Chatterjee-Kishore, H. Nguyen, and G. R. Stark. 2000. Com- the JAK-STAT signaling pathway. Cell 84:431. plex roles of Stat1 in regulating gene expression. Oncogene 19:2619. 16. Ihle, J. N., B. A. Witthuhn, F. W. Quelle, K. Yamamoto, W. E. Thierfelder, 43. ten Hoeve, J., M. de Jesus Ibarra-Sanchez, Y. Fu, W. Zhu, M. Tremblay, B. Kreider, and O. Silvennoinen. 1994. Signaling by the cytokine receptor su- M. David, and K. Shuai. 2002. Identification of a nuclear Stat1 protein tyrosine perfamily: JAKs and STATs. Trends Biochem. Sci. 19:222. phosphatase. Mol. Cell. Biol. 22:5662.

17. Ihle, J. N. 1995. Cytokine receptor signalling. Nature 377:591. 44. Osman, F., N. Jarrous, Y. Ben-Asouli, and R. Kaempfer. 1999. A cis-acting http://www.jimmunol.org/ 18. Ramana, C. V., M. P. Gil, R. D. Schreiber, and G. R. Stark. 2002. Stat1-depen- element in the 3Ј-untranslated region of human TNF-␣ mRNA renders splicing dent and -independent pathways in IFN-␥-dependent signaling. Trends Immunol. dependent on the activation of protein kinase PKR. Genes Dev. 13:3280. 23:96. 45. Brook, M., G. Sully, A. R. Clark, and J. Saklatvala. 2000. Regulation of tumour 19. Vilcek, J., and T. H. Lee. 1991. Tumor necrosis factor: new insights into the necrosis factor ␣ mRNA stability by the mitogen-activated protein kinase p38 molecular mechanisms of its multiple actions. J. Biol. Chem. 266:7313. signalling cascade. FEBS Lett. 483:57. 20. Ruddle, N. H. 1992. Tumor necrosis factor (TNF-␣) and lymphotoxin (TNF-␤). 46. Beutler, B., V. Tkacenko, I. Milsark, N. Krochin, and A. Cerami. 1986. Effect of Curr. Opin. Immunol. 4:327. ␥ interferon on cachectin expression by mononuclear phagocytes: reversal of the 21. Meltzer, M. S., M. Occhionero, and L. P. Ruco. 1982. Macrophage activation for lpsd (endotoxin resistance) phenotype. J. Exp. Med. 164:1791. tumor cytotoxicity: regulatory mechanisms for induction and control of cytotoxic 47. Lee, J. Y., and K. E. Sullivan. 2001. ␥ Interferon and lipopolysaccharide interact activity. Fed. Proc. 41:2198. at the level of transcription to induce tumor necrosis factor ␣ expression. Infect. 22. Alleva, D. G., E. B. Johnson, F. M. Lio, S. A. Boehme, P. J. Conlon, and Immun. 69:2847.

P. D. Crowe. 2002. Regulation of murine macrophage proinﬂammatory and anti- 48. Narumi, S., J. H. Finke, and T. A. Hamilton. 1990. Interferon ␥ and interleukin by guest on September 26, 2021 inﬂammatory cytokines by ligands for peroxisome proliferator-activated recep- 2 synergize to induce selective monokine expression in murine peritoneal mac- tor-␥: counter-regulatory activity by IFN-␥. J. Leukocyte Biol. 71:677. rophages. J. Biol. Chem. 265:7036. 23. Frei, K., C. Siepl, P. Groscurth, S. Bodmer, C. Schwerdel, and A. Fontana. 1987. 49. Narumi, S., J. M. Tebo, J. H. Finke, and T. A. Hamilton. 1992. IFN-␥ and IL-2 Antigen presentation and tumor cytotoxicity by interferon-␥-treated microglial cooperatively activate NF␬B in murine peritoneal macrophages. J. Immunol. cells. Eur. J. Immunol. 17:1271. 149:529. 24. Schook, L. B., H. Albrecht, P. Gallay, and C. V. Jongeneel. 1994. Cytokine 50. Kotenko, S. V., and S. Pestka. 2000. Jak-Stat signal transduction pathway regulation of TNF-␣ mRNA and protein production by unprimed macrophages through the eyes of cytokine class II receptor complexes. Oncogene 19:2557. from C57BL/6 and NZW mice. J. Leukocyte Biol. 56:514. 51. Aoki, N., S. Ueno, H. Mano, S. Yamasaki, M. Shiota, H. Miyazaki, 25. Rendon-Mitchell, B., M. Ochani, J. Li, J. Han, H. Wang, H. Yang, S. Susarla, Y. Yamaguchi-Aoki, T. Matsuda, and A. Ullrich. 2004. Mutual regulation of C. Czura, R. A. Mitchell, G. Chen, et al. 2003. IFN-␥ induces high mobility group protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by ty- box 1 protein release partly through a TNF-dependent mechanism. J. Immunol. rosine phosphorylation and dephosphorylation. J. Biol. Chem. 279:10765. 170:3890. 52. Zhao, Z., Z. Tan, J. H. Wright, C. D. Diltz, S. H. Shen, E. G. Krebs, and 26. Rose, D. M., B. W. Winston, E. D. Chan, D. W. Riches, and P. M. Henson. 1997. E. H. Fischer. 1995. Altered expression of protein-tyrosine phosphatase 2C in 293 Interferon-␥ and transforming growth factor-␤ modulate the activation of mito- cells affects protein tyrosine phosphorylation and mitogen-activated protein ki- gen-activated protein kinases and tumor necrosis factor-␣ production induced by nase activation. J. Biol. Chem. 270:11765. Fc␥-receptor stimulation in murine macrophages. Biochem. Biophys. Res. Com- 53. Schmidt, A., S. J. Rutledge, N. Endo, E. E. Opas, H. Tanaka, G. Wesolowski, mun. 238:256. C. T. Leu, Z. Huang, C. Ramachandaran, S. B. Rodan, et al. 1996. Protein- 27. Hayes, M. P., S. L. Freeman, and R. P. Donnelly. 1995. IFN-␥ priming of mono- tyrosine phosphatase activity regulates osteoclast formation and function: inhi- cytes enhances LPS-induced TNF production by augmenting both transcription bition by alendronate. Proc. Natl. Acad. Sci. USA 93:3068. and MRNA stability. Cytokine 7:427. 54. Skorey, K., H. D. Ly, J. Kelly, M. Hammond, C. Ramachandran, Z. Huang, 28. Nguyen, V. T., and E. N. Benveniste. 2002. Critical role of tumor necrosis fac- M. J. Gresser, and Q. Wang. 1997. How does alendronate inhibit protein-tyrosine tor-␣ and NF-␬B in interferon-␥-induced CD40 expression in microglia/macro- phosphatases? J. Biol. Chem. 272:22472. phages. J. Biol. Chem. 277:13796. 55. Opas, E. E., S. J. Rutledge, E. Golub, A. Stern, Z. Zimolo, G. A. Rodan, and 29. Yao, J., N. Mackman, T. S. Edgington, and S. T. Fan. 1997. Lipopolysaccharide A. Schmidt. 1997. Alendronate inhibition of protein-tyrosine-phosphatase-meg1. induction of the tumor necrosis factor-␣ promoter in human monocytic cells: Biochem. Pharmacol. 54:721. regulation by Egr-1, c-Jun, and NF-␬B transcription factors. J. Biol. Chem. 56. Endo, N., S. J. Rutledge, E. E. Opas, R. Vogel, G. A. Rodan, and A. Schmidt. 272:17795. 1996. Human protein tyrosine phosphatase-␴: alternative splicing and inhibition 30. Raabe, T., M. Bukrinsky, and R. A. Currie. 1998. Relative contribution of tran- by bisphosphonates. J. Bone Miner. Res. 11:535. scription and translation to the induction of tumor necrosis factor-␣ by lipopoly- 57. Murakami, H., N. Takahashi, S. Tanaka, I. Nakamura, N. Udagawa, S. Nakajo, saccharide. J. Biol. Chem. 273:974. K. Nakaya, M. Abe, Y. Yuda, F. Konno, et al. 1997. Tiludronate inhibits protein 31. Liu, H., P. Sidiropoulos, G. Song, L. J. Pagliari, M. J. Birrer, B. Stein, tyrosine phosphatase activity in osteoclasts. Bone 20:399. J. Anrather, and R. M. Pope. 2000. TNF-␣ gene expression in macrophages: 58. Wang, L., A. Kamath, H. Das, L. Li, and J. F. Bukowski. 2001. Antibacterial regulation by NF-␬B is independent of c-Jun or C/EBP␤. J. Immunol. 164:4277. effect of human V␥2V␦2 T cells in vivo. J. Clin. Invest. 108:1349. 32. Ohmori, Y., and T. A. Hamilton. 2001. Requirement for STAT1 in LPS-induced 59. Das, H., L. Wang, A. Kamath, and J. F. Bukowski. 2001. V␥2V␦2 T-cell recep- gene expression in macrophages. J. Leukocyte Biol. 69:598. tor-mediated recognition of aminobisphosphonates. Blood 98:1616. 33. Makkonen, N., A. Salminen, M. J. Rogers, J. C. Frith, A. Urtti, E. Azhayeva, and 60. Clezardin, P. 2002. The antitumor potential of bisphosphonates. Semin. Oncol. J. Monkkonen. 1999. Contrasting effects of alendronate and clodronate on RAW 29:33.