A proliferation-inducing ligand (APRIL) in neutrophils of patients with oral cavity squamous cell carcinoma

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1 Eur. Cytokine Netw. Vol. 23 n 3, July-August-September 212, RESEARCH ARTICLE A prolifertion-inducing lignd (APRIL) in neutrophils of ptients with orl cvity squmous cell crcinom Ew Jbłońsk 1, Ntli Wwrusiewicz-Kurylonek 2, Mrzen Grley 1, Wiolett Rtjczk-Wron 1, Bożen Antonowicz 3, Dorot Dziemińczyk-Pkieł 4, Jkub Jbłoński 5, Adm Krętowski 2, Stnisłw Zyt Grbowsk 4 1 Deprtment of Immunology, Medicl University of Bilystok, Polnd 2 Deprtment of Endocrinology, Dibetology nd Internl Medicine, Medicl University of Bilystok, Polnd 3 Deprtment of Orl Surgery, Medicl University of Bilystok, Polnd 4 Deprtment of Mxillofcil nd Plstic Surgery, Medicl University of Bilystok, Polnd 5 Deprtment of Toxicology, Medicl University of Bilystok, Polnd Correspondence: E. Jbłońsk, Deprtment of Immunology, Medicl University of Bilystok, ul. J. Wszyngton 15A Bilystok, Polnd <ew.jblonsk@umb.edu.pl> Accepted for publiction July 7, 212 To cite this rticle: Jbłońsk E, Wwrusiewicz-Kurylonek N, Grley M, Rtjczk-Wron W, Antonowicz B, Dziemińczyk-Pkieł D, Jbłoński J, Krętowski A, Grbowsk SZ. A prolifertion-inducing lignd in neutrophils of ptients with orl cvity squmous cell crcinom. Eur. Cytokine Netw. 212; 23(3): 93-1 doi:1.1684/ecn ABSTRACT. Avilble dt indicting role for neutrophils in the tumor-host rections re controversil. In 37 ptients with orl cvity squmous cell crcinom (OSCC), we investigted the expression of tumor-promoting, prolifertion-inducing lignd (APRIL) molecule by peripherl blood neutrophils isolted from blood smples collected t presenttion nd three weeks fter surgery, nd the serum levels of TGF- in the sme smples. Additionlly, we investigted the consequences of TLR4 ctivtion by for the synthesis of APRIL by those cells.the levels of mrna for APRIL nd TLR4 were mesured using rel-time PCR method. Western blot nlysis ws used to ssy the expressions of APRIL nd ERK1/2 in cell lystes. The results of the present study reveled the unfvorble fetures of the detection, in the blood, of neutrophils displying n enhnced expression of the tumor-promoting APRIL molecule. The incresed expression nd relese of APRIL ccompnying dvnced stges of disese demonstrted by these cells, combined with the incresed number of neutrophils, my be n importnt mrker of disese progression in the ptient group exmined. Simultneously, n incresed level of circulting TGF- in the serum of these ptients ppered to be ssocited with the overexpression of APRIL in their neutrophils. In contrst to the helthy controls, TLR4 expression nd the ERK1/2 signling pthwy pper to ply only minor roles in APRIL induction in the cells of ptients with cncer. The chnges presented in the current study suggest tht modultion of the expression of tumor-promoting APRIL, in ddition to TRAIL nd BAFF, might be tken into ccount in the development of new strtegies for supportive immunotherpy of OSCC disese nd possibly for other types of neoplsm s well. Key words: orl cvity squmous cell crcinom (OCSCC), prolifertion-inducing lignd (APRIL), polymorphonucler neutrophils (PMNs), Toll-like receptor 4 (TLR4), the extrcellulr signl-regulted kinses 1/2 (ERK1/2), trnsforming growth fctor (TGF- ) doi: /ecn There is good del of evidence suggesting tht chronic inflmmtion is involved in tumor promotion nd progression [1]. Avilble dt indicting role for tumor-infiltrting, inflmmtory cells, such s polymorphonucler neutrophils (PMNs), in the tumor-host rections re controversil [2, 3]. Conflicting results were lso observed in ptients with orl cvity squmous cell crcinom (OCSCC), type of hed nd neck squmous cell crcinom (HNSCC), which is the sixth most frequent cncer in the world. A fvourble nti-tumor spect of neutrophil ctivity, ssocited e.g. with cytotoxic effects of humn neutrophil peptide-1 (HNP1), hs been demonstrted [4]. Unfvourble effects my be cused by oxidtive stress or secretion of prongiogenic fctors by these cells [5, 6]. Importnt meditors in the reltionship between inflmmtion nd cncer include cytokines produced by ctivted immune cells, s well s by cncer cells themselves [1]. Neutrophils re the first cells recruited from peripherl blood to tumor, nd re source of inflmmtory cytokines, modulting the tumor microenvironment. It is known tht these cells lso hve the bility to synthesize cytokines tht cn ply direct role in the prolifertion, growth nd survivl of tumor cells in the locl nd systemic comprtments [7, 8]. The circulting cncer cells observed in ptients with OCSCC re in direct contct with neutrophils in the peripherl blood, s is the cse within the tumor [9]. Our previous study, crried out in ptients with OCSCC, reveled unfvorble fetures of peripherl blood neutrophils, ssocited with chnges in the relese of some proteins belonging to the tumor necrosis fctor (TNF) superfmily. The high secretion of soluble TNFRp75 receptor tht cn limit the vilbility of TNF- nd deficit of tumor-inhibiting TNF-relted poptosisinducing lignd (TRAIL), in ddition to overexpression

2 94 E. Jbłońsk, et l. of tumor-promoting B cell-ctivting fctor (BAFF/Blys) in these cells, my fcilitte tumor development in ptients with OSCC [1-12]. Studies by Mhwech et l. [13] in ptients with OCSCC, suggested tht tumor-infiltrting neutrophils re the min source of nother TNF-superfmily lignd, i.e. prolifertion-inducing lignd (APRIL), exhibiting tumorpromoting ctivity. APRIL, lso known s TNFSF13 or TALL-2, is unique member of the TNF fmily. APRIL exists minly s secreted, soluble lignd nd cn lso be expressed s cell surfce fusion protein with TWEAK, clled TWE- PRIL. APRIL is closely relted to BAFF/Blys. The two lignds shre two receptors: B cell mturtion ntigen (BCMA) nd trnsmembrne ctivtor nd clcium signlmodulting cyclophilin lignd interctor (TACI) [14, 15]. However, neither receptor ppers crucil for the tumorpromoting effects of APRIL. It hs been demonstrted tht the specil receptors or binding prtners for APRIL re heprn sulfte proteoglycns (HSPG), which ply role in the APRIL-medited tumor-promoting ction [16]. It hs been shown tht synthesis nd expression of some TNF superfmily proteins in neutrophils cn be modulted by exogenous nd endogenous fctors, including Toll-like receptor 4 (TLR4) lignds [17]. Toll-like receptor 4 (TLR4) is member of the pttern recognition receptor fmily (PRRs), recognizing minly the Grm-negtive component () nd C. lbicns, respirtory syncytil virus (RSV), s well s endogenous gonists, such s het shock proteins [18, 19] TLR4 plys n importnt role in the initition of signling events tht trigger the inflmmtory response, which cn influence tumor growth [18, 2]. Results from our lbortory demonstrted the role of stimultion in the enhnced secretion of pro-inflmmtory IL-1 nd TNF-, nd incresed secretion of nitric oxide (NO) by neutrophils in ptients with OSCC [6, 12]. Our previous observtions lso indicted the involvement of TLR4 ligtion by in the induction of the APRIL molecule in humn peripherl blood neutrophils. Furthermore, our results suggest tht encourges neutrophils to express APRIL through the ERK1/2 signling pthwy [21]. Among the endogenous fctors, n importnt role in the biology of neutrophils is plyed by trnsforming growth fctor (TGF- ), which seems to be mjor, proximl cytokine within tumors tht defines the tumor-ssocited neutrophils (TAN) phenotype nd skews differentition towrd the N2 protumorigenic phenotype. Friendler et l. [22] suggested tht N2 phenotype cells re the mjority of tumor-ssocited neutrophils (TANs), which my contribute to tumor growth nd immunosuppression. In ptients with OSCC, we investigted the ctivity of peripherl blood neutrophils ssocited with the expression of the APRIL molecule, in reltion to the serum levels of TGF-. We lso exmined the effect of TLR4 ligtion by on the regultion of induction of this molecule. The results obtined indicted tht the enhnced expression nd secretion of APRIL by neutrophils, nd consequently its serum concentrtions, ccompnied tumor progression in ptients with OSCC. Furthermore, the dt presented suggest tht TGF- might be one of the fctors responsible for the chnges in APRIL expression, presumbly ssocited with the polriztion of neutrophils to the N2 phenotype. In contrst to the helthy controls, TLR4 expression nd the extrcellulr signl-regulted kinses 1/2 (ERK1/2) signling pthwy pper to ply only minor roles in APRIL induction in the cells of cncer ptients. The results obtined confirm pro-tumorigenic ctivity of neutrophils, ssocited with the bility of these cells to synthesize some of the lignds belonging to the TNF superfmily. DONORS AND METHODS Ptients We exmined 37 ptients with squmous cell crcinom of orl cvity (ged 45 to 59), treted in the Deprtment of Mxillofcil nd Plstic Surgery t the Medicl University of Bilystok. Study results were nlyzed tking into ccount the clinicl stge of the disese ccording to TNM clssifiction (tble 1). Exmintions were crried out on ptients t presenttion nd three weeks fter the surgicl removl of the tumor mss. For one week fter surgery, ptients received morphine sulphte penthydrte (1 mg every 6 h for three dys, Polf Trchomin S.A, Polnd), prcetmol (1, mg every 12 h for seven dys, Perflgn; Bristol-Myers Squibb Phrmceuticls, UK) for postopertive pin, nd the ntibiotic cephlosporin (1g every 12 hours for seven dys, Trfzolin, Polf Trchomin S.A). Eighty percent of ptients were long-time tobcco users. Control subjects (n = 15) were non-smoking, helthy volunteers ged from 3 to 6 yers (men ± SD: 42.5 ± 15.3 yers). None of the ptients or control subject hd concomitnt diseses such s dibetes mellitus, liver disese, or rheumtoid rthritis. Tble 1 Ptient chrcteristics. All ptients 37 Mle 29 Femle 8 Tumor locliztion Orl cvity fundus 1 Tongue 5 Tongue + orl cvity fundus 5 Cheek mucos 6 Inferior gingivl 5 Lower lip 6 TNM clssifiction T1/2 13 T3/4 24 PMN count (%) T1/ T3/4 71.9

3 Role of neutrophils in tumor-bering host 95 Three weeks following surgery, no clinicl signs of infection were observed in ptients. Ptients hd no significntly incresed leukocytosis. The study ws pproved by the Ethics Committee of the Medicl University of Bilystok, nd ll ptients submitted their consent in writing. Isoltion nd culture of PMNs Peripherl blood neutrophils were isolted from blood smples collected t presenttion nd three weeks fter surgery. The cells were isolted from peripherl blood treted with EDTA by wy of density centrifugtion, using Polymorphprep (Axis-Shield, Oslo, Norwy) (density: g/ml). After wshing in PBS without CCl 2 nd MgCl 2 (GIBCO, Gret Britin), the PMNs were seprted by mgnetic selection. PMNs were seprted by positive selection using Midi MACS mgnetic seprtion system (Miltenyi Biotec, Germny). For the seprtion, MicroBeds conjugted to monoclonl nti-humn CD16 ntibodies were used. The MACS column ws plced in the mgnetic field of suitble MACS seprtor nd rinsed with MACS buffer. Isolted PMNs were suspended in MACS buffer (up to totl cells) nd incubted with CD16 MicroBeds for 3 min t 4-8 C. After wshing, the cells were suspended in MACS buffer. The purity of the isolted PMNs, determined by My-Grunewld-Giems-stining, ws 99%. The seprted PMNs were suspended in the culture medium (RPMI-164) to provide cells/ml nd incubted in flt-bottomed, 96-well pltes (Microtest III- Flcon, Frnklin Lkes, USA) for 4 h t 37 C in humidified incubtor with 5% CO 2 (NUAIRE ). (1 ng/ml, Sigm) ws tested to stimulte the expression of TLR4 nd APRIL in PMNs. The vibility of PMN mesured fter incubtion ws 94%. In cells from the sme blood smples, we investigted the role of ERK1/2 kinse in APRIL protein induction in PMNs t the protein level. For this purpose, p42/p44 MAPK inhibitor PD9859 (2 -mino-3 -methoxyflvone), peptide tht specificlly inhibits ERK1/2 ctivtion, ws used. After isoltion, the cells were treted with PD9859 (Clbiochem, Bd Soden, Germny) (4 M) for 1 h before nd during incubtion. The presence of inhibitor did not ffect cell vibility. RNA isoltion nd cdna synthesis For rel-time PCR, totl RNA ws isolted from 1 7 untreted nd stimulted neutrophils. An RNesy Mini Kit, Qigen, Germny, ws used to isolte totl RNA from PMNs, ccording to the mnufcturer s specifiction. The mount of RNA ws mesured by spectrophotometry (QuntGen Biophrmci). RNA integrity ws verified by 1.5% grose gel electrophoresis, identified by ethidium bromide stining, nd n OD 26/28 bsorbnce rtio >1.95. One microgrm of totl RNA ws used to prepre cdna. cdna synthesis ws performed using SuperScript TM First-Strnd Synthesis System for RT-PCR (Invitrogen) ccording to the mnufcturer s specifictions in MJ Reserch Therml Cycler (Model PTC-2, Wtertown, MA, USA). Rel-time PCR The levels of trnscripts were mesured by rel-time PCR using humn genes QuntiTec Hs_TLR4_2_SG Assy (Qigen), QuntiTec Hs_TNFSF13_2_SG Assy (Qigen) nd QuntiTec Hs_PRS18_1_SG Assy (s18) (Qigen) s normlizer. Rel-time PCR ws performed in duplicte in 2 l using the QuntiTect SYBR Green PCR Mster Mix (Qigen) following the mnufcturer s instruction, nd crried out in the Chromo4 Rel-time PCR Detector (BIO-RAD, USA). The therml cycling conditions included n initil ctivtion step t 95 C for 15 min, followed by 4 cycles of denturtion, nneling nd mplifiction (95 C for 3 s, 55 C for 3 s, 72 C for 3 s). At the end of the mplifiction phse, melting curve nlysis ws crried out on the product formed. The fluorescent dt collection ws performed during the nneling step. A stndrd curve construction ws generted employing series of four dilutions of cdna derived from unstimulted cells in rection with the house-keeping gene s18. Bsed on these curves, the levels of totl TLR4 nd TNFSF13 trnscripts were clculted fter normliztion of TLR4 nd TNFSF13 products to s18. The vlue of C T ws determined by the first cycle number t which florescence ws greter tht the set threshold vlue. To clculte our dt, we used the comprtive C T method for reltive quntifiction ( C T method). Western blot nlysis Cytoplsmic protein frctions of PMNs were nlyzed using western blotting for the presence of the APRIL protein nd the ctivtion of ERK1/2 kinses, by determining the phosphoryltion sttus of ERK1/2 (p-erk1/2). Cells were lysed directly in the presence of protese inhibitor cocktil (Sigm-Aldrich, CHEMIE GmbH P.O. Steinheim, Germny) by soniction, using Vibr- Cell Ultrsonic Processor (Sonics&Mterils, Inc., USA). Protein frctions were suspended in Lemmli buffer (Bio-Rd Lbortories, Herkules CA, USA), nd then electrophoresed on SDS-PAGE. The resolved protein ws trnsferred onto.45 m pore-sized nitrocellulose (Bio- Rd Lbortories, Hercules CA, USA). The nitrocellulose ws incubted with the primry polyclonl ntibody nti- APRIL (R&D Systems) nd nti-phospho ERK1/2 (1:1; Snt Cruz Biotechnology, Heidelberg, Germny). After wshing in.1% TBS-T, the membrne ws incubted with lkline phosphtse nti-mouse IgG Abs (Vector Lbortories, Burlingme, CA, USA). Immunorective protein bnds were visulized following the ddition of BCIP/NBT Liquid Substrte System (Sigm-Aldrich, Steinheim, Germny). Bnds intensity ws quntified using the ImgeJ method. The ntibody ginst bet-ctin (1:1; Snt Cruz Biotechnology), which detects the expression of bet-ctin in cells lystes, ws used s n internl control. Cytokine mesurement by ELISA ssys Soluble APRIL concentrtions in the culture superntnts of PMNs nd the serum were ssessed using ELISA kits by R&D Systems (Minnepolis, USA). The serum concentrtions of TGF- were mesured using n ELISA kit from BioSource Int. (Cliforni, USA).

4 96 E. Jbłońsk, et l. Sttisticl nlysis Sttisticl nlysis ws performed with sttistics pckge - Sttistic 6. softwre. The nonprmetric U Mnn- Whitney test ws used. Results were expressed s medin, minimum, nd mximum vlues. For nlysis correltion, Person s liner correltion ws used. p-vlues below.5 were considered sttisticlly significnt. RESULTS Expression of APRIL-mRNA nd TLR4-mRNA nlyzed by rel-time PCR Rel-time PCR nlysis reveled differences in the expression of APRIL-mRNA between neutrophils in OSCC ptients in erly stges of disese (T1/2) nd ptients in dvnced stges (T3/4) (figure 1). Unchnged expression of APRIL-mRNA levels in unstimulted neutrophils from ptients in erly phses of disese t presenttion ws observed. Incresed expression of this molecule in cells stimulted by ws lso observed. In contrst, in ptients in dvnced stges of disese, we found n incresed expression of APRIL-mRNA in unstimulted neutrophils. No chnges were observed fter stimultion (figure 1). Furthermore, levels of APRIL-mRNA in neutrophils from ptients in stges T3/4 were higher thn those in the cells from ptients in stges T1/2. It is importnt to note tht the expression of APRIL-mRNA in the cells of ll ptients (stges T1/2 nd T3/4) following surgicl tretment remined t the sme levels s before tretment (figure 1). Reltive expression of mrna APRIL 1,6 1,4 1,2 1, medin±min/mx n = 15 n = 13 n = 24 * * Control Before After Before After b T1/2 T3/4 Figure 1 Expression of APRIL-mRNA in neutrophils from ptients with OSCC before nd fter tretment. RT-PCR ws performed on humn neutrophils nd mononucler cells from peripherl blood, n = the number of persons studied. APRIL-mRNA level ws normlized to house-keeping gene s18. * Sttisticlly significnt differences between unstimulted nd stimulted cells (p<.5), sttisticlly significnt differences with control (p<.5), b sttisticlly differences between T1/2 nd T3/4, c sttisticlly significnt differences between before nd fter tretment. Ech vlue represents the medin±minimum nd mximum. c Reltive expression of TLR n = 15 n = 13 n = 24 * medin±min/mx + + Control Before After Before After b T1/2 T3/4 Figure 2 Expression of TLR4-mRNA in neutrophils of ptients with OSCC before nd fter tretment. RT-PCR ws performed on humn neutrophils nd mononucler cells from peripherl blood, n = the number of persons studied. The TLR4-mRNA level ws normlized to housekeeping gene s18. * Sttisticlly significnt differences between unstimulted nd -stimulted cells (p<.5), sttisticlly significnt differences with control (p<.5), b sttisticlly differences between T1/2 nd T3/4, c sttisticlly significnt differences between before nd fter tretment. Ech vlue represents the medin ± minimum nd mximum. Similrly to APRIL expression, levels of TLR4-mRNA were unchnged in unstimulted neutrophils from ll ptients before tretment (figure 2). TLR4 expression in the cells of ptients in dvnced stges, ws very similr to tht found in ptients in erly stges of disese. Furthermore, -stimultion did not hve significnt effect on TLR4 expression in the cells. After tretment, there were no significnt chnges in the expression of TLR4-mRNA in neutrophils from ptients in erly stges, s compred to results obtined in the cells of ptients before tretment. Surprisingly, the expression of TLR4-mRNA in ptients in dvnced stges ws higher thn in the cells of ptients before tretment. Expressions of APRIL nd phospho-erk1/2 in PMNs in cncer ptients, ssessed using the Western blot method The presence of APRIL protein (27 kd) nd phospho- ERK1/2 (44/42 kd) ws shown in the neutrophils exmined (figure 3). PMNs from ptients t stges T1/2 before nd fter tretment demonstrted decresed in the expression of APRIL protein in comprison with the cells of the control group. In contrst to APRIL expression, significntly incresed expression of phospho-erk1/2 kinses in comprison to the controls ws found in the lystes of PMNs from ptients before tretment. ERK1/2 expression in the cells of ptients fter tretment ws lower thn tht seen in cells of ptients before tretment. stimultion led to incresed expression of APRIL nd phospho-erk1/2 in cells of ptients before tretment. bc bc

5 Role of neutrophils in tumor-bering host 97 controls - ptients T1/2 below controls - ptients T3/4 below APRIL control n = 15 before n = 13 fter n = 13 APRIL control n = 15 before n = 24 fter n = 24 perk1/2 perk1/2 Bet-Actin Bet-Actin Arbitrry units Arbitrry units 12, 1, 8, 6, 4, 2, 12, 1, 8, 6, 4, 2, n = 15 n = 13 n = 13 * APRIL * perk1/2 n = 15 n = 13 n = 13 * c c Figure 3 Western blot nlysis of APRIL nd phospho-erk1/2 protein expression in neutrophils of ptients t stge T1/2, before nd fter tretment. A) unstimulted PMNs from controls, B) -stimulted PMNs from controls, C) unstimulted PMNs from ptients before tretment, D) -stimulted PMNs from ptients before tretment, E) unstimulted PMNs from ptients fter tretment, F) -stimulted PMNs from ptients fter tretment, n: the number of persons studied, * sttisticlly significnt differences between unstimulted nd -stimulted cells (p<.5), sttisticlly significnt differences compred to controls (p<.5), c sttisticlly significnt differences between before nd fter tretment. In ptients with dvnced stges of disese (T3/4), the expression of APRIL protein in neutrophils ws incresed in comprison with controls nd cells from ptients in erly stges of disese. In contrst, expression of phospho- ERK1/2 in neutrophils ws lower thn tht found in the controls (figure 4). stimultion did not led to n increse in the expressions of APRIL nd ERK1/2 proteins in the cells of this ptient group. APRIL protein expression in PMNs ws higher in ptients fter tretment thn in ptients before tretment. No significnt chnges in the expression of ERK1/2 protein levels in the ptients were found fter tretment. The bove observtions suggest lrgely insignificnt role of ERK1/2 kinses in APRIL induction in neutrophils of OSCC ptients. Arbitrry units Arbitrry units 12, 1, 8, 6, 4, 2, 12, 1, 8, 6, 4, 2, APRIL c n = 15 n = 24 n = 24 c perk1/2 n = 15 n = 24 n = 24 Figure 4 Western blot nlysis of APRIL nd phospho-erk1/2 protein expression in neutrophils of ptients t stge T3/4, before nd fter tretment. A) unstimulted PMNs from controls, B) -stimulted PMNs from controls, C) unstimulted PMNs from ptients before tretment, D) -stimulted PMNs from ptients before tretment, E) unstimulted PMNs from ptients fter tretment, F) -stimulted PMNs from ptients fter tretment, n: the number of persons studied, sttisticlly significnt differences compred to controls (p<.5), c sttisticlly significnt differences between before nd fter tretment. APRIL concentrtions in superntnts of cells nd serum The ELISA dt showed tht the relese of APRIL by PMNs in cncer ptients in erly stges, before nd fter surgicl tretment, remined unchnged in comprison to its relese by cells in the control group (tble 2). In ptients with dvnced stge disese, before tretment, the secretion of APRIL by PMNs ws higher thn tht seen in the control cells nd cells of ptients in erly stges of disese. After tretment, significntly decresed secretion of APRIL by cells ws observed. Next, we mesured the serum concentrtions of APRIL in ll ptients. We found incresed concentrtions of APRIL in ptients t stges T3/4 before tretment, in comprison to controls nd ptients t stges T1/2 (tble 2). Exmintions

6 98 E. Jbłońsk, et l. Tble 2 APRIL concentrtions in superntnts of PMNs, nd the serum nd TGF- concentrtions in the serum of ptients with OSCC. Control n=15 T1/2 n=13 APRIL (ng/ml) Ptients before tretment T3/4 n=24 T1/2 n=13 Ptients fter tretment T3/4 n=24 PMNs 2.45 ± ± b ± ± b ± 1.22 Serum 2.1 ± ± b ± ± ± 3.33 TGF- (ng/ml) Serum 2.47 ± ± b ± ± b ± 6.8 significnt differences with control (p<.5) b significnt differences with T1/2 (p<.5) n : number of persons studied fter tretment showed tht the serum levels of APRIL in ll ptients were t the sme levels s those in ptients before tretment (tble 2). TGF- serum concentrtions The ELISA dt lso showed incresed concentrtions of TGF- in the serum of ptients with erly nd dvnced stges of disese, s compred to helthy controls (tble 2). The concentrtions of TGF- in the serum of ptients t stges T3/4 were higher thn those in ptients t T1/2 stges. There were no significnt chnges in TGF- concentrtions in the serum of ptients fter tretment. DISCUSSION A number of clinicl nd experimentl studies hve focused on the mesurement of cytokine expression s prmeters of the immune potentil of cncer ptients. Avilble dt, including our own observtions, indicte tht the production of cytokines by humn neutrophils my hve different effects on tumor development [7, 8, 1, 11]. The results of the present study, crried out on orl cvity cncer ptients, reveled unfvorble fetures of neutrophils ssocited with the chnged bility to produce the tumor-promoting APRIL molecule. The demonstrted incresed expression nd relese of APRIL by these cells ccompnying dvnced stges of disese, combined with the incresed number of neutrophils, my be n importnt mrker of disese progression in the ptient group exmined. Results involving neutrophil counts in ptients with OCSCC re in greement with those demonstrted by Trellkis et l. [23], who lso observed systemic differences in the PMN comprtment ssocited with tumor size in HNSCC ptients with orl cvity disese. Incresed circulting peripherl blood neutrophil numbers hve lso been identified s poor prognostic fctor in other cncer diseses, e.g. renl cell crcinom [24]. Incresed expression nd secretion of APRIL by neutrophils, together with n increse in serum concentrtions of APRIL in ptients with dvnced OCSCC, observed in the present study, my directly nd/or indirectly influence tumor development vi interction with HSPG on tumor cells [13]. The direct effect ssocited with APRIL-induced tumor prolifertion ws confirmed by the inhibition of APRIL binding to HSPG, which prevented the induction of tumor cell prolifertion [16]. The indirect effect of APRIL-HSPG interctions resulting from binding of HSPG to the extrcellulr mtrix (ECM) nd/or different soluble lignds, my led to the modultion of tumor cell dhesion, their migrtion nd invsion [16, 25]. Another spect of the tumor-promoting ction of APRIL my be dependent upon the protection of the tumor cells from poptosis. It ws demonstrted tht APRIL, through the ctivtion of NF- B or MAPK kinses, leds to strong up-regultion of nti-poptotic proteins, such s Mcl-1 nd Bcl-2 belonging to Bcl-2 fmily of proteins. High expression of Mcl-1 nd Bcl-2 my significntly inhibit the mitochondril pthwy of poptosis in cncer cells [26]. Immunochemistry investigtion in ptients with orl squmous cell crcinom showed different expressions of Bcl-2 protein within the tumor [27, 28]. However, it hs been suggested tht ptients with greter Bcl-2 expression hve worse prognosis; its low level seeming to be ssocited with higher survivl rte [28, 29]. It is interesting to note tht, in contrst to neutrophils from helthy control, chnges in the expression of APRIL in cells of ptients in dvnced stges of disese, before nd fter tretment, ws ccompnied by diverse chnges in expression of the TLR4 receptor. The reltionship between these proteins suggest the presence of distinct mechnisms responsible for their ctivtion in the neutrophils of the tumor-bering host thn those in control cells. The bove suggestion ppers to be confirmed by the differences in the expression of APRIL nd perk1/2 kinses, observed in neutrophils from the ptients exmined. Reltionships between these proteins suggest tht ERK1/2 does not ply the role of criticl messenger in the induction of APRIL in these cells. It is not cler whether this is reflection of the modifiction of neutrophil function cused by tumor cells or the reson for the chnges ssocited with tumor progression. One of the possible explntions of the distinct mechnism of APRIL induction in neutrophils of cncer ptients my be the presence of circulting meditors, such s immunosuppressing TGF-. Recent studies by Jng et l. [3] on mouse mcrophges showed tht TGF- is cpble of stimulting APRIL expression in these cells. The source of TGF- in ptients with OSCC my be the neutrophils, s well s Treg, whose count significntly incresed in the circultion [31, 32]. The presence of lrge mounts of TGF- my induce the formtion of protumorigenic N2

7 Role of neutrophils in tumor-bering host 99 phenotype neutrophils, nlogous to the N2 cells presented in the microenvironment of tumor [22]. The incresed concentrtions of TGF- observed in the serum of ptients with OSCC ccording to tumor progression, pper to confirm the role of this cytokine in the modultion of APRIL expression in neutrophils nd my be one of mrkers of the N2 phenotype. Simultneously, the demonstrted, unltered expression of TLR4 in these cells suggests tht TGF- does not hve impct on the ctivity of neutrophils medited by this receptor in OSCC ptients. In conclusion, the results obtined prove tht the tumorpromoting properties of neutrophils in ptients with orl cvity cncer my be cused not only by enhnced inflmmtory ctivity, but lso by the chnges in the synthesis nd secretion of the APRIL molecule. The dt presented herein, together with previously reported ltertions in expression of other TNF superfmily lignds, pper to be n obvious indiction of the unfvorble role of these cells in ptients with OSCC nd other types of neoplsms. This might be confirmed by our studies crried out in ptients with B-cell chronic lymphocytic leukemi (B-CLL) tht reveled similr reltionships between APRIL, BAFF nd TRAIL secretion by neutrophils [33]. Bsed on the bove observtions, it cn be ssumed tht the ltertions in TNF superfmily secretion by these cells re independent of the type of neoplsm. Further investigtion will be helpful to explin fully the effective role of neutrophils in tumor-host rections. However, the chnges presented in the current study suggest tht modultion of the expression of tumor-promoting APRIL, in ddition to TRAIL nd BAFF, might be tken into ccount in the development of new strtegies for supportive immunotherpy of OSCC disese nd possibly for other types of neoplsm s well. 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