Summary. Original Article. Postepy Hig Med Dosw (online), 2016; 70: e-issn

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1 Postepy Hig Med Dosw (online), 2016; 70: e-issn Originl Article Received: Accepted: Published: Effects of Grnulocyte Colony-Stimulting Fctor on Opsonin Receptor Expression nd Neutrophil Antibcteril Activity in Mouse Model of Severe Acute Pncretitis Wpływ czynnik stymulującego tworzenie kolonii grnulocytów n ekspresję receptor opsoninowego i przeciwbkteryjną ktywność neutrofilów w mysim modelu ostrego zpleni trzustki Hong-Fng Tuo B,C,F, Yn-Hui Peng B,D, Lei Bo C,F, Wn-Xing Zhng A Deprtment of Heptobiliry Surgery, Hebei Generl Hospitl, No. 348 of Shijizhung Heping Xilu, Shijizhung, Hebei Province, Chin Summry The ntimicrobil function of neutrophils, which is dependent on opsonin receptors, deteriortes in severe cute pncretitis (SAP). Grnulocyte colony-stimulting fctor (G-CSF) puttively enhnced levels of the opsonin receptors CD11b nd CD32/16 in helthy humn subjects, nd provided protection ginst infection in niml models of SAP. A sttisticlly convincing study of the effect of G-CSF on CD32/16 expression in n SAP model is lcking. We used mouse model of SAP to investigte the ssocition between G-CSF dministrtion nd CD32/16 levels on neutrophils nd bcteril trnsloction. G-CSF or sline ws subcutneously injected into SAP-induced mice. The pncreses were histologiclly exmined, nd leukocytes were stined to count neutrophils. The expression of CD11b nd CD32/16 on neutrophils ws mesured by flow cytometry, nd bcteril trnsloction ws observed by bcteril culture. The numbers of CD11b nd CD32/16-positive neutrophils were significntly elevted in the SAP mice treted with G-CSF, nd the men fluorescence intensities of these receptors on neutrophils were significntly elevted. Bcteril trnsloctions to cvity orgns were suppressed from 17% to 6% by G-CSF tretment. Our results indicted tht the number of neutrophils significntly incresed with incresing expression of CD11b nd CD32/16 nd their men fluorescence intensities (MFIs). This inhibited bcteril trnsloction to other orgns. These results re in ccord with other studies in SAP dogs nd SAP mice. Our findings suggest tht G-CSF ws effective in protecting ginst bcteril infection in SAP mice. Keywords: CD11b CD32/16 grnulocyte colony-stimulting fctor pncretitis severe cute pncretitis mouse model 352 Postepy Hig Med Dosw (online), 2016; 70

2 Tuo HF et l. Effects of Grnulocyte Colony-Stimulting Fctor... Full-text PDF: DOI: Word count: Tbles: Figures: References: / Authors ddress: Yn-Hui Peng, Deprtment of Heptobiliry Surgery, Hebei Generl Hospitl, No. 348 of Shijizhung Heping Xilu, Shijizhung, Hebei Province, Chin ; e-mil: yhpeng @sin.com Introduction Severe cute pncretitis (SAP) cuses the filure of locl nd systemic bcteril clernce, nd the incidence rtes of bcteremi nd mortlity re proportionl to the severity of the disese [5]. Infected pncretic necrosis is primrily considered lte compliction in the course of cute pncretitis, but in lmost one of four ptients it lso occurs erly [11]. Neutrophil grnulocytes (neutrophils), which mke up the mjority of white blood cells (leukocytes), re importnt constituents of the innte immune system. There is evidence tht the defensive functions of neutrophils deteriorte in SAP. The migrtion nd phgocytic functions of neutrophils re strictly controlled by the opsonin receptors on their surfce [1,16]. The min opsonin receptors re cluster of differentition molecule 11B (CD11b) nd CD32/16 [5,7]. CD11b nd CD32/16 levels re elevted in mice with edemtous pncretitis, but ttenuted in SAP [7,13,15]. Grnulocyte colony-stimulting fctor (G-CSF) hs been shown cliniclly to stimulte the survivl, prolifertion, differentition, nd function of neutrophil precursors nd mture neutrophils [2,3,8,14]. Other studies hve endevored to improve neutrophil function through G-CSF. G-CSF enhnced the expressions of CD11b nd CD32/16 nd the phgocytic functions of neutrophils in helthy volunteers [8]. The dministrtion of G-CSF ws lso ssocited with induced CD16 expression in two ptients complicted by bcteril infections, nd these ptients recovered from or evded infection [10]. However, the smll number of humn ptients nd helthy volunteers in these studies limited their sttisticl power. In cnine model of SAP, tretment with G-CSF ws ssocited with significnt nd sustined increse in mture grnulocytes, reduced bcteril trnsloction, nd decresed rte of distnt infection [12]. In rt SAP model, dministrtion of G-CSF ppered to increse the number of neutrophils nd enhnce their ntibcteril functions [4]. However, the expression of CD32/16 ws not investigted in these two in vivo studies [4,12], nd the effect of G-CSF on CD32/16 levels is unknown. In the present study, we hypothesized tht G-CSF CSF might promote CD32/16 expression on neutrophils, thus inhibiting bcteril trnsloction to cvity orgns in SAP mice. To evlute the effects of G-CSF on the ntibcteril ctivity of neutrophils, we directly investigted the expression of CD32/16 on neutrophils nd bcteril trnsloctions in mouse model of SAP. Mteril nd Methods Animls Femle Blb/c mice (8 weeks old nd g) were supplied by Cler Corportion of Jpn (Tokyo, Jpn). The cre nd hndling of the nimls were in ccordnce with the Guidelines of the Ntionl Institutes of Helth of the United Sttes. The mice were cclimted in our niml fcility for one week prior to the experiments. The Surgicl Reserch Committee nd Animl Administrtive Advisory Committee of Kyorin University, Jpn pproved this study. Experimentl Design Thirty mice were rndomly pportioned to one of the following three groups: SAP (SAP, n = 12), SAP with G-CSF tretment (SAP-G, n = 12), or norml control group without SAP or G-CSF tretment (NC, n = 6). SAP ws induced fter the mice were fsted for 6 hours vi 13 hourly subcutneous injections of 0.2 ml of sline with 50 μg/kg body weight ceruletide (Sigm-Aldrich Jpn, Tokyo, Jpn). The G-CSF group ws treted with G-CSF (120 μg/kg body weight; Nrtogrstim from Kyow Hkko Kogyo, Tokyo, Jpn), while the NC group received the sme volume of sline, subcutneously injected into the mice every 12 hours, beginning 2 hours fter the finl injection of ceruletide. The mice were euthnized fter fsting for 6 hours in the NC group, nd 12 hours or 24 hours fter the finl injection of ceruletide in the SAP nd SAP-G groups. Lvge of the peritonel cvity ws performed with phosphte buffered sline (PBS), nd the peritonel fluid ws collected. Heprinized blood (100 units/ml) ws obtined from the inferior ven cv. The pncres, liver, nd mesenteric tissues were hrvested for pncretic histologicl exmintion nd bcteril culture t ech time point. 353

3 Postepy Hig Med Dosw (online), 2016; tom 70: Histologicl Exmintion The hrvested pncreses were fixed in 10% formldehyde for 24 hours. After hemtoxylin nd eosin stining, the pncreses were histologiclly exmined nd pncretitis ws grded bsed on the degrees of cinr cell vcuoliztion, necrosis, nd inflmmtory cell infiltrtion ccording to the method of Niederu et l. [9]. The two exminers were blinded to the tretment nd control groups. Amylse Assy Plsm mylse ctivity ws mesured 12 nd 24 hours fter the induction of cute pncretitis using CNP-G7(5) s the substrte (Knto Kgku, Tokyo, Jpn) on n utomted nlyzer (7070 Type, Hitchi, Tokyo, Jpn). Leukocyte Counting Circultory leukocytes (per mm 3 ) nd peritonel exudtive leukocytes (totl number in peritonel lvge fluid) were stined with Turcki solution (Muto Chemicl, Tokyo, Jpn) nd counted. Smple Preprtion nd Mesurement of Expression Levels of CD11b nd CD32/16 The monoclonl ntibodies used in this study were fluorescein isothiocynte-conjugted rt nti-mouse CD11b, which rects specificlly with the 170-kD α chin of Mc-1, nd r-phycoerythrin-conjugted rt nti-mouse CD32/16 immunoglobulin G, which rects specificlly with common nonpolymorphic epitope on the extrcellulr domins of mouse FcγRII nd FcγRIII receptors immunoglobulin G (Phrmingen, Sn Diego, CA). Cells were considered to stin positive for CD11b or CD32/16 if they exceeded the fluorescence of 99.95% of the isotype-mtched control (rt IgG2bκ) cells. Peritonel lvge fluids were centrifuged t 600 g for 5 minutes, nd the peritonel exudtive cells were resuspended in 400 μl of PBS. One hundred microliters of blood or peritonel exudte cell smple ws incubted with nti-cd11b or nti-cd32/16 monoclonl ntibody t 40 C in the drk for 30 minutes. Erythrocytes were then lysed with 2 ml of FACS lysis buffer (168 mm NH 4 CL, 10 mm KHCO 3, nd 10 mm EDTA-4N). The remining leukocytes were wshed with PBS supplemented with 0.1% sodium zide nd 0.1% bovine serum lbumin (Gibco Lbortories, Grnd Islnd, NY) nd fixed in 0.5 ml of 1% prformldehyde for 5 minutes. Finlly, the cells were wshed with PBS nd stored t 4 C in the drk until nlyzed with flow cytometry. Flow Cytometry Flow cytometry ws performed using FACScn (Becton Dickinson Immunocytometry Systems, Sn Jose, CA). For ech smple, 104 leukocytes were enumerted. Neu- trophils were gted using morphologic chrcteristics displyed on dot plot of forwrd light sctter versus side light sctter. Of these neutrophils, 95% were stined with the 7/4 ntibody, which is monoclonl ntibody tht rects with polymorphic neutrophil differentition ntigen present on neutrophils of specific mouse strins. The percentge of totl neutrophils or CD mrker-positive neutrophils mong circultory or peritonel exudtive leukocytes ws clculted. The bsolute number of CD mrker-positive neutrophils ws clculted s the circultory or peritonel exudtive leukocyte count multiplied by the pproprite percentge. Microbiologicl Anlysis Liver, pncres, nd mesenteric tissues were homogenized by crushing nd mixing them with serum. Petri dishes with blood gr medium were incubted t 37 C for 24 hours. Cells were stined with grm stin nd observed under light microscope. Grm-negtive bcteri were identified using McConkey gr supplemented with 10% lctose, nd grm-positive bcteri were identified using Schedler gr. Sttisticl Anlyses Results re expressed s men ± stndrd devition (SD). Sttisticl nlyses were performed using Student s t-test for vribles mesurement nd Fisher s exct test with SPSS13 softwre. Sttisticl significnce ws set t P < Results Amylse Concentrtions nd Pncretic Histology The plsm mylse concentrtion ws higher in the SAP group thn in the NC group (P < 0.05, Tble 1). The mylse concentrtion did not differ between the SAP-G nd the SAP groups, t ech corresponding time point. The histologicl exmintion of the NC group showed no inflmmtion or necrosis of the pncretic tissues (Tble 2). However, the pncreses of the SAP nd SAP-G groups showed edem of the strom, inflmmtory cell invsion, nd cinr cell necrosis, fetures tht re comptible with those of SAP. Inflmmtion ws more prominent in the SAP-G group thn in the SAP group, while neither necrosis nor vcuoliztion ws different between the two groups. Opsonin Receptor Expression on Circultory Neutrophils The numbers of CD11b-nd CD32/16-positive neutrophils mong circultory neutrophils were significntly higher in the SAP group thn in the NC group t 12 nd 24 hours fter the finl injection of ceruletide (P < 0.01; Tble 3). These vlues in the SAP-G group were significntly higher thn in the SAP group t ech time point 354

4 Tuo HF et l. Effects of Grnulocyte Colony-Stimulting Fctor... Tble 1. Plsm mylse (men ± SD, IU/L) Time fter finl ceruletide injection 0 hours 12 hours 24 hours NC 5492 ± 145 SAP ± ± 1221 SAP-G ± 6329,b 8876 ± 936,c P < 0.05 between SAP or SAP-G nd NC; b P = 0.06 between SAP nd SAP-G; c P = 0.19 between SAP nd SAP-G. Tble 2. Histologicl scores of pncreses Time points 0 h 12 h 24 h NC SAP SAP-G SAP SAP-G Necrosis Vcuoliztion Inflmmtion ± The histologicl grding of necrosis nd vcuoliztion refers to the pproximte percentge of cells involved: 0, bsent; ±, 5%; 1, 5-15%; 2, 15-35%; 3, 35-50%; nd 4, 50%. The grde of the inflmmtory ltertions refers to scle rnging from ± (the minimum) to 4 (the mximum). Tble 3. CD11b nd CD32/16 expression on circultory neutrophils (cells/mm 3 ) CD11b+ cells CD32/16+ cells 0 h 12 h 24 h 0 h 12 h 24 h NC 325 ± ± 107 SAP 1025 ± ± ± ± 327 SAP-G 4614 ± 757 b 1682 ± 206 b 4780 ± 765 b 1748 ± 509 b Results re shown s men ± SD; b P < 0.01 between the SAP nd SAP-G groups. Tble 4. CD11b nd CD32/16 expression on peritonel exudte neutrophils CD11b + cells CD32/16 + cells 0 h 12 h 24 h 0 h 12 h 24 h NC 0.6 ± ± 0.2 SAP 9.0 ± 2.0 b 1.6 ± ± 2.0 b 2.4 ± 0.3 SAP-G 30.0 ± 6.0 c 7.1 ± 0.6 c 40.0 ± 6.0 c 7.4 ± 0.6 d Number of cells ( 10 4 /totl) is shown s men ± SD; b P < 0.001, between the SAP nd NC groups; c P < 0.05 nd d P < 0.001, between the SAP nd SAP-G groups. (P < 0.01). The numbers of CD11b-nd CD32/16-positive neutrophils were bout 4-fold higher in the SAP-G group thn in the SAP group (P < 0.01); the men fluorescence intensity (MFI) of CD11b t 12 nd 24 hours (P < 0.01) nd the MFI of CD32/16 only t 24 hours were significntly greter in the SAP-G group compred with the SAP group (P < 0.05, Fig. 1). Opsonin Receptor Expression on Peritonel Exudtive Neutrophils The numbers of CD11b-positive peritonel exudtive neutrophils incresed more thn 15-fold (P < 0.001), nd CD32/16-positive peritonel exudtive neutrophils more thn 6-fold (P < 0.001), t 12 hours in the SAP group compred with the NC group, nd these incresed 3- to 4-fold (P < 0.05) in the SAP-G group compred with the SAP group (Tble 4). The MFIs of CD11b nd CD32/16 were higher in the SAP-G group thn in the SAP group (P < 0.05) 24 hours fter the finl injection of ceruletide (Fig. 2). Microbiologicl Findings The vrious tissue cultures showed three min bcteril species: Enterococcus feclis, Proteus mirbilis, nd Escherichi coli. For ech smpled orgn, there were no significnt differences in the number tht tested positive for bcteril trnsloction between the NC nd SAP groups, or between the SAP nd SAP-G groups (Tble 5).We com- 355

5 Postepy Hig Med Dosw (online), 2016; tom 70: Fig. 1. MFIs of opsonin receptor expression on circultory neutrophils. MFI is shown s men ± SD. *P < 0.05; **P < Above, MFI for CD11b; Below, MFI for CD32/16. NS denotes no sttisticlly significnt difference Fig. 2. MFIs of opsonin receptor expression on peritonel exudtive neutrophils. MFI is shown s men ± SD. *P < 0.05 nd **P < Above, MFI for CD11b; Below, MFI for CD32/16 Tble 5. Bcteril trnsloction into vrious tissues Time points 0 h 12 h 24 h Pncres NC 1/6 SAP 2/6 1/6 SAP-G 3/6 0/6 Mesenteric tissue NC 0/6 SAP 2/6 1/6 SAP-G 1/6 0/6 Liver NC 0/6 SAP 1/6 1/6 SAP-G 1/6 1/6 Totl NC 1/18 (6%) SAP 6/18 (33%) b 3/18 (17%) SAP-G 5/18 (28%) 1/18 (6%) Number of positive cses/number of exmined cses. b P< 0.05 between NC nd SAP group. 356

6 Tuo HF et l. Effects of Grnulocyte Colony-Stimulting Fctor... pred the rtes of bcteril trnsloction in three types of orgns. The positive rtes were 6% in the NC group; 33% t 12 hours nd 13% t 24 hours in the SAP group; nd 28% t 12 hours nd 6% t 24 hours in the SAP-G group. The positive rte of bcteril trnsloction t 12 hours in the SAP group ws significntly higher thn in the NC group (P < 0.05). The positive rtes of bcteril trnsloction were lower in the SAP-G group thn in the SAP group t 12 nd 24 hours, lthough the differences were not sttisticlly significnt. Discussion To evlute the effect of G-CSF on neutrophil function, we dministered G-CSF to SAP mice, nd investigted the expression levels of CD11b nd CD16/32. Our results indicted tht the number of neutrophils significntly incresed, nd these increses were ccompnied by incresed expression of CD11b nd CD32/16 in neutrophils nd their respective MFIs, fter the dministrtion of G-CSF. These results re in ccord with the observtion tht G-CSF tretment incresed the number of neutrophils nd CD11b nd CD32 expression on neutrophils in helthy volunteers nd SAP dogs nd SAP mice [4,10,12]. Our results lso showed tht G-CSF reduced bcteril trnsloction, lthough the reduction ws not sttisticlly significnt compred to the control group. In fct, it ws found previously tht G-CSF significntly incresed nd mintined grnulocytes, nd decresed the rte of distnt orgn infection in SAP dogs [12]. Further studies re necessry with lrger popultions of experi- mentl nimls, longer observtion time, nd multiple dministrtions of G-CSF, becuse the effects of G-CSF on leukocytes nd neutrophils were more effective t two dys post-injection, when dministered to swine model of intr-bdominl sepsis twice dy [4]. Interestingly, G-CSF incresed the numbers of neutrophils nd mcrophges infiltrting res of cute myocrdil infrction nd promoted the bsorption of necrotic tissues [9]. Therefore, G-CSF tretment ccelertes the heling of myocrdil infrction wounds nd the regenertion of myocrdil tissues. Accordingly, our finding tht G-CSF tretment incresed the number of neutrophils infiltrting pncretic tissues supports the need for further studies regrding whether these infiltrting neutrophils will promote the bsorption of necrotic pncretic tissues nd induce the regenertion of pncretic cinr cells. In conclusion, G-CSF tretment in SAP mice incresed the number of neutrophils, enhnced the expression of opsonin receptors of circultory nd peritonel exudtive neutrophils, nd inhibited bcteril trnsloction to cvity orgns. Our findings suggest tht G-CSF my prevent bcteril infection in SAP through the increse of the number of neutrophils nd expression of opsonin receptors. This study should promote the development of tretment, or even cure, for SAP using G-CSF. Acknowledgement I thnk Prof. Atomi Yutk nd Msnori Sugiym from Kyorin University for their direction nd ssistnce. 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