TLR3 Ligand-Induced Accumulation of Activated Splenic Natural Killer Cells into Liver

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1 Cellular & Molecular Immunology 449 rticle TLR3 Ligand-Induced ccumulation of ctivated Splenic Natural Killer Cells into Liver Jing Wang 1, Jiawei Xu 1, Weici Zang 1, Haiming Wei 1 and Zigang Tian 1, 2 It as been revealed tat poly I:C is a potent stimulator for NK cells, wic can induce NK cell rapid activation and preferential accumulation into liver. However, te process mediating te influx of NK cells remains obscure. In tis study, we found tat poly I:C administration increased te portion and absolute number of NK cells in liver, but largely decreased tose in spleen. Tere were no obvious canges of tese lympocytes in oter immune organs. Te results from splenic adoptive transfer and splenectomy sowed tat te recruited spleen NK cells contributed to te accumulation of NK cells in liver, and tis process was regulated by te production of cemokines and te presence of T cells. Tis investigation will elp to understand te enanced immune cell recruitment in liver upon viral infection. Cellular & Molecular Immunology. 25;2(6): Key Words: liver, spleen, NK, poly I:C, recruitment Introduction Te liver plays a critical role in first-line ost defense against incoming gut-derived foreign antigens tat enter via te portal veins (1). Te epatic immune system must maintain a well-controlled balance between tolerance and initiation of an immune response. Te immune constitution of liver is quite distinct from oter sites, containing a large resident macropages and innate immune lympocytes suc as natural killer (NK) cells. Tese populations provide immune surveillance against foreign antigens. NK cells are one of te cellular mediators of innate defense. Tey are lympoid cells, witout te need for immunization or pre-activation, and can recognize and kill te aberrant cells and rapidly release a large amount of soluble factors wic ave antimicrobial effects or prime te immune responses of oter cells (2). Te abundance of NK cells in liver implies a very important role of tese cells in liver biology. Several polyribonucleotides ave already been adopted in clinical trials for te treatment of cancer and viral diseases. Polyinosinic-polycytidylic acid (poly I:C) is an 1 Institute of Immunology, Scool of Life Sciences, University of Science and Tecnology of Cina, Hefei, nui, Cina; 2 Corresponding to: Dr. Zigang Tian, Institute of Immunology, Scool of Life Sciences, University of Science and Tecnology of Cina, 443 Huangsan Road, Hefei 2327, nui, Cina. Tel: , Fax: , tzg@ustc.edu.cn. Received Oct 2, 25. ccepted Nov 3, 25. Copyrigt 25 by Te Cinese Society of Immunology artificial mimic of viral RN, wic may trigger te immune response resembling viral infection (3). Te previous reports in mice demonstrated tat poly I:C potently augmented NK cell activity and accumulated tese cells into liver, suggesting tat tis molecule is an excellent augmentor of liverassociated NK activity (4). However, te origin contributing to te influx of NK cells remains obscure. In present study, we found tat poly I:C administration increased te portion and absolute number of NK cells in liver, but largely decreased tose in spleen. Tere were no obvious canges of tese lympocytes in oter immune organs. Te results from splenic adoptive transfer and splenectomy sowed tat te recruited spleen NK cells contributed to te accumulation of NK cells in liver, and tis process was regulated by te production of cemokine protein and te presence of T cells. Tis investigation will elp to understand te enanced immune cell recruitment upon viral infection. Materials and Metods nimals Male C57L/6, L/C and SCID mice wit L/C gene background (six to eigt week-old, weiging 2-24 g) were obtained from Sangai Experimental Center, Cinese cademy of Sciences and maintained at our animal facility under te specific patogen-free condition. Reagents Poly I:C was purcased from Sigma Cemical Co. and separately dissolved in pyrogen-free pospate-buffered saline (PS) at 1 mg/ml. Te mbs used in tis study included FITC-conjugated anti-dx5 and anti-nk1.1, PE- Volume 2 Number 6 December 25

2 45 Poly I:C Induced te Recruitment of NK Cells from Spleen into Liver Table 1. Tissue distribution of NK cells Liver Spleen one marrow Tymus Lung 8.55 ± ± ± ± ± ± ± ± ± ± 1.6 Te lympocytes from different tissues of C57L/6 mice were isolated at and 12 after poly I:C injection (5 μg/g), and teir penotype was detected wit anti-nk1.1 and anti-cd3 antibodies by FCS analysis. Te portions of NK cells were calculated. Eac value represents mean ± SD of results from tree mice. p <.1 vs. corresponding time point. conjugated anti-nk1.1 (eioscience), and PE-CY5-conjugated anti-cd3e (ParMingen), 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) was obtained from Sigma Cemical Co., kept as.5 mm stock in DMSO and stored at -2 o C in a desiccator box. Cell preparation Livers were passed troug a 2-gauge stainless steel mes. Wasing once wit PS, te cells were resuspended in 4% Percoll (Sigma) and ten gently overlaid on 7% Percoll and centrifuged at 2,4 rpm for 3 min at room temperature. Hepatic mononuclear cells (MNCs) were collected from te interpase and wased twice in PS. To obtain lung mononuclear cells (MNCs), te lung was minced, suspended in medium containing.5% collagenase (Sigma) and.1% DNase I (Sigma), and ten saken for 2 min in a 37 o C water bat. Tereafter, te lung specimens were passed troug a 2-gauge stainless steel mes, and MNCs were isolated wit Percoll solution as described above. Te splenocytes and tymocytes were obtained by forcing tissues troug stainless steel mes, and te bone marrow cells were isolated form mouse femur and tibia. efore using, te erytrocytes were removed from te cell suspension by lysing solution (155 mm NH 4 Cl, 1 mm KHCO 3, 1 mm EDT, and 17 mm Tris, ph7.3). Tracking te recruitment of splenic lympocytes Under sterile conditions, te spleen cells were separated and used after erytrocyte lysing. Te splenic lympocytes were wased wit ice cold PS, resuspended at cells/ml in ice cold PS and ten incubated wit.5 μm CFSE for 1 min at 37 o C. fter labeling, te cells were immediately centrifuged and wased tree times wit ice cold PS. Suspension containing cells in 1 μl PS was gently injected into te inferior tip of spleen wit emostasis obtained by ligating te injection site (5). t a given time after transfer, te epatic lympocytes were prepared, and te CFSE + cell frequency and penotype were determined. Splenectomy Splenectomy was performed as previously described (6). riefly, mice were anestetized by inalation of eter before surgery. Under sterile conditions, a left-sided laparotomy was performed, ten te spleen was gently mobilized, exteriorized and te vascular supply was cut off by two ligatures (4- resorbable suture) placed around te vessels on te upper and lower poles of te spleen. Te peritoneal wall and te skin were closed by separate sutures. Mice were allowed to recover from surgery for at least 5 days. Flow cytometry fter blocking wit anti-fcγr (eioscience), te cells were incubated wit saturating amount of te indicated fluorescencelabeled mbs in darkness at 4 o C for 3 min, and ten wased twice. Te stained cells were analyzed by FCSCalibur, (ecton Dickinson) and te data were analyzed by WinMDI 2.8 software. Detection of specific mrn expressions by RT-PCR PCR primers for detecting mrns for VL, VCM-1, LF-1, ICM-1, FKN, MCP-1, MIP-1, IP-1 and β-actin were designed by us to be nucleotides long and to ave a 1% omology wit te particular regions of te genes according to gene sequences. Te RT-PCR metod was used as previously described (7). Statistical analysis Te results were analyzed using te Student s t test or analysis of variance were appropriate. p <.5 was considered significant. Results Poly I:C injection preferentially accumulated NK cells in liver bout 8% NK cells were resident in te liver of normal C57L/6 mouse, wile poly I:C treatment significantly elevated tis portion to around 4% 12 ours after administration. Te portion of NK cells diminised largely in spleen (from 6% to 2%), but remained uncanged in bone marrow, tymus and lung (Table 1). Consistently, after poly I:C injection te number of total NK cells in liver was dramatically increased in liver and peaked at 12 wit 4-fold increase, wile tose in spleen was significantly decreased and kept steady after 18 post injection (Figure 1). s sown in Figure 1, nearly all te accumulated NK cells in liver expressed activation molecule CD69 after poly I:C administration and remained at a ig level till 24 ours, indicating a lasting activation of NK cells induced by poly I:C stimulation. Volume 2 Number 6 December 25

3 Cellular & Molecular Immunology 451 NK cell number CD liver ( 1 6 ) spleen ( 1 7 ) NK1.1 Recruited spleen NK cells contributed to te accumulation of NK cells in liver Te evidence tat te portion and absolute number of NK cells were markedly increased in liver but significantly decreased in spleen after poly I:C injection raised a possibility tat NK cells infiltrated from spleen to liver following poly I:C callenge. To furter confirm tis conclusion, we performed splenic adoptive transfer of labeled cells. Gated on CFSE positive transferred cells in liver, toug T cells were increased to nearly 2 fold of tat in PS-treated group, NK cells were te major enanced population wit about 6-fold increase (Figure 2), suggesting te recruitment of NK cells from spleen into liver. s sown in Figure 2, wen mice were splenectomized, te number of liver NK cells in poly I:C-treated mice was significantly reduced compared to sam-operated group, indicating tat spleen supplied additional NK cells to liver and largely contributed to teir recruitment into liver upon poly I:C stimulation. Since a considerable amount of NK cells were recruited into liver, it is possible tat poly I:C locally enanced te liver expression or release of some effector molecules to trap NK cells. s sown in Figure 3, poly I:C injection did rapidly induce muc greater production of FKN and a sligt increase of VCM-1 in liver, but te levels of oter detected cemokine molecules and adesion proteins were not significantly elevated Figure 1. poly I:C preferentially assembled NK cells in liver. () Te lympocytes from liver and spleen of C57L/6 mice were isolated at te indicated time points after poly I:C injection and stained wit anti-nk1.1 and anti-cd3 antibodies. Te absolute number of NK cells was calculated. Eac value represents mean ± SD of results from tree mice. () Te epatic lympocytes were prepared at te indicated time points post poly I:C administration, and ten stained wit anti-nk1.1, anti-cd69 and anti-cd3 antibodies. FSC NK cell number ( 1 5 ) Te recruitment and activation of NK cells required te presence of T cells Next, we investigated weter NK cells were solely sufficient for teir own infiltration and activation after poly I:C callenge. In T cell-deficient SCID mice, te early recruitment of NK cells was significantly reduced (Figure 4), and tis absence of T cells also blunted te expression of CD69 on NK cells as indicated by te decreased percentage of CD69 + NK cells in liver compared to control L/C mice (Figure 4), suggesting tat to some extent T cell existence was important to te effect of poly I:C on NK cells. Discussion CFSE CFSE + cells (%) splenectomy-poly I:C sam-poly I:C Liver contains an unusual population of resident lympocytes, among wic CD8 + T cells usually out-number CD4 + T cells, and bot natural killer (NK) and natural killer T (NKT) cells are enriced relative to teir proportions in oter lympoid tissues (8). Tese resident and migratory populations of lympocytes and micropages set a powerful defense line against invading antigens, and can be rapidly expanded in response to infection or injury by recruiting leucocytes from te circulation and oter lympoid organs (9) PS poly I:C Figure 2. Splenic NK cells were recruited into liver by poly I:C stimulation. () C57L/6 mice received adoptive transfer of CFSE-labeled spleen cells, and ten were injected wit poly I:C. Te portion and penotype of liver CFSE positive cells were determined by FCS analysis wit anti-nk1.1 and anti-cd3 antibodies. () Mice were splenectomized and ten treated wit poly I:C. Twelve ours later, te liver lympocytes were arvested and analyzed by FCS using anti-nk1.1 and anti-cd3 antibodies. Te absolute number of NK cells was calculated. Eac value represents mean ± SD of results from tree mice. p <.5 vs. PS-treated or sam-operated group. T NK Volume 2 Number 6 December 25

4 452 Poly I:C Induced te Recruitment of NK Cells from Spleen into Liver VL-4 VCM-1 LF-1 ICM-1 β-actin Poly I:C 3 Double-stranded RN (dsrn) and te viral RN mimic, poly I:C, are recognized by Toll-like receptor 3 (TLR3) tat mediates te innate immune response to viral infections (1). It as been well documented tat poly I:C, as a potent stimulator for NK cells, can igly activate and accumulate tese innate immune cells into liver, and also enance teir natural cytotoxic activity and cytokine production (4). Consistent wit te previous observations, our results sowed tat poly I:C did preferentially accumulate NK cells in liver, wit a rapid increases of absolute number counting and cell portion in total epatic lympocytes. Interestingly, NK cell distribution in spleen was significantly reduced after poly I:C injection, but no measurable alterations were detected in oter lympoid tissues. Considering te close location of spleen and liver in pysiology, te results raised a possibility of te recruitment of NK cells from spleen to liver. Cell-trafficking experiment, wit fluorescence-labeled cells, directly sowed tat compared to T cells, poly I:C induced a more greater influx of spleen NK cells into liver. Te furter results from splenectomized mice supported our conclusion: liver recruited splenic NK cells upon poly I:C stimulation. ut te splenectomy did not completely inibit poly I:C-induced accumulation of NK cells in liver, suggesting tat tere probably exist oter sources supplying NK cells to liver, suc as te periperal circulation. Our preliminary experiment sowed a sligt decrease of NK cells in periperal blood from poly I:C-treated mice (data not sown). Te recruiting process is dependent on te ability of lympocytes to recognize, bind to and migrate across te endotelial cells tat line te vasculature. Liver sinusoidal endotelial cells constitutively express intercellular adesion molecule-1 (ICM-1) and leukocyte function associated antigen (LF-1) (1, 9, 11), and can be induced to express vascular cell adesion molecule (VCM) to retain te target cells wit VL expression (9, 12, 13). Tus, epatic endotelium could trap te circulating lympocytes troug te interaction of tese adesion proteins. We only detected a 3 FRK IP-1 MCP-1 MIP-1 β-actin Figure 3. Te expression of effector molecules may direct te infiltration of NK cells. Te mrn were extracted from liver tissue at te indicated time points after poly I:C injection, and te expressions of te indicated genes were determined using RT-PCR. Total NK ( 1 4 ) ctivated NK ( 1 4 ) L/C SCID sligt increase of poly I:C-induced VCM-1 expression in liver, wic is partially consistent wit te observation of Fogler WE, et al. (14). ut te expressions of oter adesion molecules sowed no distinct canges compared to tose of control group. In addition, cemokines ave great potential to promote NK cell cemoattraction and migration. Some studies ave sown tat expression of many CC cemokines including macropage inflammatory protein 1 (MIP-1) and monocyte cemotactic protein 1 (MCP-1), as well as CXC cemokines suc as interferon γ-inducible protein 1 (IP-1) in te liver (15, 16). newly identified CX3C-cemokine, fractalkine, expressed on activated endotelial cells also plays an important role in leukocyte adesion and migration (17, 18). Fractalkine plays an important role not only in te binding of NK cells to endotelial cells (19) or cemoattraction of NK cells into tumor sites (2), but also activation of NK cells (21, 22), all of wic play important roles in elimination of patogens and cancer cells. Our results sowed tat a dramatic elevation of fractalkine was induced by poly I:C injection, wile oter cemokines remained uncanged. Tis pattern of cemokine expression provided evidence for specific patways of poly I:C-caused NK cell recruitment into liver. Furtermore, te absence of T cells markedly retarded te L/C SCID Figure 4. Te presence of T cells assisted NK cell recruitment and activation. Te liver lympocytes from L/C and SCID mice were isolated at te indicated time points after poly I:C injection, and ten stained wit anti-dx5, anti-cd69 and anti-cd3 antibodies. Te number of total NK cells () and CD69 + NK cells () were calculated. Eac value represents mean ± SD of results from tree mice. p <.5 vs. te corresponding time point. Volume 2 Number 6 December 25

5 Cellular & Molecular Immunology 453 early liver recruitment of NK cells, and also downregulated NK cell activation in liver, suggesting tat te infiltration and activation of NK cells induced by poly I:C was T celldependent. Our preliminary experiment also found tat pre-activated T cells could boost NK cell activation and finally lead to an aggravated liver injury (our unpublised data). In conclusion, te studies presented in tis work demonstrated tat poly I:C induced te migration of NK cells from spleen into liver wit elevated fractalkine production and te presence of T cells in liver. Tese trafficking events may serve to promote ost immune responses against viral infection. References 1. Peters MG. nimal models of autoimmune liver disease. Immunol Cell iol. 22;8: Colucci F, Caligiuri M, Di Santo JP. Wat does it take to make a natural killer? Nat Rev Immunol. 23;3: igger JE, Tomas III C, terton SS. NK cell modulation of murine cytomegalovirus retinitis. J Immunol. 1998;16: Twilley T, Mason L, Talmadge JE, Wiltrout RH. Increase in liver-associated natural killer activity by polyribonucleotides. Nat Immun Cell Growt Regul. 1987;6: Ponder KP, Gupta S, Leland F, et al. Mouse epatocytes migrate to liver parencyma and function indefinitely after intrasplenic transplantation. Proc Natl cad Sci U S. 1991;88: Jon E. Coligan, da M. Kruisbeek, David H. Margulies, Etan M. Sevac, Warren Strober. Current protocols in immunology. oston, M: Jon Wiley & Sons, Inc.; Tian Z, Sen X, Feng H, Gao. IL-1β attenuates IFN-α β-induced antiviral activity and STT1 activation in te liver: involvement of proteasome-dependent patway. J Immunol. 2;165: Norris S, Collins C, Doerty DG, et al. Resident uman epatic lympocytes are penotypically different from circulating lympocytes. J Hepatol. 1998;28: Lalor T, dams D. Te liver: a model of organ-specific lympocyte recruitment. Expert Rev Mol Med. 22;22: Scmidt KN, Leung, Kwong M, et al. PC-independent activation of NK cells by te Toll-like receptor 3 agonist double-stranded RN. J Immunol. 24;172: Crispe IN. Hepatic T cells and liver tolerance. Nat Rev Immunol. 23;3: dams DH, urra P, Hubscer SG, Elias E, Newman W. Endotelial activation and circulating vascular adesion molecules in alcoolic liver disease. Hepatology. 1994;19: Yoong KF, McNab G, Hubscer SG, dams DH. Vascular adesion protein-1 and ICM-1 support te adesion of tumor-infiltrating lympocytes to tumor endotelium in uman epatocellular carcinoma. J Immunol. 1998;16: Fogler WE, Volker K, McCormick KL, Watanabe M, Ortaldo JR, Wiltrout RH. NK cell infiltration into lung, liver, and subcutaneous 16 melanoma is mediated by VCM-1/VL-4 interaction. J Immunol. 1996;156: iron C, Nguyen K, Pien GC, Cousens LP, Salazar-Mater TP. Natural killer cells in antiviral defense: function and regulation by innate cytokines. nnu Rev Immunol. 1999;17: Morris M, Ley K. Trafficking of natural killer cells. Curr Mol Med. 24;4: azan JF, acon K, Hardiman G, et al. new class of membrane-bound cemokine wit a CX3C motif. Nature. 1997;385: Pan Y, Lloyd C, Zou H, et al. Neurotactin, a membraneancored cemokine upregulated in brain inflammation. Nature. 1997;387: Yoneda O, Imai T, Goda S, et al. Fractalkine-mediated endotelial cell injury by NK cells. J Immunol. 2;164: Lavergne E, Combadiere, onduelle O, et al. Fractalkine mediates natural killer-dependent antitumor responses in vivo. Cancer Res. 23;63: Robinson L, Nataraj C, Tomas DW, et al. Te cemokine CX3CL1 regulates NK cell activity in vivo. Cell Immunol. 23;225: Guo J, Cen T, Wang, et al. Cemoattraction, adesion and activation of natural killer cells are involved in te antitumor immune response induced by fractalkine/cx3cl1. Immunol Lett. 23;89:1-7. Volume 2 Number 6 December 25