嗜中性粒細(xì)胞通過分泌IL-22促進(jìn)結(jié)腸上皮細(xì)胞的修復(fù)研究

趙海晴，謝子英，魏春華，賴?yán)m(xù)文

廣州軍區(qū)廣州總醫(yī)院消化內(nèi)科，廣東 廣州 510010

嗜中性粒細(xì)胞通過分泌IL-22促進(jìn)結(jié)腸上皮細(xì)胞的修復(fù)研究

趙海晴，謝子英，魏春華，賴?yán)m(xù)文

廣州軍區(qū)廣州總醫(yī)院消化內(nèi)科，廣東 廣州 510010

目的探討嗜中性粒細(xì)胞通過分泌白介素-22(Interleukin-22，IL-22)刺激腸道上皮細(xì)胞分泌黏液蛋白的作用。方法分離小鼠骨髓來源的嗜中性粒細(xì)胞，不同濃度的IL-23刺激中性粒細(xì)胞，檢測IL-22 mRNA及蛋白合成；收集經(jīng)IL-23刺激后中性粒細(xì)胞上清，將其作為條件培養(yǎng)基培養(yǎng)小鼠腸道上皮細(xì)胞CMT-93，檢測CMT-93合成Reg3及MUC2的變化，并用IL-22中和抗體驗(yàn)證IL-22是否在條件培養(yǎng)基中起主要作用。結(jié)果50 ng/ml IL-23即可顯著誘導(dǎo)嗜中性粒細(xì)胞合成IL-22 mRNA，ELISA檢測培養(yǎng)6 h的嗜中性粒細(xì)胞上清，IL-22蛋白量為(92±19)pg/ml。條件培養(yǎng)基培養(yǎng)CMT-93細(xì)胞24 h，Reg3和MUC2的mRNA合成倍數(shù)與對(duì)照組相比分別上升(11±3)和(14±5)倍，與單用IL-22誘導(dǎo)無顯著差異，但使用IL-22阻斷抗體后，條件培養(yǎng)基誘導(dǎo)CMT-93細(xì)胞合成抗菌肽的倍數(shù)分別降低至(2.4±0.8)和(2.1±0.5)倍。結(jié)論嗜中性粒細(xì)胞通過分泌細(xì)胞因子IL-22在促進(jìn)腸道上皮細(xì)胞分泌抗菌肽中起重要作用。

嗜中性粒細(xì)胞；白介素-22；腸道上皮細(xì)胞；黏液蛋白

白介素-22(Interleukin-22，IL-22)屬于IL-10家族成員，參與多種自身免疫性疾病，包括炎癥性腸病(inflammatory bowel disease，IBD)、哮喘及銀屑病[1-3]。大量研究認(rèn)為IL-22可通過促進(jìn)腸道上皮細(xì)胞增殖、遷移及產(chǎn)生黏液蛋白來維持腸道內(nèi)環(huán)境穩(wěn)態(tài)[4-5]。IBD包括潰瘍性結(jié)腸炎和克羅恩病，其特點(diǎn)是會(huì)引起腸黏膜下固有免疫和適應(yīng)性免疫反應(yīng)。在小鼠腸炎模型中，腸道固有免疫反應(yīng)包括募集的巨噬細(xì)胞和粒細(xì)胞，它們既可以起促炎作用也可以起抗炎作用[6]。固有免疫細(xì)胞失控，產(chǎn)生大量的活性氧會(huì)導(dǎo)致組織損傷，然而更多的研究表明嗜中性粒細(xì)胞在急性腸炎中起保護(hù)性作用[7-8]。本研究通過使用腸炎中的主要細(xì)胞因子IL-23對(duì)嗜中性粒細(xì)胞作體外刺激，檢測中性粒細(xì)胞分泌IL-22的能力；利用IL-22的阻斷抗體證明嗜中性粒細(xì)胞主要通過分泌IL-22促進(jìn)腸道上皮細(xì)胞分泌黏液蛋白。

1 材料與方法

1.1 細(xì)胞培養(yǎng)

1.1.1 分離培養(yǎng)原代C57BL/6小鼠骨髓來源嗜中性粒細(xì)胞：頸椎脫臼法處死小鼠，取出小鼠的股骨和脛骨，放入含有D-Hanks液的平皿中。剪去股骨和脛骨的兩端，用吸取了D-Hanks液的注射器將骨髓沖洗至另一無菌平皿。40 μm孔徑過濾篩除去可見固體顆粒后離心，去上清，2 ml PBS重懸細(xì)胞。按54%、64%、72%的Percoll分離液依次加入15 ml離心管后，再將細(xì)胞懸液加入，2 400 r/min，梯度離心30 min。小心吸取72%與64%兩層Percoll之間的細(xì)胞于新試管中，向試管中加入足量PBS，輕柔混勻后，1 000 r/min，離心5 min。棄上清，紅細(xì)胞裂解液于冰上裂解3 min，加入足量PBS，1 000 r/min，離心5 min。棄上清，適量RPMI 1640完全培養(yǎng)基(含10%胎牛血清，青霉素鏈霉素)重懸，即為所需要的小鼠骨髓來源的嗜中性粒細(xì)胞，37 ℃，5%二氧化碳細(xì)胞培養(yǎng)箱培養(yǎng)。

1.1.2 小鼠結(jié)腸上皮細(xì)胞系CMT-93：取對(duì)數(shù)生長期的鼠結(jié)腸癌細(xì)胞株CMT-93細(xì)胞，接種于含10%FBS及青霉素鏈霉素的DMEM培養(yǎng)基的12孔細(xì)胞培養(yǎng)板，每孔細(xì)胞數(shù)為1×106，培養(yǎng)24 h，待細(xì)胞貼壁生長，進(jìn)行刺激。

1.2 檢測指標(biāo)及方法TRIZOL法(Invitrogen)提取細(xì)胞總mRNA，TOYOBO反轉(zhuǎn)錄試劑盒將其反轉(zhuǎn)成cDNA后用SYBR法做實(shí)時(shí)定量PCR(見表1)，檢測細(xì)胞中IL-22、Reg3b、MUC2的拷貝數(shù)。酶聯(lián)免疫吸附法(ELISA，eBioscience)檢測細(xì)胞上清中IL-22的蛋白表達(dá)量。

表1 實(shí)時(shí)定量聚合酶鏈反應(yīng)引物

Tab 1 Primer sequence of realtime-PCR

基因序列(5’-3’)IL-22FCATGCAGGAGGTGGTACCTTIL-22RCAGACGCAAGCATTTCTCAGReg3FATGGCTCCTACTGCTATGCCReg3RGTGTCCTCCAGGCCTCTTMUC2FGCTGACGAGTGGTTGGTGAATGMUC2RGATGAGGTGGCAGACAGGAGACActinFCTTCTTTGCAGCTCCTTCGTTActinRAGGAGTCCTTCTGACCCATTC

1.3 分組方法設(shè)置0～50 ng/ml濃度梯度的細(xì)胞因子IL-23刺激嗜中性粒細(xì)胞，通過realtime-PCR和ELISA檢測IL-22的變化。分別收集0 ng/ml和50 ng/ml IL-23刺激后的嗜中性粒細(xì)胞上清，培養(yǎng)CMT-93小鼠結(jié)腸癌上皮細(xì)胞，檢測其CMT-93產(chǎn)生黏液蛋白的影響；在此基礎(chǔ)上，再分別使用IL-22阻斷抗體和同型對(duì)照，驗(yàn)證IL-22的作用。

1.4 統(tǒng)計(jì)學(xué)處理采用SPSS 13.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行分析，計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示，組間比較采用單因素方差分析，P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 IL-23刺激嗜中性粒細(xì)胞產(chǎn)生IL-22不同濃度的IL-23作用于嗜中性粒細(xì)胞6 h時(shí)，各濃度組IL-22 mRNA和蛋白水平與空白對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。50 ng/ml IL-23刺激嗜中性粒細(xì)胞6 h，IL-22 mRNA水平較空白對(duì)照組升高(570±89)倍，IL-22蛋白含量為(92±19)pg/ml，較空白對(duì)照組(5±2)pg/ml顯著上升(見圖1)。

圖1 中性粒細(xì)胞在IL-23刺激下產(chǎn)生IL-22Fig 1 IL-22 production in neutrophils induced with IL-23

2.2 嗜中性粒細(xì)胞通過IL-22促進(jìn)腸道上皮細(xì)胞分泌黏液蛋白取IL-23刺激后的嗜中性粒細(xì)胞上清作為條件培養(yǎng)基，培養(yǎng)CMT-93細(xì)胞24 h時(shí)，提取CMT-93細(xì)胞總mRNA，realtime-PCR檢測黏液蛋白R(shí)eg3和MUC2的表達(dá)較空白對(duì)照組顯著上升，分別為(11±3)和(14±5)倍，與單獨(dú)用IL-22刺激無顯著差異。為驗(yàn)證中性粒細(xì)胞是通過表達(dá)IL-22促進(jìn)腸道上皮細(xì)胞分泌黏液蛋白，在嗜中性粒細(xì)胞條件培養(yǎng)基中加入IL-22的阻斷抗體；中和IL-22后，CMT-93合成Reg3b和MUC2的基因水平較同型對(duì)照抗體組顯著下調(diào)，分別為(2.4±0.8和2.1±0.5)倍(見圖2)。

圖2 嗜中性粒細(xì)胞通過IL-22促進(jìn)腸道上皮細(xì)胞分泌黏液蛋白Fig 2 Neutrophils induced intestinal epithelial produce mucin via IL-22

3 討論

在慢性腸炎中，IL-23通過結(jié)合免疫細(xì)胞表面上的受體復(fù)合物(IL-12Rb1和IL-23R構(gòu)成)激活Stat信號(hào)傳導(dǎo)通路, 誘導(dǎo)IL-22的產(chǎn)生。本研究發(fā)現(xiàn)，IL-23可在體外刺激骨髓來源中性粒細(xì)胞分泌IL-22，為粒細(xì)胞可增強(qiáng)上皮細(xì)胞防御功能的理論奠定基礎(chǔ)。與本研究一致的是，研究者[9]發(fā)現(xiàn)，缺失趨化因子受體CXCR2的小鼠受到檸檬酸桿菌C.rodentium的攻擊時(shí)不能有效募集嗜中性粒細(xì)胞趨化至腸道，而該類細(xì)菌的清除依賴于細(xì)胞因子IL-23誘導(dǎo)的IL-22表達(dá)[10-11]。因此，嗜中性粒細(xì)胞在腸炎早期具有雙重作用：直接清除入侵的病原微生物及間接通過IL-22/IL-22R信號(hào)通路增強(qiáng)上皮細(xì)胞分泌抗菌肽等防御功能。

早期研究發(fā)現(xiàn)IL-22主要來源于Th17細(xì)胞[12]，Th22細(xì)胞[13]、NK細(xì)胞[14]等，隨著對(duì)先天樣淋巴細(xì)胞群的深入研究，證實(shí)腸道系統(tǒng)中的ILC3細(xì)胞群是分泌IL-22的主要細(xì)胞群[15]。然而，NK細(xì)胞在DSS誘導(dǎo)腸炎后約第4天分泌IL-22，到第6～8天到達(dá)峰值，Th17、Th22、ILC3s細(xì)胞則在第10～12天分泌IL-22，這些適應(yīng)性免疫細(xì)胞亞群在腸炎后期才能被大量活化進(jìn)而調(diào)節(jié)宿主防御系統(tǒng)[16-18]。而嗜中性粒細(xì)胞在腸炎發(fā)生的早期即可浸潤到腸黏膜處發(fā)揮作用[19]，由此可見，固有性免疫和適應(yīng)性免疫系統(tǒng)在腸炎發(fā)生后按先后順序通過合成IL-22調(diào)節(jié)腸道內(nèi)環(huán)境穩(wěn)態(tài)。在接下來的研究中，還需明確固有性免疫系統(tǒng)和適應(yīng)性免疫系統(tǒng)合成IL-22時(shí)是否有相互作用，它們被募集到結(jié)腸的先后順序，它們的來源及探索表達(dá)IL-22受體的細(xì)胞能否成為潛在的治療靶點(diǎn)。

與其他可產(chǎn)生IL-22的細(xì)胞不同，嗜中性粒細(xì)胞在腸黏膜損傷后的短時(shí)間內(nèi)快速被大量募集到結(jié)腸。在IL-23的刺激下，嗜中性粒細(xì)胞不僅表達(dá)IL-22，還表達(dá)IL-17(未公開數(shù)據(jù))，而這兩種細(xì)胞因子為Th17細(xì)胞特異性表達(dá)。有趣的是，Th17細(xì)胞在腸炎發(fā)生時(shí)產(chǎn)生GM-CSF和G-CSF，直接或間接增強(qiáng)中性粒細(xì)胞的募集[20]。Th17-嗜中性粒細(xì)胞的反饋軸可能是增強(qiáng)IL-22依賴的上皮細(xì)胞防御功能及清除入侵外源微生物的重要機(jī)制。

[1]Schmechel S, Konrad A, Diegelmann J, et al. Linking genetic susceptibility to Crohn’s disease with Th17 cell function: IL-22 serum levels are increased in Crohn’s disease and correlate with disease activity and IL23R genotype status [J]. Inflamm Bowel Dis, 2008, 14(2): 204-212.

[2] Zhao Y, Yang J, Gao YD, et al. Th17 immunity in patients with allergic asthma [J]. Int Arch Allergy Immunol, 2008, 151(4): 297-307.

[3] Cochez PM, Michiels C, Hendrickx E, et al. AhR modulates the IL-22 producing cell proliferation/recruitment in imiquimod-induced psoriasis mouse model [J]. Eur J Immunol, 2016, 46(6): 1449-1459.

[4] Brand S, Beigel F, Olszak T, et al. IL-22 is increased in active Crohn’s disease and promotes proinflammatory gene expression and intestinal epithelial cell migration [J]. Am J Physiol Gastrointest Liver Physiol, 2006, 290(4): G827-G838.

[5] Sugimoto K, Ogawa A, Mizoguchi E, et al. IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis [J]. J Clin Invest, 2008, 118(2): 534-544.

[6] Williams IR, Parkos CA. Colonic neutrophils in inflammatory bowel disease; Double-edged swords of the innate immune system with protective and destructive capacity [J]. Gastroenterology, 2007, 133(6): 2049-2052.

[7] Spehlmann ME, Dann SM, Hruz P, et al. CXCR2-dependent mucosal neutrophil influx protects against colitis-associated diarrhea caused by an attaching/effacing lesion-forming bacterial pathogen [J]. J Immunol, 2009, 183(5): 3332-3343.

[8] Lebeis SL, Bommarius B, Parkos CA, et al. TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to citrobacter rodentium [J]. J Immunol, 2007, 179(1): 566-577.

[9] Spehlmann ME, Dann SM, Hruz P, et al. CXCR2-dependent mucosal neutrophil influx protects against colitis-associated diarrhea caused by an attaching/effacing lesion-forming bacterial pathogen [J]. J Immunol, 2009, 183(5): 3332-3343.

[10]Mangan RR, Harrington LE, O’Quinn DB, et al. Transforming growth factor-beta induces development of the T (H)17 lineage [J]. Nature, 2006, 441(7090): 231-234.

[11] Zheng Y, Valdez PA, Danilenko DM, et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens [J]. Nat Med, 2008, 14(3): 282-289.

[12] Kleinschek MA, Boniface K, Sadekova S, et al. Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation [J]. J Exp Med, 2009, 206(3): 525-534.

[13] Eyerich S, Eyerich K, Pennino D, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling [J]. J Clin Invest, 2009, 119(12): 3573-3585.

[14] Goto M, Murakawa M, Kadoshima-Yamaoka K, et al. Murine NKT cells produce Th17 cytokine interleukin-22 [J]. Cell Immunol, 2009, 254(2): 81-84.

[15] Basu R, O’Quinn DB, Silberger DJ, et al. Th22 cells are an important source of IL-22 for host protection against enteropathogenic bacteria [J]. Immunity, 2012, 37(6): 1061-1075.

[16] Zenewicz LA, Yancopoulos GD, Valenzuela DM, et al. Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease [J]. Immunity, 2008, 29(6): 947-957.

[17] Monk JM, Jia Q, Callaway E, Weeks B, et al. Th17 cell accumulation is decreased during chronic experimental colitis by (n-3) PUFA in Fat-1 mice [J]. J Nutr, 2012, 142(1): 117-124.

[18] Sonnenberg GF, Fouser LA, Artis D. Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22 [J]. Nat Immunol, 2011, 12(5): 383-390.

[19] Williams IR, Parkos CA. Colonic neutrophils in inflammatory bowel disease: double-edged swords of the innate immune system with protective and destructive capacity [J]. Gastroenterology, 2007, 133(6): 2049-2052.

[20] Acciani TH, Suzuki T, Trapnell BC, et al. Epidermal growth factor receptor signalling regulates granulocyte-macrophage colony-stimulating factor production by airway epithelial cells and established allergic airway disease [J]. Clin Exp Allergy, 2016, 46(2): 317-328.

(責(zé)任編輯：王豪勛)

Study on neutrophils contribute the reparation of colonic epithelial cells via IL-22 production

ZHAO Haiqing, XIE Ziying, WEI Chunhua, LAI Xuwen

Department of Gastroenterology, Genenral Hospital of PLA Guangzhou Military Area, Guangzhou 510010, China

Objective To investigate the up-regulation of mucin by IL-22 produced by neutrophils in mouse colonic epithelial reparation.Methods IL-23 stimulated mouse bone marrow derived neutrophils, the mRNA and protein levels of IL-22 production were measured;the supernatant of neutrophils treated with IL-23 was collected, mouse colonic epithelial cells CMT-93 with this conditional culture medium was administrated, the synthesis of Reg3 and MUC2 mRNA level were detected. Furthermore, IL-22-blocking antibody to neutralize IL-22 in the conditional culture medium was used, the Reg3 and MUC2 mRNA levels were observed. Results IL-22 mRNA level was significantly enhanced by IL-23 treatment, the production of IL-22 protein was (92±19) pg/ml at 6 h after IL-23 treatment. The mRNA fold of Reg3 and MUC2 in CMT-93 cells were markedly up-regulated by neutrophils-conditional culture medium compared with control group, which were(11±3) and (14±5) respectively, however, with IL-22-blocking antibody treatment, the mRNA fold of Reg3 and MUC2 in CMT-93 cells was decreased to (2.4±0.8) and (2.1±0.5).Conclusion IL-22-producing neutrophils may play a critical role in mouse colonic epithelial cells production of antimicrobial mucin.

Neutrophils; Interleukin-22; Colonic epithelial cells; Mucin

趙海晴，本科，技師，研究方向：慢性結(jié)腸炎的發(fā)病機(jī)制及治療。E-mail:1912hanxiup@sina.com

10.3969/j.issn.1006-5709.2016.09.020

R574

A 文章編號(hào)：1006-5709(2016)09-1044-03

2016-04-18