摘要
[背景]
在原发性干燥综合征(primary Sjogren's syndrome, pSS)中约1/5患者的唇腺病理出现异位生发中心(Ectopic germinal centers, EGCs),其病理作用目前尚不完全清楚,有待深入研究。对EGCs的研究有助于了解SS患者受累靶器官炎症细胞的招募和脏器损伤过程。本研究通过唇腺病理免疫组化(Immunohistochemistry, IHC) CD21染色、HE染色、流式细胞计数(Flow cytometry, FC)及酶联免疫吸附试验(Enzyme-linked immunosorbent assay, ELISA)比较pS患者唇腺病理有无异位生发中心形成者唇腺中淋巴细胞的浸润、B淋巴细胞自我活化、T淋巴细胞调节炎症程度的差异以及两者在外周血相应淋巴细胞的分布、迁移及临床病情的差异。
[目的]
研究原发性干燥综合征唇腺异位生发中心形成的发生机制。
[方法]
研究通过免疫组化染色CD21标记异位生发中心周围树突状细胞网络结构观察pSS患者唇腺EGCs形成的比例。把受试者按唇腺病理免疫组化(IHC) CD21染色及HE染色结果分成4组,0级组(GO)为正常唇腺,1级组(G1)唇腺病理中可见淋巴细胞分散浸润、2级组(G2)唇腺病理中可见导管旁淋巴细胞浸润,3级组(G3)唇腺病理中可见异位生发中心形成。通过唇腺病理免疫组化检测未成熟B淋巴细胞标记CD24、T调节细胞Th17标记IL17,相关趋化因子CXCL13,用光密度法及评分法对照各组患者的淋巴细胞分化、调节及趋化情况,并用细胞流式计数法对照各组患者外周血CD24+CD19+B淋巴细胞、TH17细胞及CXCR4、CXCR5细胞外周血比例,ELISA法对照各组患者的趋化因子CXCL13、CXCL12浓度,临床统计各组患者的系统受累指标的差异。
[结果]
统计pSS患者唇腺病理38例CD21染色,8例患者(21.1%)有异位生发中心形成。
1.GO(正常对照)唇腺病理1例CD24、IL17、CXCL13仅在唇腺导管上皮细胞染色。G1、G2组患者的CD24、IL17、CXCL13除出现在唇腺导管上皮细胞,还出现在淋巴浸润灶,G3组患者的各指标分布有分区现象,CD24主要分布于EGCs内部及边缘区、IL17主要分布在淋巴浸润灶外周,CXCL13主要在滤泡区周围表达。IL17和CD24高度正相关。
2.G3组患者唇腺病理中,有异位生发中心形成的淋巴浸润灶CD24.IL17.CXCL13与无异位生发中心形成的淋巴浸润灶平均光密度值均无显著差异。
3.唇腺病理不同组的pSS患者CD24、IL17、CXCL13平均样品光密度及IL17、CXCL13评分法比较,CD24:G3>G2>G1, IL17:G3>G2>G1, CXCL13:G3>G1>G2。G3组患者上述三项指标明显高于其他组,62.5%G3组患者高于这些指标的95%置信区上限。
4.与正常对照比pSS患者外周血中Th17细胞及CXCR4日性细胞比例都显著增高,血浆中CXCL13水平显著增高,外周血Th17细胞与血浆中CXCL13水平呈正相关。
5.唇腺病理不同组的患者CD24、Th17和CXCL13以及配体(CXCR4、CXCR5)在外周血的表达比较,表达CD24阳性细胞和IL17阳性细胞均为G3组>G2组>G1组,外周血CD19+CD24+B淋巴细胞G3组G1组>G3组：唇腺病理CXCL13阳性细胞G3组>G1组>G2组而外周血血浆中CXCL13G2组>G1组>G3组,外周血CXCR5阳性细胞G1组>G3组>G2组；外周血血浆中CXCL12G1组>G3组>G2组,CXCR4阳性淋巴细胞G2组>G3组>G1组。
6.除G2组患者在SICCA角膜染色评分上比G1组患者明显增高外,其它主要各项指标各组间无明显差异。
[结论]
1.原发性干燥综合征患者唇腺病理中约21%有异位生发中心形成,且均出现在中重度淋巴细胞浸润的患者中。
2.有异位生发中心形成的患者CD24阳性B淋巴细胞从外周血往唇腺迁移的能力较强
3.Th17细胞参与了pSS患者唇腺淋巴浸润和异位生发中心形成。
4.淋巴细胞趋化因子CXCL13及受体CXCR5参与淋巴细胞向唇腺迁移、浸润以及异位生发中心的形成。
Background. About1/5salivary glands biopsy in primary Sjogren's syndrome (pSS) patients have Ectopic germinal centers (EGCs), their pathological role is still unclear. Our preliminary study found by HE staining that12.5%of pSS patients with the EGCs. To investigate the role of EGCs in pSS, we detected in the way of immunohistochemistry (IHC), cell flow analysis (FC) and enzyme-linked immunosorbent assay (ELISA) to find out the role of EGCs in local infiltration, circulatory migration, B lymphocytes differentiation or self-activation and T lymphocytes regulation of inflammation in pSS patients.
Methods. CD21is stained to identify the dendritic cells network structure around the ectopic germinal centers through immunohistochemical method (IHC). All the subjects were divided into4groups:grade0group is normal control, grade1group (G1) with unorganized diffuse infiltrates in labila glands biopsy, grade2group (G2) with focal periductal T cell and B cell aggregates, grade3group (G3) with germinal center-like structures. To find out the role of EGCs, the differences of the B cells marker CD24, Th17cells marker IL17and chemokine CXCL13in labial glands paraffin sections among different groups, while CD24+CD19+B cells, IL17+CD4+cells and CXCR4, CXCR5cells in peripheral blood were also analyzed by FC and the concentration of the chemokine CXCL13, CXCL12were analyzed by method of ELISA. The clinical information of the pSS patients was evaluated simultaneously.
Results.
1.8cases (21.1%) of38pSS patients have ectopic germinal centers
2. CD24, IL17, CXCL13were only expressed by acinar and ductal epithelial cells in control and G1group. CD24, IL17, CXCL13were also found in the area of lymphocytes infiltration focus in the G2group. In G3group CD24was mainly in the follicular and marginal zones, IL17was mainly in the circumference of lymphocytes infiltration focus, while CXCL13was mainly surround the follicular zones.
3. In patients with EGCs, there was no difference in expression of CD24, IL17and CXCL13between EGCs and the other inflamed focus in the labial glands. IL17and CD24were highly positive correlated.
4. The average optical density of CD24, IL17, CXCL13and positive cell scores of IL17, CXCL13.are respectively as follow:CD24:G3> G2> G1, IL17:G3> G2> G1, CXCL13:G3> G1> G2.62.5%G3patients have higher scores and average optical density than upper limit of95%Cl of all3indexes.
5. By comparison of the circulatory indexes with normal controls, Th17cells, CXCR4cells and plasma CXCL13were significantly higher in pSS patients. Th17cells and plasma CXCL13were positive correlated.
6.Compare the relative indexes in the labial inflamed focus and peripheral blood in different groups:CD24and Th17cells in labial glands G3> G2> G1, peripheral blood CD19+CD24+B cells G3 G1>G3; CXCL13positive cells in labial glands G3> G2> G1, plasma CXCL13G2> G1> G3, peripheral blood CXCR5+cells G1> G3> G2; plasma CXCL12G1> G3> G2, peripheral blood CXCR4+cells G2G2> G3> G1.
7. G2group have higher SICCA corneal staining score than the G1group, but the other indexes were no significant differences among the groups.
Conclusion,
1. Ectopic germinal centers occur in moderate to severe focal lymphocytic infiltration of inflammation.
2. The pSS patients with ectopic germinal center can mobilize more CD24-positive B lymphocytes from peripheral blood to the labial gland
3. Th17is involved into the lymphocytes infiltration formation of ectopic germinal centers.
4. CXCL13and the acceptor CXCR5participates the migration of lymphocytes to the labial glands as well as the formation of ectopic germinal centes.
引文
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[1]Salomonsson S, Jonsson MV, Skarstein K, et al. Cellular basis of ectopic germinal center formation and autoantibody production in the target organ of patients with μμ's syndrome. Arthritis Rheum 2003,48:3187-3201.
[2]Jonsson MV, Szodoray P, Jellestad S, et al. Association between circulating levels of the novel TNF family members APRIL and BAFF and lymphoid organization in primary Sjogren's syndrome. J Clin Immunol 2005,25:189-201.
[3]Wise CM, Woodruff RD. Minor salivary gland biopsies inpatients investigated for primary Sjogren's syndrome. Areview of 187 patients. J Rheumatol 1993,20:1515-1518.
[4]Hansen A, Jacobi A, Pruss A, et al. Comparison of immunoglobulin heavy chain rearrangements between peripheral and glandular B cells in a patient with primary Sjogren's syndrome. Scand J Immunol 2003.57:470-479.
[5]Jacobi AM, Hansen A, Kaufmann O, et al. Analysis of immunglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjogren's syndrome. Arthritis Res 2002.4:R4
[6]Hansen A, Odendahl M, Reiter K, et al. Diminished peripheral blood memory B cells and accumulation of memory B cells in the salivary glands of patients with Sjogren's syndrome. Arthritis Rheum 2002.46:2160-2167.
[7]Le Pottier L, Devauchelle V, Fautrel A, et al. Ectopic germinal centers are rare in Sjogren's syndrome salivary glands and do not exclude autoreactive B cells. J Immunol. 2009.182(6):3540-7.
[8]Bombardieri M, Barone F, Humby F, et al. Activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large B cells supports functionality of ectopic lymphoid neogenesis in autoimmune sialoadenitis and MALT lymphoma in Sjogren's syndrome. J Immunol.2007.1; 179(7):4929-38.
[9]Kay R, Rosten PM, Humphries RK, et al.CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor.J Immunol.1991.15; 147(4):1412-6.
[10]Suzuki, T., N. Kiyokawa, T. Taguchi, et al. CD24 induces apoptosis in human B cells via the GEM/rafts-mediated signaling system.2001.J. Immunol.166:5567.
[11]Baumhoer D, Riener MO, Zlobec I, et al. Expression of CD24, P-cadherin and S100A4 in tumors of the ampulla of Vater. Mod. Pathol.2009.22 (2):306-13.
[12]Lee JH, Kim SH, Lee ES, Kim YS.CD24 overexpression in cancer development and progression:a meta-analysis.. Oncol. Rep.2009.22 (5):1149-56.
[13]Vesuna F, Lisok A, Kimble B,, et al. Twist modulates breast cancer stem cells by transcriptional regulation of CD24 expression. Neoplasia.2009.11 (12):1318-28.
[14]Jin-Qing Liu, Joseph W. Carl, Jr., Pramod S. Joshi, et al.CD24 on the Resident Cells of the Central Nervous SystemEnhances Experimental Autoimmune Encephalomyelitis. J. Immunol.2007.178; 6227-6235
[15]Chen G-Y, Tang J, Zheng P, et al. CD24 and Siglec-10 selectively repress tissue damage-induced immune responses.Science.2009.323(5922):1722-5.
[16]Sanchez E, Abelson AK, Sabio JM, et al. German Systemic Lupus Erythematosus Study Group, Alarcon-Riquelme ME, Martin J. Association of a CD24 gene polymorphism with susceptibility to systemic lupus erythematosus. Arthritis Rheum. 2007.56(9):3080-6.
[17]Ronaghi M, Vallian S, Etemadifar M. CD24 gene polymorphism is associated with the disease progression and susceptibility to multiple sclerosis in the Iranian population. Psychiatry Res 2009.170 (2-3):271-2.
[18]Weaver CT, Hatton RD.Interplay between the TH17 and TReg cell lineages:a (co-)evolutionary perspective.Nat Rev Immunol.2009. (12):883-9.
[19]Stockinger B, Veldhoen M. Differentiation and function of Th17 T cells. Current Opinion in Immunology 2007.19 (3):281-6.
[20]Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type I and 2 lineages. Nature Immunology 2005.(6):1123-32.
[21]Cuong Q. Nguyen, Min H.Salivary Gland Tissue Expression of Interleukin-23 and Interleukin-17 in Sjogren's Syndrome. Arthritis Rheum 2008. (58):734-743
[22]Sakai A, Sugawara Y, Kuroishi T, et al. Identification of IL-18 and Th17 cells in salivary glands of patients with Sjogren's syndrome, and amplification of IL-17-mediated secretion of inflammatory cytokines from salivary gland cells by IL-18.J Immunol. 2008.181(4):2898-906.
[24]Barone F, Bombardieri M, Manzo A, et al. Association of CXCL13 and CCL21 expression with the progressive organization of lymphoid-like structures in Sjogren's syndrome. Arthritis Rheum 2005.52:1773-1784.
[25]Hansen A, Reiter K, Ziprian T, et al. Dysregulation of chemokine receptor expression and function by B cells of patients with primary Sjogren's syndrome. Arthritis Rheum 2005.52:2109-2119.
[21]C Vitali, S Bombardieri, R Jonsson. Classification criteria for Sjogren's syndrome:a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002.61:554-558
[22]D.J. Aframian, R. David, H. Ben-Bassat. Characterization of Murine Autologous Salivary Gland Graft Cells:A Model for Use with an Artificial Salivary Gland. Tissue Engineering.2004,10(5-6):914-920.
[23]Rie Takagi, Takehiro Higashi, Kumiko Hashimoto. B Cell Chemoattractant CXCL13 Is Preferentially Expressed by Human Th17 Cell Clones1. J. Immunol.2008. 181; 186-189
[23]Jin-Qing Liu, Joseph W. Carl, Jr., Pramod S. Joshi, et al.CD24 on the Resident Cells of the Central Nervous SystemEnhances Experimental Autoimmune Encephalomyelitis. J. Immunol.2007.178; 6227-6235
[24]Blair PA, Norena LY, Flores-Borja F, et al. CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients.Immunity.2010.32(1):129-40.
[25]Jonsson MV, Szodoray P, Jellestad S, et al. Association between circulating levels of the novel TNF family members APRIL and BAFF and lymphoid organization in primary Sjogren's syndrome. J Clin Immunol 2005.25:189-201.
[26]Barone F, Bombardieri M, Rosado MM, et al. CXCL13, CCL21, and CXCL12 expression in salivary glands of patients with Sjogren's syndrome and MALT lymphoma: association with reactive and malignant areas of lymphoid organization.J Immunol.2008. 180(7):5130-40.
