J Clin Immunol (2013) 33:938–946 DOI 10.1007/s10875-013-9883-4

ORIGINAL RESEARCH

Characterization of Th1- and Th2-associated Expression in Spleens of Patients with Immune Thrombocytopenia

Shu-Fen Zhou & Ji Ma & Hui-Ting Qu & Zong-Tang Liu & Wei-Dong He & Juan-Dong Wang & Ai-Xia Dou & Ni Zhang & Jun-Li Liu & Cheng-Shan Guo & Yan Shi & Ming Hou & Jun Peng

Received: 12 December 2012 /Accepted: 4 March 2013 /Published online: 14 March 2013 # Springer Science+Business Media New York 2013

Abstract were investigated by real-time polymerase chain reaction Purpose In view of the numerous clinical observations and (RT-PCR). laboratory studies that suggest a critical role for the spleen in Results Reactive hyperplasia could be seen in follicles of immune thrombocytopenia (ITP) pathophysiology, we the white pulp, the germinal central zone was enlarged and aimed to characterize Th1-associated chemokine receptors the marginal zone was thickened, the central arteries were CXCR3 and CCR5 and Th2-associated thickened and fibrotic, and the density of the capillary vessel CCR3 in spleens of ITP patients and assess the significance was increased in ITP patients. ITP group displayed a higher of their differential expression in the clinical setting. rate of expression of Th1-associated chemokine receptors Methods The histopathology of spleens was observed using CXCR3 and CCR5 (83.3 % vs. 75 %, 100 % vs. 83.3 %) but hematoxylin-eosin staining (HE), and the positive rate of lower rate of expression of Th2-associated chemokine recep- CXCR3, CCR5 and CCR3 expression in spleens of 24 ITP tor CCR3 (50 % vs. 66.7 %) compared with the controls (P< patients and 12 patients with traumatic splenic rupture as 0.05, respectively). Western blot analysis revealed that normal controls was detected by immunohistochemistry CXCR3 and CCR5 expression was significantly in- using the SP method. CXCR3, CCR5 and CCR3 protein creased in ITP patients while CCR3 was significantly reduced expression was analyzed by Western blot and mRNA levels (P<0.05, respectively). Meanwhile, ITP patients displayed increased mRNA levels of CXCR3 and CCR5 but decreased gene expression of CCR3 (P<0.05, respectively). Conceived and designed the experiments: CSG, JP. Performed the Conclusion experiments: SFZ, JM, HTQ, ZTL. Analyzed the data: SFZ, JM. The data suggested that the abnormal expres- Contributed reagents/materials/analysis tools: WDH, JDW, AXD, NZ. sion of Th1/Th2 chemokine receptors may participate in Wrote the paper: SFZ, CSG, MH, JP. splenic immune disorder in patients with ITP. Using corre- S.-F. Zhou : H.-T. Qu : Z.-T. Liu : J.-D. Wang : A.-X. Dou : sponding inhibitors may inhibit Th1-dominant expression N. Zhang : C.-S. Guo (*) and mitigate the progress of the disease. Department of Hematology, Second Hospital, Shandong University, 250033, Jinan, China Keywords Immune thrombocytopenia . Th cell . chemokine e-mail: [email protected] receptor . spleen J. Ma : Y. Shi : M. Hou : J. Peng Department of Hematology, Qilu Hospital, Shandong University, 250033, Jinan, China Introduction W.-D. He Department of Component Blood, Immune thrombocytopenia (ITP) is an acquired organ- Blood Center of Shandong Province, 250014, Jinan, China specific autoimmune disorder characterized by a low periph- eral blood platelet count. The pathogenesis of thrombocyto- J.-L. Liu Central Molecular Biology Lab, Second Hospital, penia in ITP is complicated, involving not only impaired Shandong University, 250033, Jinan, China platelet production but also -mediated immunity J Clin Immunol (2013) 33:938–946 939 disorder [1, 2]. The abnormal Th cell could direct (T1D) [24], autoimmune thyroiditis and Graves’ disease autoreactive B cells to differentiate and secrete IgG autoan- [25]. tibodies [3], resulting in platelet destruction in the reticulo- However, the status of these and chemokine endothelial system. The spleen, serving as the largest receptors in spleens of ITP patients remains unknown. In lymphoid organ, is the main site of autoreactive B cell this study, as well as detected the basic pathological activation, antiplatelet antibody production and platelet de- changes, we further analyzed CXCR3, CCR5 and CCR3 struction. Splenectomy is an effective treatment in ITP with protein and gene expression through immunohistochemis- a success rate of about 66 % [1, 4]. More interestingly, ITP try, western blotting and quantitative real-time polymerase is now considered a Th1 disease, characterized by the chain reaction to analyze the significance of chemokine oligoclonal accumulation of Th cells [5]. Given their differ- receptor abnormalities in the spleens of ITP patients. ent effector functions, Th1 and Th2 are differentially recruited to peripheral sites of inflammation [6]. It has been shown that Th1, but not Th2, expresses a functional ligand Methods for P- and E- and therefore is selectively recruited to sites where Th1 immune responses occur [7]. In summary, Patients Th cell migration is a complicated process in which a series of cytokines are involved. Twenty-four patients with active ITP in the Second Hospital Chemokines are small chemotactic cytokines that play a of Shandong University from July 2011 to June 2012 were role in a myriad of immune functions such as angiogenesis, enrolled. All cases met the diagnostic criteria of ITP as hematopoiesis, mast cell degranulation, T cell differentiation described in the international consensus report in 2010 [1]. and leukocyte trafficking. The diverse biological effects of The median age was 27.5 years with a range of 12–48 years, chemokine-induced signaling are communicated through and 18 patients were female. The clinical features of the seven trans-membrane G-protein-coupled receptors. patients are shown in Table I. All patients had been treated Chemokines and their receptors are essential elements that with glucocorticoid before splenectomy. The platelet counts regulate the positioning of T cells and their partners for ranged between 4 and 24×109/L, with a median count of priming and Th1- or Th2-mediated responses. More than 17×109/L. Spleens removed from 12 patients with traumatic 50 chemokines have been described to date, and been clas- splenic rupture (nine males and three females; median age sified into four major subfamilies according to their struc- 25.5 years with a range of 16–39 years) were studied as ture, namely CC, CXC, CX3C and XC chemokines [8]. controls. Written informed consent was signed by all partic- However, only two of these groups have been extensively ipants and the study obtained the approval of the ethics characterized, namely, the CC and CXC chemokines [9]. committee of the Second Hospital of Shandong University Chemokine receptors are selectively expressed on T cell in accordance with the Helsinki declaration. surfaces, depending on their antigenic experience and the type of polarization. CXC chemokine receptor CXCR3 and Preparation for the Staining Protocol CC chemokine receptor CCR5 are expressed on activated T cells, especially the Th1 subset [10], and are thus associated Spleen specimens were fixed in 10 % formalin, paraffin- with a Th1 phenotype [11–13]. In contrast, CCR3 is embedded and cut into 4-μm thick sections, and after expressed mainly on Th2 cells [14]. Even though these mounting on slides, were heated at 60 °C for 60 min. chemokine receptors are not exclusive for these cell types, Before proceeding with the staining protocol, the slides they can be used as markers to differentiate Th1/Th2 cells were immersed into two changes of xylene for 5 min each from CD4 cells [15]. Chemokines of the CC subfamily are and rehydrated in decreasing concentrations (100 %, 95 %, generally chemoattractant for T lymphocytes, monocytes 85 %, 70 %) of ethanol for 3 min each. Then the slides were and natural killer (NK) cells while CXC chemokines attract kept in water until the following hematoxylin-eosin staining neutrophils and promote their adherence to endothelial cells or immunohistochemistry. [16–18]. In addition to their role in cell recruitment, chemokines have been reported to play an essential part in Hematoxylin-eosin Staining of the Spleens immunoregulatory activities, such as cytokine production and Th1/Th2 cell induction [19]. The strong chemoattractant Hematoxylin-eosin staining was carried out to observe the activity of chemokine CCL10, along with its receptor histopathology of the spleens. The above slides were stained CXCR3, has been reported in several autoimmune diseases, with hematoxylin for 10 min. After washing with distilled such as rheumatoid arthritis (RA)[20], systemic lupus water for 3 min, the slides were differentiated in 0.3 % acid erythematosus (SLE) [21], systemic sclerosis (SS) [22], alcohol and then rinsed in water to blue up. After staining (MS) [23], mellitus with eosin for 2 min, the slides were washed in tap water 940 J Clin Immunol (2013) 33:938–946

Table I Clinical characteristics 9 of patients with active ITP Patient Gender Age Platelet count (×10 ) Major therapy Bleeding symptoms before splenectomy 1 F 28 8 GC PT EC ET 2 F 24 9 GC EC EP 3 F 24 9 GC PT 4 M 48 13 GC PT EC 5 F 23 18 GC EC GUH 6 F 12 11 GC PT GIH 7 F 28 15 GC EC GUH 8 M 23 14 GC PT 9 F 26 18 GC EC GH 10 F 41 23 GC EC GIH 11 M 27 19 GC EC 12 F 30 23 GC EC GIH 13 F 29 10 GC EC GUH 14 F 34 19 GC EC 15 F 28 16 GC EC 16 M 27 17 GC EC 17 F 36 18 GC PT EC 18 F 31 22 GC PT 19 F 21 24 GC PT 20 M 28 13 GC PT 21 F 16 15 GC PT 22 M 22 21 GC EC 23 F 19 17 GC PT PT petechiae, EC ecchymoses, EP epistaxis, GUH genitouri- 24 F 26 16 GC EC GUH nary hemorrhage, GH gingival Median 27.5 17 hemorrhage, GIH gastrointesti- Range 12–48 4–24 nal hemorrhage and dehydrated in increasing concentrations (70 %, 85 %, purchased from ZSJQ Bio, Beijing, China. The primary 95 %, and 100 %) of ethanol. The slides were cleared and antibodies diluted in this process included CXCR3 mounted in neutral resin, followed by observation under the (ab71864) (1:150), CCR5 (ab65850) (1:100) and CCR3 microscope. (ab36827) (1:100). The role of nonspecific staining of pri- mary antibody was analyzed by substitution of the primary Immunohistochemistry SP Method antibody with PBS.

After heat-induced epitope retrieval using a microwave ov- Western Blot Analysis en, the slides were incubated with 3 % hydrogen peroxide for 10 min at room temperature to block endogenous per- Total were extracted from the spleens using a Total oxidase and rinsed three times in TBS plus 0.025 % Triton Protein Extraction Kit DBI-1017(DBI Bioscience), and the X-100 (TBST) with gentle agitation for 10 min each. Then concentration was tested using the BCA Protein Assay Kit. the slides were incubated overnight at 4 °C with primary The proteins were mixed with electrophoresis sample buffer antibody diluted in PBS. The slides were then washed in and then boiled for 10 min, and subsequently detected by TBST and subsequently incubated with Polymer Helper for SDS-PAGE, and then transferred to a nitrocellulose mem- 60 min. The slides were washed in TBST again, followed by brane (Boster) using a DYCZ-24D blotting apparatus. After 90-min incubation in poly-HRP anti-Rabbit IgG. After rins- blocking with 5 % non-fat milk for 60 min, the membrane ing in TBST three times, the slides were immersed in the was incubated overnight at 4 °C with primary antibody enzyme substrate DAB solution. After that, the slides were diluted in Primary Antibody Dilution Buffer (Beyotime). counterstained with hernatoxylin and dehydrated, cleared The antibodies involved in this process were CXCR3 and mounted with cover slips. Polink-2 plus(R) Polymer (ab71864) (abcam), CCR5 (ab65850) (abcam), CCR3 HRP Detection System for Rabbit Primary Antibody was (ab36827) (abcam) and GAPDH (Beyotime). The dilution J Clin Immunol (2013) 33:938–946 941 radio was 1:500 for the CCR5 antibody, and 1:1000 for the light microscope were as follows: the reactive hyperplasia others. After rinsing with TBST three times for 10 min per could be seen in follicles of the white pulp, and the germinal wash, the nitrocellulose membrane was immersed in dilute central zone was enlarged and the marginal zone was thick- HRP-conjugated goat anti-rabbit IgG (1:5000, ZSJQ Bio, ened. The splenic histopathology showed dilatation and Beijing, China) at room temperature for 90 min. The pro- congestion of the splenic sinusoid, infiltration of neutrophil- teins were visualized using ECL Western blotting detection ic leukocytes in the red pulp, and macrophage, red cell and reagents (Millipore) and exposed to clear-blue X-ray film. platelet accumulation in the splenic cord. The central arter- Protein expression in patients and controls was quantified ies were thickened and fibrotic and the density of the cap- using Image J. illary vessels was increased (as shown in Fig. 1).

Quantitative Real-time Polymerase Chain Reaction Analysis Immunohistological Changes in CXCR3, CCR5 and CCR3 in ITP Total RNA of the spleens was extracted using TRIzol re- agent (Invitrogen Life Technologies). Amplification and the The positive rate of CXCR3 together with CCR5 expression reverse transcription reactions were performed using an in spleens of ITP patients was increased and that of CCR3 Eppendorf Realplex2 PCR Detection System according to was decreased compared with trauma patients (as shown in the manufacturer’s instructions. The reagents were the RT Fig. 2). The positive rate of CXCR3 expression in spleens of reagent and SYBR Premix Ex Taq (Takara). RT-PCR ITP patients was 83.3 %, which was higher than that in primer sequences were as follows: CXCR3 forward: 5′- controls (75 %, p<0.05), and the positive rate of CCR5 was CCTACTGCTATGCCCACATCCT-3′, reverse: 5′- also higher in ITP patients (100 % vs. 83.3 %) (p<0.05). AAAGCGCCCAGGTCC ATGAG-3′; CCR5 forward: 5′- However, the positive rate of CCR3 was lower in ITP GAAGAGCTGAGACATCCGTTCC-3′, reverse: 5′- patients than that in controls (50 % vs. 66.7 %) (p<0.05). ACACCAG TGAGTAGAGCGGAG -3′;CCR3forward: 5′-TGCTGAGTTGTATT GGAGAAGTG-3′, reverse: 5′- AGTGAACACCAGGGAGTACAG-3′ and β-actin internal control forward: 5′-CACTCTTCCA GCCTTCCTTCC-3′, reverse: 5′-AGGTCTTTGCGGATGTCCAC-3′.ThePCR reaction parameters were set as follows: 95 °C for 30s followed by 40 cycles of PCR reacting at 95 °C for 15 s, and 60 °C for 30s. The relative quantification (RQ) of gene expression was calculated using the following equation: RQ ¼ 2ΔΔct.

Statistical Analysis

Values are presented as means ± SD. The data on chemokine receptor protein levels and mRNA levels in ITP groups and controls were normally distributed. Thus the independent- samples t test was performed to compare chemokine recep- tor protein levels and mRNA levels and 2-tailed Fisher exact test was used to compare the positive rate of expression between two groups using the Statistical Package for the Social Sciences version 17.0 (SPSS Inc., Chicago, IL). Values of p<0.05 were considered statistically significant.

Results

The Morphological Characteristics of Spleens in ITP Patients Fig. 1 The histopathology of the spleens (HE×200). Reactive hyper- plasia could be seen in follicles of the white pulp. The germinal central The surface of the spleen in ITP was smooth, without zone was enlarged and the marginal zone was thickened. The central arteries were thickened and fibrotic, and the density of the capillary thickness in capsule, and the splenic nodule was clear, vessel was increased. b The morphology of spleens from trauma without hemorrhage or necrosis. Characteristics under the patients is presented in Fig. 1b 942 J Clin Immunol (2013) 33:938–946

Fig. 2 Immunohistological differences in CXCR3, CCR5 and CCR3 expression in ITP (SP method×200). 10 % formalin-fixed, paraffin- embedded spleen specimens were cut into 4-μm thick sections, and immunohistochemically analyzed using the SP method with anti-CXCR3, anti-CCR5 and anti-CCR3. The slides were observed under a light microscope. a CXCR3 in ITP patients; b CXCR3 in traumatic spleens; c CCR5 in ITP patients; d CCR5 in traumatic spleens; e CCR3 in ITP patients; f CCR3 in traumatic spleens

Variation in Protein Expression of the Chemokine Receptors receptor CCR3 was significantly downregulated. CXCR3 in ITP mRNA expression was about 4-fold higher than that in controls (3.85±0.26 vs. 1.09±0.21, p<0.05) and CCR5 Th1-associated chemokine receptors CXCR3 and CCR5 mRNA level was also increased in spleens of ITP patients expression levels were increased in spleens of ITP patients, (1.71±0.03) compared with that in controls (1.01±0.01, p< while Th2-associated chemokine receptor CCR3 expression 0.05) while CCR3 mRNA level was lower (0.75±0.03) in was decreased (as shown in Fig. 3). ITP patients displayed patients than that in normal controls (1.01±0.01, p<0.05). increased expression of CXCR3 (1.74±0.18) compared with controls (0.58±0.07, p<0.05). CCR5 expression in spleens was also significantly higher in ITP patients (0.93±0.09) Discussion than that in controls (0.79±0.06, p<0.05). However, Th2- associated chemokine receptor CCR3 expression level was The study of chemokine receptors and their ligands is a new significantly lower (0.33±0.04, p<0.05) in spleens of ITP research focus in the immunologic field. In various human patients compared with that in the trauma patients (1.42± autoimmune diseases, such as rheumatoid arthritis, multiple 0.11, p<0.05). sclerosis, glomerulonephritis, inflammatory bowel disease and solid organ allograft rejection, the increased expression Relative Quantification of Chemokine mRNA in ITP of CXCR3 and CCR5 ligands is followed by the infiltration of CXCR3+ and CCR5+ T cells, suggesting a critical role for As shown in Fig. 4, mRNA levels of Th1-associated che- these chemokine receptors in T cell-induced tissue damage. mokine receptors CXCR3 and CCR5 were increased in T cell-induced immune disorder together with platelet de- spleens from ITP patients while Th2-associated chemokine struction plays an important role in the pathogenesis of ITP, J Clin Immunol (2013) 33:938–946 943

Fig. 3 The protein expression of CXCR3, CCR5 and CCR3 in ITP. CXCR3 and CCR5 were significantly upregulated whereas Th2-asso- The balance of Th1/Th2 chemokine receptor protein expression in ITP ciated chemokine receptor CCR3 was downregulated in spleens from was disrupted. Total proteins were extracted from the spleens using a ITP patients. a Representative Western blots of CXCR3, CCR5 and Total Protein Extraction Kit, and western blot was performed using CCR3 in spleens from ITP and controls. b CXCR3 expression was anti-CXCR3, anti-CCR5 and anti-CCR3. GAPDH was used as an about 3-fold higher than that in traumatic spleens. Expression of internal control. Bands were scanned, and volumes were calculated CCR5 was about 20 % higher, but CCR3 expression was 92.5 % by densitometry. Quantification was based on the ratio between che- lower (* P<0.05) mokine receptors and GAPDH. Th1-associated chemokine receptors and platelets are mainly destroyed in the spleen in immune expression has yet to be characterized in spleens of patients thrombocytopenia. However, CXCR3, CCR5 and CCR3 with ITP. Although the polarized immune activity detected in PBMC was thought to reflect the nature of the localized autoimmune process taking place in the target organs of ITP [26], this should be confirmed in the spleen. In this study, reactive hyperplasia in follicles of the white pulp, enlargement of the germinal central zone together with the thickness of the marginal zone underlined the key role of B cells, as well as Th cells, in ITP pathogenesis. Furthermore, the infiltration of neutrophilic granulocytes in the red pulp, and accumulation of macrophage, red blood cell and platelet in the splenic cord as well as the thickness and fibrosis of central arteries and the increase in density of Fig. 4 The relative quantification of CXCR3, CCR5 and CCR3 gene the capillary vessels indicated an inflammatory response in expression in spleens. Total RNA of the spleens was extracted using spleens of ITP. In addition to platelet destruction in the TRIzol reagent and RT-PCR was performed to analyze mRNA of spleen, we found that the spleen of ITP patients presented β CXCR3, CCR5 and CCR3. -actin served as the internal control. a higher positive rate of Th1-associated chemokine receptor The relative quantification (RQ) of gene expression was calculated by the following equation: RQ ¼ 2ΔΔct . Spleens from ITP patients CXCR3 and CCR5 expression but a lower positive rate of displayed significantly enhanced gene expression of Th1-associated Th2-associated chemokine receptor CCR3 expression, chemokine receptors CXCR3 and CCR5 and reduced mRNA expres- according to immunohistochemistry using the SP method. sion of CCR3. mRNA of CXCR3 in ITP patients was about 4-fold Meanwhile, CXCR3, CCR5 and CCR3 expression was higher than that in controls. CCR5 expression increased by 70 %, but the relative quantification of CCR3 gene expression decreased by quantified by RT-PCR and western blot analysis, and the 26.1 % (* P<0.05) results showed that spleens from the ITP group had 944 J Clin Immunol (2013) 33:938–946 significantly higher CXCR3 and CCR5 expression com- model of spontaneous abortion was significantly lower than pared to that in the control group, but significantly lower that in the normal pregnant model, suggesting that lower CCR3 expression. expression of CCR3 may cause impairment of Th2 cell dom- By interacting with their chemotactic ligands, chemokine inance, and may be linked to embryo loss [36]. receptors play significant parts in the leukocyte migration In our study, we found a Th1 polarized phenomenon in process. Chemokine receptor expression is an essential and spleens of ITP patients. The higher expression of CXCR3 critical factor in determining how chemokines work. and CCR5 together with the relatively lower level of CCR3 Generally speaking, chemokine receptors combine with mo- in ITP patients may raise the possibility that the abnormality re than one ligand. For example, CXC receptors bind CXC in chemokine receptor expression may be one of the key chemokines, while CC receptors exclusively combine with factors in the pathogenesis of ITP. The upregulated expres- CC chemokines. Chemokine receptors are selectively sion of CXCR3 and CCR5 promotes the proliferation of a expressed on Th1 and Th2 effector cells, leading to the large number of Th0 cells into Th1 cells and the recruitment distribution of these cells in their particular tissue environ- of more Th1 cells to immune response sites. Meanwhile, the ments. The expression of chemokine receptors is not only reduced expression of CCR3 suggests lower production of significant in T cell migration but also in T cell proliferation Th2 cells. Subsequently, the balance between Th1 and Th2 and differentiation, directly or indirectly. Increasing evi- cells is disturbed, resulting in the accumulation of Th1 cells, dence demonstrates that T cell homing potentials and effec- more type 1 cytokines, and ultimately ITP. That is to say, the tor functions are coordinately regulated during the high level of CXCR3 and CCR5 may contribute to Th1 bias, differentiation process [27]. Naive T cells, when stimulated and lower expression of CCR3 may weaken Th2 cell dom- by specific antigens and cytokines, enhance the expression inance. Moreover, our results are in accordance with the of adhesion molecules as well as chemokine receptors on consensus that ITP is a kind of Th1-dominant disease. their membrane, which helps them to infiltrate inflamed Targeting of the Th1-associated chemokine receptors tissues. CXCR3 and CCR3 are preferentially induced in CXCR3 and CCR5 as a therapeutic strategy has been stud- Th1 and Th2 priming conditions, respectively [11, 28]. For ied in a variety of human and experimental autoimmune example, developing Th1 cells acquire the ability to express diseases [32]. Based on the altered expression of Th1/Th2 chemokine receptors such as CXCR3, CCR5 and CXCR6 as chemokine receptors in spleens and the critical role of the well as IFN-γ, which are required to migrate at sites of spleen in ITP pathophysiology, pharmacological interven- delayed-type hypersensitivity reactions. Conversely, devel- tions targeting CXCR3 and CCR5 alongside the use of oping Th2 cells acquire the capacity to produce IL-4 and neutralizing antibodies or antagonists may effectively pre- express CCR3, CCR4 and CCR8 and the prostaglandin D2 vent Th1 cell migration in the spleen and impair Th1 polar- receptor CRTH2, and to transfer them to sites of allergic ization. Importantly, these results indicate that splenectomy reactions [28–30]. Changes in chemokine receptor quantity may be an effective treatment of ITP for correction of the or type are key factors in controlling Th1 and Th2 cell imbalance between Th1 and Th2 chemokine receptors, thus activation and polarization. CXCR3 and CCR5 have been normalizing the Th1/Th2 ratios, which are commonly in- found predominantly expressed on Th1 cells secreting creased in ITP patients [2, 5, 26, 37]. IFN-γ [9], while CCR3 is expressed on Th2 cells producing In summary, the data suggest that the abnormal expres- IL-4 [13]. In addition, cytokine production by T cells is sion of Th1/Th2 chemokine receptors may participate in regulated by the chemokine receptors expressed on their splenic immune disorder in patients with ITP and that using surface [31]. The abnormality in expression of these three corresponding inhibitors may inhibit Th1-dominant expres- chemokine receptors may result in changes in type and/or sion and mitigate the progress of the disease. quantity of T cell accumulation in the spleen tissue and the peripheral blood, and disturb the balance of Th1/Th2 cells, Acknowledgements We are grateful to Prof. Jing-Jie Zhao and Feng which can cause a cascade of Th1 or Th2 cells, leading to Kong (Central Molecular Biology Lab, Second Hospital, Shandong University, Jinan, China) for their assistance in the laboratory. We also Th1-dominant disease or Th2-dominant disorder. A growing thank Dr.s Ying-Xue Wang, Zhe Yin, Zhi-Lun Wang, Yong-Jing Wang, number of studies have demonstrated the importance of Ai Li, De-Xiao Kong, Qi Zhang and Cheng-Yun Zhen (Department of CXCR3 and CCR5 receptor-ligand interaction in Th1- Hematology, Second Hospital, Shandong University, Jinan, China) for dominant autoimmune diseases [32]. For instance, in pa- clinical help. tients with rheumatoid arthritis and multiple sclerosis, the Funding This work was supported by grants from the National levels of the CXCR3 ligands CXCL9 and CXCL10 [33] and Natural Science Foundation of China (No. 81270577, No. 30971278, the CCR5 ligands CCL3, CCL4 and CCL5 [33–35]are No. 81070411), and National Science Fund for Distinguished Young significantly higher than those observed in healthy controls, Scholars (No. 81125002); Key projects of Natural Science & + + Technology Research (No. Z2008C08), Tackle Key Program in accordingly leading to an accumulation of CXCR3 CCR5 T Science and Technology (No. 2009 GG10002020), Projects of cells [32]. More interestingly, CCR3 expression in the mouse Natural Science & Technology Research (No. ZR2011HQ051), J Clin Immunol (2013) 33:938–946 945

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