The Study Of Regulatory T Cells Functional Status In UC Patients Peripheral Blood

IntroductionInflammatory bowel disease (IBD) is a group of autoimmune diseases that involve gastro-intestine tract. Ulcerative colitis and Crohn's disease are the two common forms of IBD. Recently, the incidence of IBD, especially the incidence of UC, shows rising tendency. Its etiology and pathogenesis are still unknown. It is considered to related to the interaction of immune, environment, infection and heredity. The present investigations are improving our understanding of the pathogenesis of immune-mediated enteropathies and will hopefully lead to new approaches to the management of these disorders.Inflammatory bowel disease (IBD) is mediated by hyper-immunological reaction and T lymphocytes play an important role. The balance between regulatory T cells and pathogenic T lymphocytes is one of the important regulatory patterns of body immunological reaction. Its disorder may be the pathogenesis of many autoimmune diseases. Regulatory T cells (Tregs) form an essential arm of peripheral tolerance. Defective Treg function is thought to contribute to the pathogenesis of autoimmune and other inflammatory diseases. It is worth studying whether the morbidity of IBD is related to Treg antigen specific immunosuppressive functional defect.We collect and separate lymphocytes and monocytes from peripheral blood of the normal and UC patients and use autologous enteric microbial population as antigen. To detect Treg phenotype CD25,CD152,CD45RB (CD45RO), transcription factor FOXP3 and cytokine IL-6, TNF-αIL-10, IFN-γand IL-4 etc after mixed culture In vitro in order to study Treg functional status in UC pheriphral blood.Material and methodsMateril All people are from endoscopic center of china medical university Shengjing hospital. Control group were15 persons and UC group were 23 patients.Reagents and instruments Human Regulatory T Cell Staining Kit,CD4(7E14)-FITC/CD25(1TYV)-PE Cocktail, anti-human CD45RO (UCHL1) /Biotin and CD152/Biotin, ELISA Kit etc. Flow cytometry, CO₂ incubator, ultrasonic cell disintegration appearance, refrigerated centrifuge, visible range spectrophotometer etc.Lymphocytes and monocytes isolation10ml peripheral blood was collected into heparinized tube from each person and within 10 minutes diluted with 10ml RPMI-164. Then put it into 2 15ml-centrifuge tubes and added slowly 4ml Lympholyte-H (1.077 g/ml) to diluted blood, centrifuged for 20 minutes at 1200rpm. Sucked grayish-white layer monocytes suspension and washed them with PBS twice time. 4ml low density Percoll (1.068g/ml, 335mOsm) and 2ml high density Percoll(1.080 g/ml, 335mOsm)were spread under cell suspension with 5ml RPMI-1640(contain 10% fetal bovine, HEPES and di-Ab suspended cells and centrifuged for 15 minutes at 2000rpm. Cells suspension from low-density Percoll midpiece to high-density Percoll midpiece was collected and was regarded as monocytes and lymphocytes respectively.Preparation of enteric bacterial antigenBacteria on colon surface were acquired through biopsy and were inoculated in 50ml cooked meat medium. Sealed with liquid paraffin and cultured under 370C for 48 hours. Centrifuged bacterial fluid at 4000rpm for 20 min. Added 1.5ml deionized water and 100 U DNase I suspension into precipitate and performed ultrasonic breaking. Filtration sterilization and assayed total protein concentration by Coomassie brilliant blue method. Cell culture Medium is RPMI-1640 that contains about 10% self-serum.Maximal medium 40ml contains 10ml first centrifuge fluid and RPMI-1640 including double Ab and HEPES. Adding 1×10⁵ monocytes per hole into 96 shadow-mask, 20μg/ml self enteric Ag and 8×10⁴ self-lymphocytes per hole. Counting and collecting lymphocytes to perform flow cytometry on 14th day after mixed culture.Flow cytometry to detect the expressions of CD4, CD25, FOXP3 CD45RO,CD152 of lymphocytes on 2nd day after separation and CD4, CD25, CD25^hi and FOXP3 on 14th day after mixed culture according to Kit directions.ELISA To measure cytokines of IL-6, TNF-α, EL-10, IFN-γand IL-4 more dicto of ELISAStatistical analysis Mean±SD are given and finished by SPSS 11.5. Forcomparison of matched-pairs samples the t test was applied and a P < 0.05 was considered significant.Results1. Separation results and identification of lymphocytes and monocytesLymphocytes and monocytes count were above 96%. Purity of lymphocytes and monocytes were above 95% and 85% respectively. We acquired 1.23±0.33×10⁷ lymphocytes and 1.76±0.71×10⁶ monocytes.2. The expressions of CD4, CD25 and FOXP3 unchallenged in vitro.There were no significant difference in the expressions of CD4, CD25, CD25^hi and FOXP3 beween nomrmal control and UC group. About 60% of CD25⁺ and 80% of CD25^hi T cells expressed FOXP3 simultaneously. There were highly significant correlations in the expressions of CD25^hiand FOXP3 between the UC group and the normal control (P<0.001) .3. The expressions of CD4, CD25, CD45RO and CD 152 unchallenged in vitro.About 91% of CD4⁺CD25⁺T cells expressed CD45R0 simultaneously and CD152 less expressed on the surface of CD4⁺T cells (about 1%). There was no significant difference in the expressions of CD45R0 and CD152 on the freshly isolated lymphocytes in UC group constract with normal control.4. The change of lymphoc ytes after activationAfter in vitro challenged by autologous bacterial antigens, the total number of lymphocytes slightly increase (elevated 52% for UC group and 21% for normal control as compared with unchallenged cells, both P<0.001), The frequency of CD4+T cells elevated (elevated 66.48% for UC group and 67.09% for normal control as compared with unchallenged cells, both P<0.001) while the percentage of FOXP3⁺, CD25⁺FOXP3⁺and CD25^hiFOXP3+ T cells did not change significantly among both UC group and normal control. The CD4⁺CD25⁺FOXP3⁺ T cells/CD4⁺CD25⁺ T cells or CD4⁺CD25^hiFOXP3⁺ T cells/CD4⁺CD25hi T cells ratio decreased for both group(P<0.05).5. The frequency of peripheral blood lymphocytes challenged by autologous bacterial antigens in vitro elevated in UC group more than that in normal control (P<0.001) . Both FOXP3⁺/ CD25⁺ T cells and FOXP3⁺/CD25^hi T cells ratio in UC group after activation were obviously lower than those in normal control (40.18%vs 52.3% or 53.90% vs 77.85%, both P<0.001) .6. Both IL-6 and TNF-αexpressed by monocytes after mix-culture with autologous enteric anaerobic antigens in UC group were ignificantly higher than those in normal control (P<0.01) . DiscussionImmunological self-tolerance, i.e. unresponsiveness to self-constituents, is maintained not only by deletion of self-reactive lymphocytes in the central lymphoid organs but also by the control of their activation and expansion in the periphery. As a key mechanism of such peripheral self-tolerance, naturally occurring regulatory T (Treg) cells suppress the expansion/activation of self-reactive T cells which have escaped thymic negative selection. The majority of these Treg cells constitutively express CD25 (IL-2R [alpha]-chain), IL-2βR(CD122),CILA-4(CD152),GITR, high-level CD44 and moderate/low-level CD45RB and constitute 5-10% of peripheral CD4+ T cells in mice and humans. These Treg cells have been found and studied in humans and rodents. However, to date, the characterization of these cells has been hampered by a lack of specific molecular markers. In mice, the forkhead/winged helix family protein FoxP3 has very recently been shown to be expressed predominantly in Treg cells and to be critical for their generation and function.In our study there were no significant difference in the expressions of CD4,CD25,CD25hi,FOXP3 in peripheral blood lymphocytes among UC group and control group. It showed that CD4+CD25+Treg possessed their inhibitory activity in IBD peripheral blood. About 60% CD25+T cells expressed FOXP3 and 85% CD25hiT cells expressed FOXP3. It showed that there were highly significant correlations in the expressions of CD25hiand FOXP3 both the UC group and the normal control. We have presented data showing that the transcriptional regulator FoxP3 is expressed predominantly, if not exclusively, in Treg cells and that it may serve as a master regulator of this cell population and provide novel pathway for investigate the development and function of CD4⁺CD25⁺Treg.CD45RO⁺ represent functioning cell that has been activated by antigens, called memory T cell. In order to probe the rlation of CD4⁺CD25⁺Treg and memory T cell, we detect the expressions of CD4,CD25 and CD45RO simultaneously. We found that 91% of CD4⁺CD25⁺T expressed CD45RO simultaneously. It showed that CD4⁺CD25⁺Treg mainly resided in memory T cell pool and be challenged by antigens. The expression of CD45RO⁺ of memory T lymphocytes elevates in active IBD patients intestinal mucosa. The activity and immunoreactivity of intestinal mucosal T cells upgrade in IBD patients.Because the mucosal immune system is continuously exposed to a myriad of potentially harmful environmental antigens, it frequently reacts with antiinflammatory/regulatory T cell responses driven by TGF-β-producing T[H]3 cells and IL-10-producing regulatory T cells. Intestinal inflammation in patients with inflammatory bowel diseases is thought to result from an overwhelming uncontrolled activation of the mucosal immune system induced by antigens of the normal luminal flora in genetically susceptible individuals. Inflammatory bowel disease appears to be mediated by subsets of CD4⁺T lymphocytes or NK T cells secreting high levels of proinflammatory cytokines such as TNF-α. The increased expression of integrins/addressins in the inflamed gut and the increased expression of adhesion molecules on T cells facilitate migration of these pathogenic T cell subsets into the lamina propria. Additionally, the local activation of antiapoptotic pathways in pathogenic T lymphocytes leads to a further accumulation of these cells in the lamina propria, causing perpetuation and chronicity of inflammatory bowel disease. Taken together, distinct T cell subsets appear to act as mediators or guardians of inflammatory bowel disease, and thus they play a central role in controlling the delicate balance between proinflammatory and antiinflammatory immune responses in the gut.In our study after in vitro challenged by autologous bacterial antigens, the total number of lymphocytes slightly increase. The frequency of CD4⁺T cells elevated while the percentage of FOXP3⁺, CD25⁺FOXP3⁺and CD25^hiFOXP3+ T cells did not change significantly both UC group and normal control. The activation/proliferation of CD4⁺CD25⁺FOXP3^- T reactive cells predominate CD4⁺CD25⁺FOXP3⁺T regulatory cells in response to the challenge of autologous bacterial antigens, especially in UC patients.The CD4⁺CD25⁺FOXP3⁺ T cells/CD4⁺CD25⁺ T cells or CD4⁺CD25^hiFOXP3⁺ T cells/CD4⁺CD25hi T cells ratio decreased for both group, especially in UC group (P<0.05) . The pathogenesis of UC may be related to insufficient differentiation and functional disturbance of Treg. IL-6 deficient mouse.Both lymphocytes and macrophages activated by enteric antigens can synthesize and secrete some inflammatory mediators including proinflammatory and anti-inflammatory cytokines. Both IL-6 and TNF-αare major proinflammatory cytokines. IL-6 mainly participate the activation of Tand B lymphocytes and TNF-αpromote inflammatory cells aggregation and formation of granulation tissue. Both IL-6 and TNF-αexpressed by monocytes after mix-culture with lymphocutes challenged by autologous enteric anaerobic antigens in UC group were significantly higher than that in normal control. Intestinal Inflammatory reaction of UC partly mediated by IL-6 and TNF-α.Conclusions1. There were no significant difference in the expressions of CD4, CD25, CD25^hi FOXP3 and CD45R0, CD152 in peripheral blood lymphocytes beween nomrmal control and UC group. Peripheral blood CD4⁺CD25⁺Treg remain the inhibitory activity in UC patients2. The expressions of CD25^hi and FOXP3 in peripheral blood CD4⁺CD25⁺Treg were highly significant correlated between the UC group and the normal control. FOXP3 is the specific molecular marker of CD4⁺CD25⁺Treg and is necessary to the development and the function of CD4⁺CD25⁺Treg.3. After in vitro challenged by autologous bacterial antigens, the total number of lymphocytes slightly increase. The frequency of CD4⁺T cells elevated while the percentage of FOXP3⁺, CD25⁺FOXP3⁺and CD25^hiFOXP3⁺ T. cells did not change significantly both UC group and normal control. The activation/proliferation of CD4⁺CD25⁺FOXP3^- T reactive cells predominate CD4⁺CD25⁺FOXP3⁺T regulatory cells in response to the challenge of autologous bacterial antigens, especially in UC patients.The CD4⁺CD25⁺FOXP3⁺ T cells/CD4⁺CD25⁺ T cells or CD4⁺CD25^hiFOXP3⁺ 4. Both IL-6 and TNF-αexpressed by monocytes after mix-culture with lymphocutes challenged by autologous enteric anaerobic antigens in UC group were significantly higher than that in normal control. Intestinal Inflammatory reaction of UC partly mediated by IL-6 and TNF-α.