| Background: Interleukin22(IL-22) is a kind of inflammatory cytokine which wasreported in2000. IL-22is secreted by different tissues and immune cells and plays animportant regularation role for inflammatory disease and autoimmune disease. Althoughit has recently been reported that Th17cells which character as secreting IL-17andIL-22are closely related to the presence and development of inflammatory diseases andautoimmune diseases, the more recently reported findings show that a new subset of Thcells, Th22cells which character as secreting IL-22may activate other immune cellsand inhibit inflammation so as to defense against infection when host infected by virusand bacterial. Recent work in this laboratory demonstrated the amount of IL-17producing γδ T cells in peripheral blood of patients with pulmonary tuberculosis (TB)increased. However the mechanism how Th22and Th17play a role in active pulmonarytuberculosis remains unclear. Also whether there exist IL-22producing γδ T cells andhow they play a role in immunity to TB infection and related to γδ T17cells remainunclear.Objective: To detect and compare the proportions of IL-22producing and IL-17producing αβ T cells, γδ T cells, B cells and NK cells among peripheral bloodlymphocyte subsets of healthy donors and the patients with active pulmonarytuberculosis. To detect the productions of IL-22among activated γδ T cells which werestimulated with M.tb heat-resistant antigen (Mtb-HAg), and to explore the role ofdeferent lymphocyte subsets in the immune response against M.tb and pathogenesis oftuberculosis.Methods:1. Peripheral blood samples of22TB patients and18health donors (HD)were obtained and stimulated with PMA and ionomycin for2hours and followed byaddition of monensin and incubation for more4hours. Cells were collected and stainedwith different fluorochrome-conjugated mAbs for staining of surface molecule andintracellular cytokines. The proportions of IL-22producing cells and IL-17producingcells were detected by flow cytometry；2. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation, stimulated withMtb-HAg, and cultured in rIL-2containing media to generate effective γδ T cellsenriched cell line that called as Mtb-AT for7to10days of culture, after cells werecollected PMA/Ionomycin and monensin or Mtb-HAg and monensin were added toMtb-AT to stimulate for6hours of culture. Interleukin22producing γδ T cells weredetected by flow cytometry；3.The proportions of IL-22producing lymphocytesubpopulation were detected in whole blood and PBMC from the same individuals；4.Whole blood samples were collected and PBMC isolated from the same individualswere stimulated with PMA and Ionomycin for6hours, and the supernatants werecollected to detect the concentration of IL-22and IL-17by ELISA assay；Theconcentrations of IL-22and IL-17from the plasma of TB patients and HD weredetected by ELISA.5. PBMC were isolated from peripheral blood of HD or TB patients,the expression of mRNA of IL-22was detected by RT-PCR.Results:1The proportions of IL-22+cells among peripheral blood lymphocyte，which werejust only a little，were3.16%in TB patients and2.69%in HD group，respectively. Andmajority of IL-22+cells were CD3+T cells，the percentage of which in TB patients was54.56%and in HD group64.10%，respectively.2. The percentage of γδ T cells among IL-22+lymphocytes in TB was (6.45%)significantly higher than that in HD group (2.73%, p<0.05).However，the percentages ofeach lymphocytes subsets among IL-22+lymphocytes in TB were not distinctlydifferent from that in HD group.3. For TB patients, the proportions of IL-22producing cells among CD3+T, CD4+T,CD8+T, γδ T, NK cells and B lymphocytes were respectively1.24%,1.58%,0.66%,7.37%,4.13%and0.54%；For HD，that were respectively1.83%,2.12%,0.67%,1.81%,1.96%and1.09%. It was suggested that the percentage for IL-22+cells among γδ Tcells in TB patients (7.37%) was distinctly higher than that in HD (1.81%, P﹤0.05).4. Co-expression of IL-22and IL-17double positive cells were detected among each Tsubsets；the percentages of Th22cells and Th17cells were respectively1.87%,3.78%inTB patients；while those were respectively2.10%,3.65%；neither the proportion ofTh22cells nor that of Th17cells was distinctly different between TB patients and HD.5. For the effective γδ T (Mtb-AT),the percentages of IL-22+cells in TB patients （0.75%，n=3）were not distinctly different from that in HD（1.41%，n=5）.6. No matter TB patients or HD，the percentages for IL-22+cells of γδ T cells in M.tb-ATthat were re-stimulated with M.tb-HAg did not change in both TB patients and HDwhen compared to that without M.tb-HAg in the presence of monensin alone.7. The percentages of IL-22+T cells subpopulations measured in whole blood cells weresignificantly higher than that in PBMC；while the percentages of IL-22+NK cells and Bcells measured in whole blood cells were significantly lower than that in PBMC.8.The amounts of IL-22and IL-17detected in supernatant of whole blood cells culturewere significantly higher than those in PBMC by ELISA; The amounts of IL-22andIL-17from the plasmas of TB were significantly higher than those from HD by ELISA.9. The expression of IL-22mRNA was detected by RT-PCR. For TB, the net photodensity value of IL-22/GAPDH was distinctly higher than in HD.Conclusion：1. T cells were the uppermost source of IL-22in human peripheral bloodlymphocytes, and CD4T cells were also one of the primary sources of IL-22..2. The proportion of IL-22producing cells of γδ T cells in peripheral bloodincreased in TB patients and this increasing did not relate to the increased proportion ofIL-17producing cells of γδ T cells of TB.3. neither the proportion of Th22cells nor that of Th17cells was distinctlydifferent between TB patients and HD.4. Mtb-HAg is able to specifically trigger the production of IFN-γ but not IL-22inγδ T cells.5. The percentages of IL-22producing each T subpopulation measured in wholeblood cells is significantly higher than that in PBMC, but the percentages of IL-22producing NK cells,B cells measured in whole blood is lower than that in PBMC.6. The concentrations of IL-22and IL-17from the plasmas of TB weresignificantly higher than those from HD... |
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