Research On Quantities And Function Of NK Cells In The Immuno-related Pancytopenia

Part 1 Research on quantities and function of NK cells in Immno-related pancytopeniaObjective: To test NK cell quantities and function in the blood cytopenia patients with Immno-related pancytopenia(IRP) and discuss how NK cell participate in the progress of this diseaseBackground: Blood cytopenia patients with Immune-related pancytopenia(IRP) is regarded as an autoimmune disease caused by unkown autoantibodies, which will damage(suppress) bone marrow hemopoietic cells, finally lead to clinical performance of different degree of anemia, bleeding and infection. Natural killer cells is peripheral blood large granule lymphocytes, essential part of the body's natural immune system, can eliminate pathgens and tumor cells by secreting cytokines and(or) cytotoxicity. Recent study discovered NK cells in autoimmune diseases such as systemic lupus erythematosus(systemic lupus erythematosus, SLE), rheumatoid arthritis(rheumatoid arthritis) have abnormal number and function, and most studies believe the protection role of NK cells in autoimmune disease, but mechanism is not clear, so we use flow cytometry to detect NK cell number and function in patients with IRP.Materials and Methods: The percentages of NK cells(CD3-CD56+)in peripheral blood lymphocytes and its subgroup( CD3-CD56 bright CD16-)( CD3-CD56 dim CD16+), the expression of activating receptor of NKG2 D ? NKp46 and NKp44,the expression of inhibiting receptor CD158 a and CD158 b,the expression of cytoplasmic protein perforin and granzyme-? was detected among 22 newly diagnosed IRP patients,20 remitted IRP patients and 12 healthy volunteers with flow cytometry. The correlation between these changes and clinical data, including hemoglobin, thrombocyte, neutronphil granulocyte(ANC%), reticulocyte absolute value(Ret) in peripheral blood and serum immunoglobulin Ig G and Ig M,complement C3 and C4, bilirubin, LDH level were analyzed. The relationshipbetween these data and B cell subgroup, T cell subgroup and DC cell subgroup were also analyzed.Results:1. The variation of NK cells and it's subsets in peripheral blood lymphocytes in IRP patients(1)Variation of NK cell percentages NK cell percentage in peripheral lymphocytes in newly diagnosed, remitted IRP patients were(10.04±5.33)%?(11.62±6.80)%separately, which is significantly decreased to health control(19.94±7.38)(p<0.05).No difference was observed between newly diagnosed and remitted IRP patients(p>0.05).(2) Variation of NK cell subgroup CD56 bright CD16- Median percentage of CD56 bright CD16- subgroup in newly diagnosed patients was 0.35, which is significantly decreased than remitted patients and control 0.64, 1.31(p<0.05).(3) Variation of NK cell subgroup NK cell subgroup of CD56dimCD16+in newly diagnosed and remitted IRP patients were(9.20±5.22)%?(10.31±7.18)% separately,which is significantly decreased than healthy control(17.23±6.94)%(p<0.05).2. Expression of NK cell receptor(1) Expression of activating receptor NKG2 D in newly diagnosed IPR patients was( 74.03±18.24) %, significantly higher than that in remitted patients and control(45.97±29.45)%?(41.89±15.34)%(p<0.05), no difference is observed between remitted patients and control(p>0.05);Expression of activating receptor NKp46 were(67.14±16.80)%?(74.98±18.39)%?(68.06±19.57)% separately, no difference was observed(p>0.05); Median expression of activating receptor NKp44 were 0.58?0.71?0.40 separately, no difference detected(p>0.05).(2) Expression of inhibiting receptor CD158 a in newly diagnosed IRP patients was3.72,which was significantly decreased than remitted patients and control 16.10?11.04( p < 0.05), no difference between remitted group and control; Median expression of inhibiting receptor CD158 b in newly diagnosed, remitted IRP patients and control were 22.65?8.11?20.69 separately, no difference was observed(p>0.05).3. Expression of cytoplasmic protein perforin and granzyme-?Expression of perforin in newly diagnosed and remitted patients were(75.71±10.14)%?(77.88±22.82)% separately, which is significantly higher than control(60.22±14.58)(p<0.05), no difference detected between newly diagnosed and remitted IRP patients; Expression of granzyme-? in newly diagnosed and remitted IRP patients and control were(83.22±13.78)%?(85.58±12.96)%?(82.07±19.27)% separately, no difference was observed(p>0.05).4. The correlations between NK cell percentage, receptor, cytoplasmic protein and clinical and laboratory data(1)The correlations between NK cell percentage, receptor and cytoplasmic protein and clinical data. The expression of perforin was positively correlated with hemoglobin levels(r=0.452,p<0.01), no correlation was observed between NK cell numbers and hemoglobin, thrombocyte, neutronphil granulocyte(ANC%), reticulocyte absolute value(Ret) in peripheral blood and serum immunoglobulin Ig G and Ig M,complement C3 and C4, bilirubin, LDH level.(2)The correlations between NK cell percentage, receptor and cytoplasmic protein and laboratory data NK cell percentage was positively correlated with B cell subgroup(CD5+CD19+/CD19+)(r=0.487,p<0.05);Negatively correlated with T cell subgroup( CD3+CD4+/CD3+CD8+) and DC cell subgroup(Lin-HLA-DR+CD123+/Lin-HLA-DR+CD11c+)(p>0.05);Negatively correlated with BMMNC-Ab positive rate(p>0.05).Conclusion: With decreased Percentage of NK cells in IRP patients, NK cell function of immunological surveillance also receded, thus can't maintain the balance of immune system and lead to morbidity. NK cells maight play a protective role in disease progress. Besides, increased expression of activating receptor, decreased expression of inhibiting receptor and increased cytoplasm protein might be a compensation way for the decreased number of NK cells.Part 2 Detect Tim-3 molecular expression on peripheral nature killer cell in Immuno-related pancytopeniaObjective: To detect Tim-3 expression on peripheral nature killer cell in the patients with Immuno-related pancytopeniaMaterials and Methods: The expression of Tim-3 molecular on nature killer cell(NK cell CD3-CD56+)on patients(newly diagnosed and remitted) and healthy volunteers were measured with flow cytometry. NK cells were separated with MACS. The expression of Tim-3 m RNA detected with Real time PCR.Results:(1) Expression of Tim-3 on nature killer cells by FCM Tim-3 molecule expression on(CD3-CD56+)NK cell in newly diagnosed IRP patient was(70.10±13.87%),which is significantly decreased than that in remitted IRP patient(88.95±9.84%)and health control(85.62±9.03%)(p<0.05)The expression of Tim-3 on(CD56bright CD16-) NK cell in newly diagnosed and remitted IRP patients and health control were(71.85±24.83%)(78.62±27.20%)(62.64±12.06%)separately. No statistical significance between three groups(p>0.05).The expression of Tim-3 on(CD56dim CD16+) NK cell in newly diagnosed was(71.58±14.10%),which is significantly decreased compared to remitted IRP patient and health control(89.32±9.27%)(83.83±10.59%)(p<0.05)(2) Seperation of nature killer cell Successfully separated nature killer cells,which purity up to 96.54%(3) Detect the expression of Tim-3 m RNA by Real Time PCR The expression of Tim-3 m RNA level were(0.76±0.11)(1.03±0.25)(3.76±2.53)separately in new dignosed and remitted IRP patient and heath control. The expression level in newly diagnosed IRP paittient is significantly than that in remitted IRP patient or health control(p<0.05).Conclusion:Successfully enrich NK cells by the method of MACS,which purity up to 96.54%;The expression of Tim-3 on NK cells(espacially on CD56 dim CD16+NK cell)dcreased in new diagnosed IRP patients,that means activated NK cell may be over functioned and more cytotoxic in disease state,which may finally push the disease progression. However,the mechanism of augmented Tim-3 level remain unclear, more research needed.