The Role Of VLA-4 And CX3CR1 On Recruitmentof T Cells Into Bone Marrow In Patientswith Acquired Aplastic Anemia

Background and objective:Aplastic anemia (AA), a typical example of the human bone marrow failure syndromes, is characterized by peripheral blood cytopenia and bone marrow empty. The key pathogenesis is decreased number and aberrant function of hematopoietic stems. Nowadays, acquired AA is believed an organ-specific autoimmune disease localized in the bone marrow. T-cell-mediated destruction and apoptosis of hematopoietic stem and progenitor cells is considered the root cause for bone marrow failure. However, the T-cells recruitment into bone marrow to take immunologic effect is intimately related to expression of certain adhesion molecules and chemokine receptors on the surface of T cells. VLA-4 and CX3CR1 are known respectively as an adhesion molecule and chemokine receptor, which play important roles in T cell migration.This study investigated the contribution of the molecules involved into T cell migration on pathogenesis of acquired aplastic anemia by assaying the expression of VLA-4 and CX3CR1 on T cell subsets as well as the levels of respective ligand, VCAM-1 and Fractalkine, expecting to exploit new target of treatment for AA.Method:1. To analyze the percentage of T cell subsets and the expression of VLA-4 and CX3CR1 on it in peripheral blood and bone marrow from 46 AA patients (20 SAA and 26 NSAA) and 21 controls by flow cytometry.2. To evaluate the correlation of granzyme B with CX3CR1 expression on lymphocytes in bone marrow from 11 randomly selected AA patients (5 SAA and 6 NSAA) and 7 randomly selected controls by flow cytometry. 3. To confirm the expression of VLA-4 and CX3CR1 on T cells, confocal scanning microscopic analysis of double-immunoflourescence staining was performed on bone marrow biopsies which obtained at the time of initial diagnosis and before treatment from 15 randomly selected SAA patients and 10 randomly selected controls.4. To assay the CX3CR1 mRNA levels in peripheral blood and bone marrow from 46 AA patients (20 SAA and 26 NSAA) and 21 controls by real-time quantitative PCR.5. To investigate the levels of VCAM-1 and Fractalkine in blood and bone marrow plasma from 46 AA patients (20 SAA and 26 NSAA) and 21 controls using Enzyme-linked immunosorbent assay.Result:1.1. The change of T cell subsets in AA patients:In peripheral blood, there was no statistically significant difference in percentage of CD3+cells (P> 0.05), but an increased number of CD8+cells and a reduced number of CD4+cells were found both in SAA and NSAA patients (P< 0.01). In bone marrow, the percentage of CD3+cells and CD8+cells were all increased in the patients (P< 0.01), and a significantly decreased percentage of CD4+cells was only found in SAA patients (P< 0.05).1.2. The expression of VLA-4 and CX3CR1 on T cells in AA patients:Both in blood and bone marrow, there was no statistically significant difference in expression of VLA-4 on T cell subsets in SAA patients, NSAA patients, and controls (P> 0.05). However, the percentage of CD3+/CX3CR1+, CD3+/CD4+/CX3CR1+, and especially CD3+/CD8+/CX3CR1+cells were increased in the patients compared with controls (P< 0.01).2. The correlation of granzyme B with CX3CR1 expression on lymphocytes of bone marrow in AA patients:Both in the patients and controls, the expression of CX3CR1 was highly correlated with intracellular content of granzyme B in CD8+or CD4+cells from bone marrow, as most CX3CR1-positive cells containing granzyme B. 3. The expression of VLA-4 and CX3CR1 on T cells by confocal scanning microscopic analysis of double-immunoflourescence stained bone marrow biopsies: There are no statistically significant difference in the MFI of VLA-4 on CD4+or CD8+cells, but an increased percentage of CD4+/CX3CR1+or CD8+CX3CR1+ cells were found in paraffin-embedded bone marrow slices from SAA patients compared with controls (P< 0.01), which were consistent with the results of flow cytometry.4. CX3CR1 mRNA levels in T cells of AA patient:The expression of CX3CR1 mRNA tended to be elevated both in CD4+or CD8+T cells from blood and bone marrow (P<0.05), and especially in CD8+cell of bone marrow (P<0.01), in AA patients compared with controls.5. The levels of VCAM-1 and Fractalkine in AA patients:The levels of VCAM-1 in blood or bone marrow plasma were no statistically significant difference between AA patients and controls (P>0.05), but the plasma levels of Fractalkine were significantly higher in SAA patients' blood and all the patients'bone marrow (P< 0.05)Conclusion:Acquired AA is a T-cell-mediated immune disease, and presents abnormal cellular immunity, as decreased T helper cells (CD3+/CD4+) and increased cytotoxic T cells (CD3+/CD8+). T cells appear to relocate from peripheral blood and accumulate in bone marrow to destruct hematopoietic stem and progenitor cells, probably due to elevated surface expression of CX3CR1. Blocking the interaction of CX3CR1 and Fractalkine may be as a new target of treatment for AA.