Differential expression of T-cell genes in blood and bone marrow between ITP patients and controls

Summary

Primary immune thrombocytopenia (ITP) is an autoimmune disease characterised by premature platelet destruction in spleen, liver and bone marrow and a diminished production of platelets. T-cells are important in all forms of autoimmunity including ITP; however, very little is known about T-cells in organs where platelets are destroyed. Our aim was to investigate differences in gene expression in peripheral blood-derived T-cells and bone marrow-derived T-cells between ITP patients and controls. T-cells and subsequent RNA were isolated from blood and bone marrow from chronic ITP patients and healthy controls followed by DNA microarray analysis. There were 1554 differentially expressed genes in peripheral blood-derived T-cells and 976 in bone marrow-derived T-cells between ITP patients and controls and three genes were verified with real-time PCR. Using Gene Ontology functional enrichment analysis we found that genes involved in growth, development, migration, chemotaxis, adhesion and apoptosis were enriched in bone marrow-derived T-cells in ITP. Immune-related genes involved in T-helper cell differentiation, T-cell chemotaxis, migration, immunoglobulin-mediated immune response and classical and alternative pathway complement activation were also enriched in bone marrow-derived T-cells in ITP. Only 213 T-cell genes were differentially expressed in both blood and bone marrow between ITP patients and controls. In conclusion, our findings show that genes involved in major pathophysiologic pathways in ITP such as T-helper cell differentiation, autoantibody response and complement activation are altered in bone marrow-derived T-cells in ITP patients compared with controls. This further supports the concept that bone marrow is an important compartment in ITP.

Keywords

Autoimmunity - bone marrow - T-cell - cytotoxicity - gene expression

• References

• 1 Harrington WJ, Minnich V, Hollingsworth JW. et al. Demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura. J Lab Clin Med 1951; 38: 1-10.
• 2 Aster RH. Platelet sequestration studies in man. Br J Haematol 1972; 22: 259-263. DOI:10.1111/j.1365-2141.1972.tb05671.x.
• 3 Heyns AD, Lotter MG, Badenhorst PN. et al. Kinetics and sites of destruction of 111Indium-oxine-labeled platelets in idiopathic thrombocytopenic purpura: a quantitative study. Am J Hematol 1982; 12: 167-177. DOI:10.1002/ajh.2830120209.
• 4 Semple JW, Milev Y, Cosgrave D. et al. Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity. Blood 1996; 87: 4245-4254.
• 5 Semple JW, Freedman J. Increased antiplatelet T helper lymphocyte reactivity in patients with autoimmune thrombocytopenia. Blood 1991; 78: 2619-2625.
• 6 Kuwana M, Kaburaki J, Ikeda Y. Autoreactive T cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura. Role in production of anti-platelet autoantibody. J Clin Invest 1998; 102: 1393-1402. DOI:10.1172/JCI4238.
• 7 Kuwana M, Kaburaki J, Kitasato H. et al. Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura. Blood 2001; 98: 130-139. DOI:10.1182/blood.V98.1.130.
• 8 Sukati H, Watson HG, Urbaniak SJ. et al. Mapping helper T-cell epitopes on platelet membrane glycoprotein IIIa in chronic autoimmune thrombocytopenic purpura. Blood 2007; 109: 4528-4538. DOI:10.1182/blood-2006-09-044388.
• 9 Olsson B, Andersson PO, Jernas M. et al. T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nat Med 2003; 09: 1123-1124. DOI:10.1038/nm921.
• 10 Zhao C, Li X, Zhang F. et al. Increased cytotoxic T-lymphocyte-mediated cytotoxicity predominant in patients with idiopathic thrombocytopenic purpura without platelet autoantibodies. Haematologica 2008; 93: 1428-1430.
• 11 Chow L, Aslam R, Speck ER. et al. A murine model of severe immune thrombocytopenia is induced by antibody- and CD8+ T cell-mediated responses that are differentially sensitive to therapy. Blood 115: 1247-1253.
• 12 Gernsheimer T, Stratton J, Ballem PJ. et al. Mechanisms of response to treatment in autoimmune thrombocytopenic purpura. N Engl J Med 1989; 320: 974-980. DOI:10.1056/NEJM198904133201505.
• 13 Chang M, Nakagawa PA, Williams SA. et al. Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro. Blood 2003; 102: 887-895. DOI:10.1182/blood-2002-05-1475.
• 14 McMillan R, Nugent D. The effect of antiplatelet autoantibodies on megakaryocytopoiesis. Int J Hematol 2005; 81: 94-99. DOI:10.1532/IJH97.04168.
• 15 Ballem PJ, Segal GM, Stratton JR. et al. Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura. Evidence of both impaired platelet production and increased platelet clearance. J Clin Invest 1987; 80: 33-40. DOI:10.1172/JCI113060.
• 16 Li S, Wang L, Zhao C. et al. CD8+ T cells suppress autologous megakaryocyte apoptosis in idiopathic thrombocytopenic purpura. Br J Haematol 2007; 139: 605-611. DOI:10.1111/j.1365-2141.2007.06737.x.
• 17 Provan D, Stasi R, Newland AC. et al. International consensus report on the investigation and management of primary immune thrombocytopenia. Blood 2010; 115: 168-186. DOI:10.1182/blood-2009-06-225565.
• 18 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-159. DOI:10.1016/0003-2697(87)90021-2.
• 19 Brazma A, Hingamp P, Quackenbush J. et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet 2001; 29: 365-371. DOI:10.1038/ng1201-365.
• 20 Therneau TM, Ballman KV. What does PLIER really do?. Cancer Inform 2008; 06: 423-431.
• 21 Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004; 03: Article3.
• 22 Benjamini Y, Drai D, Elmer G. et al. Controlling the false discovery rate in behavior genetics research. Behavioural Brain Res 2001; 125: 279-84. DOI:10.1016/S0166-4328(01)00297-2.
• 23 Oliveira AP, Patil KR, Nielsen J. Architecture of transcriptional regulatory circuits is knitted over the topology of bio-molecular interaction networks. BMC Syst Biol 2008; 02: 17. DOI:10.1186/1752-0509-2-17.
• 24 Patil KR, Nielsen J. Uncovering transcriptional regulation of metabolism by using metabolic network topology. Proc Natl Acad Sci USA 2005; 102: 2685-2689. DOI:10.1073/pnas.0406811102.
• 25 Diehl AD, Lee JA, Scheuermann RH. et al. Ontology development for biological systems: immunology. Bioinformatics 2007; 23: 913-915. DOI:10.1093/bioinformatics/btm029.
• 26 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408. DOI:10.1006/meth.2001.1262.
• 27 Jacobson DL, Gange SJ, Rose NR. et al. Epidemiology and estimated population burden of selected autoimmune diseases in the United States. Clin Immunol Immunopathol 1997; 84: 223-243. DOI:10.1006/clin.1997.4412.
• 28 Olsson B, Ridell B, Carlsson L. et al. Recruitment of T cells into bone marrow of ITP patients possibly due to elevated expression of VLA-4 and CX3CR1. Blood 2008; 112: 1078-1084. DOI:10.1182/blood-2008-02-139402.
• 29 Houwerzijl EJ, Blom NR, van der Want JJ. et al. Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thrombocytopenic purpura. Blood 2004; 103: 500-506. DOI:10.1182/blood-2003-01-0275.
• 30 McMillan R, Wang L, Tomer A. et al. Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP. Blood 2004; 103: 1364-1369. DOI:10.1182/blood-2003-08-2672.
• 31 Olsson B, Andersson PO, Jacobsson S. et al. Disturbed apoptosis of T-cells in patients with active idiopathic thrombocytopenic purpura. Thromb Haemost 2005; 93: 139-144.
• 32 Najaoui A, Bakchoul T, Stoy J. et al. Autoantibody-mediated complement activation on platelets is a common finding in patients with immune thrombocytopenic purpura (ITP). Eur J Haematol 2012; 88: 167-174. DOI:10.1111/j.1600-0609.2011.01718.x.
• 33 Melenhorst JJ, Scheinberg P, Chattopadhyay PK. et al. High avidity myeloid leukemia-associated antigen-specific CD8+ T cells preferentially reside in the bone marrow. Blood 2009; 113: 2238-2244. DOI:10.1182/blood-2008-04-151969.
• 34 Letsch A, Keilholz U, Assfalg G. et al. Bone marrow contains melanoma-reactive CD8+ effector T cells and, compared with peripheral blood, enriched numbers of melanoma-reactive CD8+ memory T cells. Cancer Res 2003; 63: 5582-5586.
• 35 Palendira U, Chinn R, Raza W. et al. Selective accumulation of virus-specific CD8+ T cells with unique homing phenotype within the human bone marrow. Blood 2008; 112: 3293-3302. DOI:10.1182/blood-2008-02-138040.