Cellular reactions of CD3+ CD4+ CD45RO+ T-lymphocytes on dexamethason in in normal patients and in patients with with rheumatoid arthritis in vitro

The aim of the study was to analyze the influence of glucocorticoid (GC) dexamethasone (Dex) on changes in CD4+ T-cells expressing the surface molecule of activation (CD25, CD71, HLA-DR and CD95) and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes obtained from healthy donors and patients with rheumatoid arthritis in vitro.

Materials and methods. The study included 50 patients and 20 healthy donors. T-cell cultures (CD3+ CD45RO+) were obtained from mononuclear leukocytes of immunomagnetic separation (MACS® technology). As an activator of T-lymphocytes, antibiotic particles with biotinylated antibodies against CD2+, CD3+, CD28+, which simulate the process of costimulation of T cells by antigen-presenting cells, were used. The following concentrations of dexamethasone (2, 8, 16, 32, 64 mg) were used in the experiment. The change in the immunophenotype of T-lymphocytes was analyzed by flow cytofluoometry. The secretion of CD3+CD45RO+ T-cells of proinflammatory cytokines IL-2, IFNγ, TNFα, IL-17 and IL-21 was evaluated by enzyme-linked immunosorbent assay.

Results. The general suppressor effect of Dex on CD3+CD45RO+ T-cell cultures mediated by a decrease in the number of CD4 + T cells expressing activation molecules (CD25) and proliferation (CD71), as well as inhibition of the production of inflammatory mediators: IFNγ, IL-2 and TNFα. It is shown that against the background of TCR activation Dex increases the number of CD4+CD95+HLA-DR+ cells in CD3+CD45RO+ cultures obtained from RA patients and does not change their content in the control. The correlations between the number of proinflammatory factors (IL-17, IL-21 and TNFα) in CD4+CD45RO+CD95+HLA-DR+ T cells in supernatants of cell cultures in RA patients indicate the presence of a pro-inflammatory potential of this population of T cells. We assume that the resistance of CD4+CD45RO+CD95+HLA-DR+ T cells in RA patients to the suppressor effect of GC generally leads to the preservation and enhancement of the functionality of autoreactive cells in the pathogenesis of RA. 

1. Matsuki F., Saegusa J., Miyamoto Y., Misaki K., Kumagai S., Morinobu A. CD45RA-Foxp3(high) activated/effector regulatory T cells in the CCR7+CD45RA-CD27+CD28+ central memory subset are decreased in peripheral blood from patients with rheumatoid arthritis // Biochem. Biophys Res. Commun. 2013; 438 (4): 778–783. DOI: 10.1016/j.bbrc.2013.05.120.

2. Sava F., Toldi G., Treszl A., Hajdú J., Harmath Á., Tulassay T., Vásárhelyi B. Expression of lymphocyte activation markers of preterm neonates is associated with perinatal complications // BMC Immunol. 2016; 17 (1): 19. DOI: 10.1186/s12865-016-0159-7.

3. Сохоневич Н.А. Роль цитокинов, имеющих общую γ-цепь рецепторов (IL-2, IL-7, IL-15), в регуляции функциональной активности Т-лимфоцитов: автореф. дис. … канд. мед. наук. Томск, 2015: 24. Sohonevich N.A. Rol’ citokinov, imeyushchih obshchuyu γ-cep’ receptorov (IL-2, IL-7, IL-15), v regulyacii funkcional’noj aktivnosti T-limfocitov [The role of cytokines, contains a common γ-chain of receptors (IL-2, IL-7, IL15), in the regulation of the functional activity of T-lymphocytes]: аvtoref. … dis. kand. med. nauk. Tomsk, 2015: 24 (in Russian).

4. Cho B.A., Sim J.H., Park J.A., Kim H.W., Yoo W.H., Lee S.H., Lee D.S., Kang J.S., Hwang Y.I., Lee W.J., Kang I., Lee E.B., Kim H.R. Characterization of effector memory CD8+ T cells in the synovial fluid of rheumatoid arthritis // J. Clin. Immunol. 2012; 32 (4): 709–720. DOI: 10.1007/ s10875-012-9674-3.

5. Van Amelsfort J.M., Jacobs K.M., Bijlsma J.W., Lafeber F.P., Taams L.S. CD4(+)CD25(+) regulatory T-cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid // Arthritis Rheum. 2004; 50 (9): 2775–2785. DOI: 10.1002/art.20499.

6. Gattorno M., Prigione I., Morandi F., Gregorio A., Chiesa S., Ferlito F., Favre A., Uccelli A., Gambini C., Martini A., Pistoia V. Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis // Arthritis. Res. Ther. 2005; 7 (2): R256–257. DOI: 10.1186/ar1485.

7. Okada R., Kondo T., Matsuki F., Takata H., Takiguchi M. Phenotypic classification of human CD4+ T cell subsets and their differentiation // Int. Immunol. 2008; 20 (9): 1189–1199. DOI: 10.1093/intimm/dxn075.

8. Baschant U., Tuckermann J. The role of the glucocorticoid receptor in inflammation and immunity // J. Steroid Biochem. Mol. Biol. 2010; 120 (2–3): 69–75. DOI: 10.1016/j.jsbmb.2010.03.058.

9. Bischof F., Melms A. Glucocorticoids inhibit CD40 ligand expression of peripheral CD4+ lymphocytes // Cell Immunol. 1998; 187 (1): 38–44. DOI: 10.1006/ cimm.1998.1308.

10. Gutsol A.A., Sokhonevich N.A., Iurova K.A., Haziakhmatova O.G., Shupletsova V.V., Litvinova L.S. Dose-dependent effects of dexamethasone on functional activity of T-lymphocytes different grade of differentiation // Mol. Biol. (Mosk). 2015; 49 (1): 149–157.

11. Pandolfi J., Baz P., Fernández P., Discianni Lupi A., Payaslián F., Billordo L.A., Fainboim L., Arruvito L. Regulatory and effector T-cells are differentially modulated by dexamethasone // Clin. Immunol. 2013; 149 (3): 400–410. DOI: 10.1016/j.clim.2013.09.008.

12. Ayroldi E., Macchiarulo A., Riccardi C. Targeting glucocorticoid side effects: selective glucocorticoid receptor modulator or glucocorticoid-induced leucine zipper? A perspective // Faseb. J. 2014; 28 (12): 5055–5070. DOI: 10.1096/fj.14-254755.

13. Кремер Н.Ш. Теория вероятностей и математическая статистика. М.: ЮНИТИ-ДАНА, 2004: 573. Kremer N.Sh. Teoriya veroyatnostej i matematicheskaya statistika [Theory of Probability and Mathematical Statistics]. M.: YUNITI-DANA Publ., 2004: 573 (in Russian).

14. Reddy M., Eirikis E., Davis C., Davis H.M., Prabhakar U. Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function // J. Immunol. Methods. 2004; 293 (1–2): 127–142. DOI: 10.1016/j.jim.2004.07.006.

15. Tang X., Yocum D.E., Dejonghe D., Nordensson K., Lake D.F., Richard J. Increased activation-induced cell death in peripheral lymphocytes of rheumatoid arthritis patients: the mechanism of action // Immunology. 2004; 112 (3): 496–505. DOI: 10.1111/j.1365-2567.2004.01888.x.

16. Matsuki F., Saegusa J., Nishimura K., Miura Y., Kurosaka M., Kumagai S., Morinobu A. CD45RA-Foxp3(low) non-regulatory T cells in the CCR7-CD45RA-CD27+CD28+ effector memory subset are increased in synovial fluid from patients with rheumatoid arthritis // Cell Immunol. 2014; 290 (1): 96–101. DOI: 10.1016/j.cellimm.2014.05.011.

17. Boumpas D.T., Anastassiou E.D., Older S.A., Tsokos G.C., Nelson D.L., Balow J.E. Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription // J. Clin. Invest. 1991; 87 (5): 1739–1747. DOI: 10.1172/ JCI115192.

18. Liberman A.C., Druker J., Perone M.J., Arzt E. Glucocorticoids in the regulation of transcription factors that control cytokine synthesis. Cytokine Growth // Factor Rev. 2007; 18 (1–2): 45–56. DOI: 10.1016/j.cytogfr.2007.01.005.

19. Shipkova M., Wieland E. Surface markers of lymphocyte activation and markers of cell proliferation // Clin. Chim. Acta. 2012; 413 (17–18): 1338–1349. DOI: 10.1016/j.cca.2011.11.006.

20. Paulsen M., Janssen O. Pro- and anti-apoptotic CD95 signaling in T-cells // Cell. Commun. Signal. 2011; 9: 7. DOI: 10.1186/1478-811X-9-7.

21. Paulsen M., Valentin S., Mathew B., Adam-Klages S., Bertsch U., Lavrik I., Krammer P.H., Kabelitz D., Janssen O. Modulation of CD4+ T-cell activation by CD95 co-stimulation // Cell. Death. Differ. 2011; 18 (4): 619–631. DOI: 10.1038/cdd.2010.134.

22. Liberman A.C., Refojo D., Antunica-Noguerol M., Holsboer F., Arzt E. Underlying mechanisms of cAMPand glucocorticoid-mediated inhibition of FasL expression in activation-induced cell death // Mol. Immunol. 2012; 50 (4): 220–235. DOI: 10.1016/j.molimm.2012.01.008.

23. Baumann S., Dostert A., Novac N., Bauer A., Schmid W., Fas S.C., Krueger A., Heinzel T., Kirchhoff S., Schütz G., Krammer P.H. Glucocorticoids inhibit activation-induced cell death (AICD) via direct DNA-dependent repression of the CD95 ligand gene by a glucocorticoid receptor dimer // Blood. 2005; 106(2): 617–625. DOI: 10.1182/blood-2004-11-4390.

24. Banuelos J., Lu N.Z. A gradient of glucocorticoid sensitivity among helper T-cell cytokines // Cytokine Growth Factor Rev. 2016; 31: 27–35. DOI: 10.1016/j.cytogfr.2016.05.002.

25. Spreafico R., Rossetti M., Whitaker J.W., Wang W., Lovell D.J., Albani S. Epi polymorphisms associated with the clinical outcome of autoimmune arthritis affect CD4+ T-cell activation pathways // Proc Natl Acad Sci USA. 2016; 113 (48): 13845–13850. DOI: 10.1073/ pnas.1524056113.

26. Bertho N., Drénou B., Laupeze B., Berre C.L., Amiot L., Grosset J.M., Fardel O., Charron D., Mooney N., Fauchet R. HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC // J. Immunol. 2000; 164 (5): 2379–2385.

27. Muehling L.M., Mai D.T., Kwok W.W., Heymann P.W., Pomés A., Woodfolk J.A. Circulating Memory CD4+ T-сells target conserved epitopes of rhinovirus capsid proteins and respond rapidly to experimental infection in humans //J. Immunol. 2016; 197 (8): 3214–3224. DOI: 10.4049/jimmunol.1600663.

28. Spreafico R., Rossetti M., van Loosdregt J., Wallace C.A., Massa M., Magni-Manzoni S., Gattorno M., Martini A., Lovell D.J. Albani S. A circulating reservoir of pathogenic-like CD4+ T cells shares a genetic and phenotypic signature with the inflamed synovial micro-environment // Ann. Rheum. Dis. 2016; 75 (2): 459–465. DOI: 10.1136/ annrheumdis-2014-206226.

29. Astry B, Harberts E, Moudgil KD. A cytokine-centric view of the pathogenesis and treatment of autoimmune arthritis // J. Interferon Cytokine Res. 2011; 31 (12): 927–940. DOI: 10.1089/jir.2011.0094.

30. Konya C., Paz Z., Apostolidis S.A., Tsokos G.C. Update on the role of Interleukin 17 in rheumatologic autoimmune diseases // Cytokine. 2015; 75 (2): 207–215. DOI:10.1016/j.cyto.2015.01.003.

31. Elshabrawy H.A., Chen Z., Volin M.V., Ravella S., Virupannavar S., Shahrara S. The pathogenic role of angiogenesis in rheumatoid arthritis // Angiogenesis. 2015; 18 (4): 433–448. DOI: 10.1007/s10456-015-9477-2.

32. Gharibi T., Majidi J., Kazemi T., Dehghanzadeh R., Motallebnezhad M., Babaloo Z. Biological effects of IL21 on different immune cells and its role in autoimmune diseases // Immunobiology. 2016; 221 (2): 357–367. DOI: 10.1016/j.imbio.2015.09.021.

33. Koetz K., Bryl E., Spickschen K., O’Fallon W.M., Goronzy J.J., Weyand C.M. T-cell homeostasis in patients with rheumatoid arthritis // Proc. Natl. Acad. Sci. USA. 2000; 97 (16): 9203–9208.

34. Liberman A.C., Refojo D., Druker J., Toscano M., Rein T., Holsboer F., Arzt E. The activated glucocorticoid receptor inhibits the transcription factor T-bet by direct protein-protein interaction // Faseb. J. 2007; 21 (4): 1177– 1188. DOI: 10.1096/fj.06-7452com.

35. Ratman D., Vanden Berghe W., Dejager L., Libert C., Tavernier J., Beck I.M., De Bosscher K. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering // Mol. Cell. Endocrinol. 2013; 380 (1–2): 41–54. DOI: 10.1016/j.mce.2012.12.014.

36. Altonsy M.O., Sasse S.K., Phang T.L., Gerber A.N. Context-dependent cooperation between nuclear factor κB (NF-κB) and the glucocorticoid receptor at a TNFAIP3 intronic enhancer: a mechanism to maintain negative feedback control of inflammation // J. Biol. Chem. 2014; 289 (12): 8231–8239. DOI: 10.1074/jbc.M113.545178.

37. Banuelos J., Cao Y., Shin S.C., Lu N.Z. Immunopathology alters Th17 cell glucocorticoid sensitivity // Allergy. 2016; 72 (3): 331–341. DOI: 10.1111/all.13051.

38. Glass C.K., Saijo K. Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells // Nat. Rev. Immunol. 2010; 10 (5): 365–376. DOI: 10.1038/nri2748.

39. Hermann-Kleiter N., Baier G. NFAT pulls the strings during CD4+ T helper cell effector functions // Blood. 2010; 115 (15): 2989–2997. DOI: 10.1182/blood-2009-10-233585.

40. Noack M., Ndongo-Thiam N., Miossec P. Evaluation of anti-inflammatory effects of steroids and arthritis-related biotherapies in an in vitro coculture model with immune cells and synoviocytes // Front Immunol. 2016; 7: 509. DOI: 10.3389/fimmu.2016.00509.

41. Baschant U., Frappart L., Rauchhaus U., Bruns L., Reichardt H.M., Kamradt T., Bräuer R., Tuckermann J.P. Glucocorticoid therapy of antigen-induced arthritis depends on the dimerized glucocorticoid receptor in T-cells // Proc. Natl. Acad. Sci. USA. 2011; 108 (48): 19317– 19322. DOI: 10.1073/pnas.1105857108.