research paper
Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients
Nikolaos C. Giannakoulas,1 Marina Karakantza,2 Georgios L. Theodorou,2 Maria Pagoni,3 Athanasios Galanopoulos,4 Theodora Kakagianni,1 Alexandra Kouraklis-Symeonidis,1 Panagiota Matsouka,1 Alice Maniatis2 and Nicholas C. Zoumbos1 1
Haematology Division, Department of Internal
Medicine, University Hospital of Patras, Rion, Patras, Greece, 2Laboratory Hematology and Transfusion Medicine, Medical School, University of Patras, Patras, Greece, 3Department of Haematology and Lymphomas, ‘Evangelismos’ General Hospital, Athens, Greece, and 4
Haematology Department, District General
Hospital of Athens ‘G. Gennimatas’, Athens, Greece 2004 Blackwell Publishing Ltd, British Journal of Haematology, 124, 97–105
Summary Immune dysfunction, which leads to the suppression of haemopoiesis by cytokines that are secreted by activated T lymphocytes, is considered to play a key role in the pathogenesis of acquired aplastic anaemia (AAA). We investigated the intracytoplasmic expression of type-1 [interferon c (IFN-c), interleukin (IL)-2] and type-2 (IL-4, IL-10) cytokines in CD4+ and CD8+ T cells before and after in vitro activation in 16 patients with AAA and 17 normal controls. Untreated or refractory patients had a significantly higher proportion of unstimulated CD4+ and CD8+ T cells that produced IFN-c and IL-2 whereas the IL-4 and IL-10 producing T cells did not differ from that of controls, resulting in a shift of IFN-c/IL-4 ratio towards a type-1 response. Patients in remission had also increased proportion of IFN-cproducing unstimulated CD4+ and CD8+ cells, with a parallel rise of IL-4and IL-10-producing cells and normal IFN-c/IL-4 ratio. These data indicate that, in newly diagnosed and refractory patients with AAA, CD4+ cells are polarized towards a type-1 response that in turn leads to activation of cytotoxic CD8+ cells and finally to haemopoietic stem cell destruction. The type-1 response persists in patients in remission although this effect is compensated by the increase of IL-4 and IL-10 production.
Received 26 June 2003; accepted for publication 17 September 2003 Correspondence: Nikolaos C. Giannakoulas MD,
Keywords: aplastic anaemia, CD4+ cells, CD8+ cells, type-1/type-2 cytokines, interferon c.
Haematology Division, Department of Internal Medicine, University Hospital of Patras, Rion, Patras GR-26500, Greece. E-mail:
[email protected]
Acquired aplastic anaemia (AAA) is a rare disease characterized by hypocellular bone marrow and peripheral blood pancytopenia. Although the exact mechanisms responsible for its pathogenesis remain unclear, possible causes include a primary stem cell defect, an immune mediated inhibition of haemopoiesis, a defective bone marrow microenviroment or a combination of these. Clinical and laboratory data suggest that the immune system, especially T lymphocytes, have an important role in the development of AAA (Young & Maciejewski, 1997; Young, 2000). High remission rates in clinical trials after combination treatment with immunosuppressive drugs, such as antilymphocyte globulin (ALG) or antithymocyte globulin (ATG) and cyclosporine, have strongly
suggested the crucial role of the immune system in the pathophysiology of aplastic anaemia (Bacigalupo et al, 1995; Marsh & Gordon-Smith, 1995; Rosenfeld et al, 1995; Young & Barrett, 1995). The exact immune mechanisms involved in the pathogenesis of AAA are still unclear. There are data showing that patients’ T cells overproduce interferon c (IFN-c) and tumour necrosis factor a (TNF-a), two cytokines that suppress the proliferation of early and late haemopoietic progenitor cells (Laver et al, 1988; Nakao et al, 1992; Nistico & Young, 1994; Selleri et al, 1995, 1996). Both cytokines induce the expression of Fas receptor and initiate apoptosis. In patients with AAA, it has been shown that haemopoietic stem cells express Fas receptors
ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 124, 97–105
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N. C. Giannakoulas et al and that their marrow contains increased numbers of apop1 totic cells (Maciejewski et al, 1995a, b; Phillpot et al, 1995; Killick et al, 2000). In addition, it has been shown that the blood and marrow of patients with AAA contains an increased number of activated CD8+ cytotoxic lymphocytes (Zoumbos et al, 1985). The above data have led to the proposal of a model for the pathogenesis of AAA, in which activated cytotoxic CD8+ cells produce cytokines that induce Fas expression in haemopoietic stem cells and eventually lead to their destruction. The role of CD4+ T cells in the immune mediated destruction of stem cells in AAA is unknown. Recent data show that, in patients with AAA, peripheral blood CD4+ T cells after mitogenic stimulation are polarized towards a type-1 immune response (Tsuda & Yamasaki, 2000). Another study showed that CD4+ and CD8+ T lymphocytes from bone marrow of paediatric AAA patients were polarized towards a type-1 immune response, whereas this polarization was not observed in peripheral blood lymphocytes (PBL) (Dufour et al, 2001). A recent study with a large number of AAA patients showed that the in vivo type-1 response of T cells parallels the activity of the disease (Sloand et al, 2002). In this study, patients that demonstrated in vivo polarization of T cells towards a type-1 response were good responders to immunosuppressive treatment and it was suggested that measurement of the intracytoplasmic IFN-c might be a useful marker for the prediction of response to treatment (Sloand et al, 2002). The purpose of our study was to investigate the type-1 and type-2-producing CD4+ and CD8+ T cells in patients with AAA at diagnosis and during haematologic remission after successful immunosuppressive treatment. For this purpose we evaluated the percentages of CD4+ and CD8+ T cells producing Type 1 [IFNc, interleukin (IL)-2] and Type 2 (IL-4, IL-10) cytokines before and after mitogenic activation, and estimated the balance between type-1/type-2 immune response. We also evaluated the immunophenotypic characteristics of peripheral blood circulating lymphocyte subpopulations.
Patients and methods Patients and controls The diagnosis of aplastic anaemia was made according to the accepted criteria (Camitta et al, 1976; Marsh et al, 1999). Severe AAA was defined by the combination of low bone marrow cellularity (
