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Summary. Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which activated T-helper (Th) cells and different Th-cell cytokines might play an important role. We have recently reported that chronic ITP patients in remission had elevated plasma levels of the Th3 cytokine transforming growth factor-β1 (TGF-β1), possibly as a part of a bystander immune suppression. In the present study we found that, in ITP patients with active disease [platelet count (plc) < 50 × 109/l], mitogen-stimulated peripheral blood mononuclear cells (PBMC) had a significantly reduced production of TGF-β1 (444 ± 178 pg/ml; n = 6) compared with patients with plc 50–150 × 109/l (1293 ± 374 pg/ml; n = 9; P < 0·05), patients with plc > 150 × 109/l (1894 ± 244 pg/ml; n = 12; P < 0·005) and healthy controls (1698 ± 241 pg/ml; n = 10; P < 0·01). Nineteen per cent of ITP patients expressed a platelet-induced PBMC proliferation. Surprisingly, 22% of the ITP patients had a PBMC proliferation below the normal range, i.e. a suppressed proliferation in the presence of platelets; five of these six patients had active disease. In summary, this study demonstrated that chronic ITP patients with active disease had reduced PBMC production of the Th3 cytokine TGF-β1. This result gives further support to the theory that chronic ITP in active phase is associated with a downregulated Th3-response.
Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which the antibodies produced cause platelet destruction and enhanced bleeds (McMillan, 1981). Although the humoral immune response in chronic ITP has been extensively studied, the cellular immunology is less well known. Nevertheless, previous studies have shown an increased T-lymphocyte activation (Mizutani et al, 1987; Semple & Freedman, 1991; Garcia-Suarez et al, 1993b) and T helper (Th)-cell cytokine patterns, indicating that Th cells may play an important role in this disorder (Garcia-Suarez et al, 1993a; Haznedaroglu et al, 1995; Nomura et al, 1995; Crossley et al, 1996; Semple et al, 1996; Erduran et al, 1998). Furthermore, a platelet-induced Th cell (CD4+) proliferation with a concomitant interleukin 2 (IL-2) production in the majority of children with chronic ITP has been reported (Semple & Freedman, 1991; Semple et al, 1996). Recently, Kuwana et al (1998) described chronic ITP patients that had autologous T-cell reactivity against platelet membrane glycoprotein (GP) IIb/IIIa and a concomitant in vitro anti-GPIIb/IIIa IgG production. Taken together, these findings indicate that, in ITP T-lymphocytes, probably Th cells, are activated by antigen-presenting cells, thereby initiating the B-cell autoantibody production.
A bystander immune suppression has been demonstrated in experimental models of oral immune tolerance induction, a phenomenon associated with expression of the Th3 cytokine transforming growth factor-β1 (TGF-β1) (Miller et al, 1991). We have recently reported (Andersson et al, 2000) that chronic ITP patients in remission had elevated plasma levels of TGF-β1, possibly as a part of a bystander immune suppression. In the present work we studied the platelet-induced reactivity and the Th-cytokine profile in mononuclear cell cultures from chronic ITP patients.
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In the present study PHA-stimulated PBMC from chronic ITP patients with ‘active’ disease had a significantly lower production of TGF-β1 than patients with stable disease, patients ‘in remission’ and healthy controls. Similarly, patients with ‘stable disease’ had a lower TGF-β1 production than patients ‘in remission’ and healthy controls.
Chronic ITP is an autoimmune disorder in which activated Th cells and different Th-cell cytokines might play an important role. Th cells are divided into three main subsets, Th1 , Th2 and Th3 cells. Th1 cells produce IL-2 and IFN-γ whereas Th2-cells release IL-4 and IL-10 (Romagnani et al, 1997). A Th1 cytokine profile has been suggested in chronic ITP. However, the results reported have not been concordant (Ware & Howard, 1993; Haznedaroglu et al, 1995; Crossley et al, 1996; Semple et al, 1996; Erduran et al, 1998). A characteristic of Th3 cells are their production of the immune-modulating cytokine TGF-β1 (Chen et al, 1994; Fukaura et al, 1996). TGF-β1 has been found to be an important inhibitor of both B-cell proliferation and immunoglobulin secretion (Kehrl et al, 1986a, 1989; Cross & Cambier, 1990). Also, it inhibits T-cell proliferation (Kehrl et al, 1986b) and suppresses some Th1 and Th2 cell-mediated autoimmune diseases (Kehrl et al, 1986a; Holter et al, 1994; Mosmann & Sad, 1996; Bridoux et al, 1997). Most recently it was shown that TGF-β1 is one of the key negative regulators of immune homeostasis and its absence leads to activation of a self-targeted immune response (Gorelik & Flavell, 2000).
We have previously reported that patients with chronic ITP in remission had significantly higher plasma levels of TGF-β1 than patients with active disease and healthy controls (Andersson et al, 2000). This finding is further supported by the results from the present study, demonstrating a reduced TGF-β1 production by PBMC from ‘active’ ITP patients. Thus, chronic ITP in active phase appears to be associated with a downregulated Th3 response. Presumably, a remission might be induced by upregulation of the Th3 response. It is known that platelets are a rich source of TGF-β1, stored in the α-granules (Fava et al, 1990). The differences we observed for the TGF-β1 production by PBMC cultures could not, however, be explained by in vitro platelet degranulation, as the mean platelet count in the PBMC suspensions was similar in all groups. Hence, our data indicates that the severity of the disease is reflected by an inability to produce the Th3 cytokine TGF-β1, i.e. high disease activity seems to be associated with a downregulated Th3 response. In addition, it has previously been reported that systemic lupus erythematosus (SLE), a disorder of generalized autoimmunity and T-cell dysfunction, is associated with a decreased production of TGF-β by lymphocytes (Ohtsuka et al, 1998). Taken together, these findings suggest that decreased production of this cytokine could be important in the maintenance of B-cell hyper-reactivity in autoimmune disorders.
Furthermore, others have previously demonstrated that 21–68% of ITP patients display a platelet-induced Th-cell proliferation (Semple & Freedman, 1991; Ware & Howard, 1993; Kuwana et al, 1998). Only 19% of our ITP patients expressed a platelet-induced PBMC proliferation; the majority of our ‘reactive’ patients were in stable disease and none was in active stage. This finding seems to be contradictory to the results of Kuwana et al (1998); all their ‘reactive’ patients had an active disease. Also, Semple and Freedman (1991) and Ware and Howard (1993) reported a platelet count between 50 and 70 × 109/l in their study population; however, the platelet count was not specifically given for ‘reactive’ patients. Conversely, we found that most patients with plc < 50 × 109/l had a suppressed PBMC proliferation in the presence of platelets. A larger proportion of our patients with active disease were on oral corticosteroids at time of study. Basal PBMC proliferation were, however, similar in all groups, indicating that corticosteroid treatment did not interfere with the unstimulated PBMC proliferation in vitro. The explanation for the platelet-induced suppression of PBMC proliferation is unclear. However, the PBMC cultures that contained platelets and which were used for cytokine analysis, had significantly higher TGF-β1 concentrations than the control cultures without platelets. This difference in TGF-β1 concentrations is at least partly explained by TGF-β1 being released from the platelets added to the cultures. TGF-β1 is known to inhibit proliferation of both B and T cells and to induce apoptosis in B cells (Inman & Allday, 2000). Furthermore, Kehrl et al (1986b) has reported that activated T cells have increased number of TGF-β receptors. Indeed, several investigators have found an elevated percentage of activated T cells in ITP patients with active disease (Mizutani et al, 1987; Semple & Freedman, 1991; Garcia-Suarez et al, 1993b; Andersson et al, 2000). Thus, it could be hypothesized that PBMC from patients with active ITP might be more sensitive to TGF-β1 and the suppression of PBMC proliferation might be mediated by this immune modulating cytokine.
Furthermore, recent reports have shown that the effect of certain immunosuppressive agents such as cyclosporin A, tacrolimus and rapamycin is mediated, at least in part, by TGF-β production (Khanna et al, 1994, 1999a,b; Dodge et al, 2000). Even though not commonly used, cyclosporin A has been reported to be effective in some patients with refractory chronic ITP (Kelsey et al, 1985; Emilia et al, 1996; Blanchette et al, 1998; Kappers-Klunne & van't Veer, 2001). In the view of our data, one possible explanation for the cyclosporine effect in ITP could be a TGF-β1-mediated immune suppression.
In conclusion, the present study demonstrated that chronic ITP patients with active disease had a reduced PBMC production of the Th3 cytokine TGF-β1. This finding gives further support to the hypothesis that chronic ITP in active phase is associated with a downregulated Th3 response. Thus, TGF-β1 might be an important regulator of both B- and T-cell proliferation in chronic ITP. Further studies should focus on the effect of different therapeutic measures in ITP and their effect on the Th3 response.