Advances in immunopathogenesis of adult immune thrombocytopenia

Abstract

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by immune-mediated accelerated platelet destruction and/or suppressed platelet production. Although the development of autoantibodies against platelet glycoproteins remains central in the pathophysiology of ITP, several abnormalities involving the cellular mechanisms of immune modulation have been identified, and the pathways behind the immune-mediated destruction of platelets have opened new avenues for the design of specific immunotherapies in an attempt to reduce the platelet destruction. This review is primarily focused on the recent literature with respect to immunopathological mechanisms in patients with ITP.

Keywords

primary immune thrombocytopenia / B lymphocytes / T lymphocytes / antigen-presenting cells / cytokines

Introduction

Primary immune thrombocytopenia (ITP) is an autoimmune disorder defined currently as isolated thrombocytopenia (peripheral blood platelet count<100×10⁹/L) in the absence of conditions known to cause thrombocytopenia [1]. Manifestations of ITP vary from asymptomatic to localized hemorrhage in skin or mucous membranes, even severe bleeding events such as intracranial hemorrhage. Traditionally ITP is regarded as an antibody-mediated disease in which the patient’s immune system reacts with a platelet autoantigen(s) resulting in thrombocytopenia due to autoantibody-mediated platelet destruction and/or suppression of platelet production [2]. According to population-based studies, the overall incidence of ITP ranges from 3.2-12.1 per 10⁵ adults each year [3-5], and recent epidemiologic data suggest nearly equal incidence for the sexes except in women of childbearing age [3]. Childhood ITP differs from adult chronic ITP in pathogenesis, diagnosis, management, and prognosis. This article addresses current understanding of immune dysregulation in adult ITP only.

Increased platelet destruction

Recent years have witnessed great progress in the efforts to determine the molecular and cellular details of platelet destruction in ITP, including autoantibody-mediated platelet clearance and CTL-mediated platelet lysis. Abnormalities of multiple cell types, including T- and B-lymphocytes, monocytes, macrophages, natural killer (NK) cells and dendritic cells (DCs), contribute to the cellular immune dysregulation in ITP.

Autoantibody-mediated platelet clearance

In 1951, Harrington demonstrated that plasma from ITP patients could induce thrombocytopenia in healthy recipients, and further studies revealed that the thrombocytopenic effect was mediated by GP-specific autoantibodies [6]. Since then, autoantibody-mediated platelet clearance has been considered as the canonical mechanism of platelet destruction in ITP. Most of these autoantibodies are IgG subtype, but IgM and IgA could be detected in some patients [7]. Platelets coated by GP-specific autoantibodies were phagocytized by macrophages via Fc receptors and prematurely cleared in the reticuloendothelial system. The humoral immune response underlying ITP pathogenesis involves a complex interaction between macrophages/DCs, T cells and B cells, which first takes place when antigens interacts with antigen-presenting cells (APCs).

Antiplatelet autoantibodies in ITP frequently appear to be directed against GPIIb/IIIa and GPIb/IX. By light and heavy chain restriction analysis, autoantibodies were shown to be clonally restricted to a limited antigenic repertoire [8]. Several features of the platelet-reactive autoantibodies indicate that they arise as part of an antigen-driven clonal expansion rather than being the result of polyclonal B cell activation triggered by nonspecific stimuli. The destruction of B cell equilibrium in ITP could be partially mediated by the elevated expression of B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) [9,10], which has been shown to support the survival and differentiation of B cells, and our recently published work have has shown that targeting BAFF-R with BR3-Fc might be valuable for the alleviation of ITP [10].

CTL as a major player in platelet lysis

Although the presence and specificity of autoantibodies in chronic ITP is well characterized, it could not account for all observations made in this disorder. Some ITP patients reported no detectable antigen-specific autoantibodies, and remission in ITP could occur despite the presence of platelet autoantibodies. In addition, several CD8⁺ T-lymphocyte abnormalities have also been demonstrated. These phenomena indicated the presence of other mechanisms in ITP.

APCs also present platelet antigen associated with MHC class I molecules to CD8⁺ T cells and activate the cytotoxic T lymphocytes (CTLs). Olsson et al. reported that several cytotoxic genes, such as Apo-1/Fas, granzyme B and perforin, together with genes involved in the type 1 T-helper cell (Th1) response, showed increased expression in ITP patients in the active phase. In addition, several members of the killer-cell immunoglobulin-like receptor (KIR) family such as KIR3DLL and KIR3DL2, which could downregulate CTL and NK cell responses by binding to major histocompatibility complex (MHC) class I molecules, were demonstrated to be elevated in remission ITP patients compared with patients with active disease and healthy controls [11]. Interaction of upregulated FasL and TNFα with their respective receptors on the surface of target cells may result in apoptosis of autologous platelets. An in vitro assay of platelet lysis showed CTL-mediated lysis of autologous platelets in patients with active ITP but not in patients in remission [11-13]. These results suggested that the cell-mediated toxicity might mediate or participate in the pathogenesis of ITP.

Other immune cells involved in ITP

T cells

(1) Oligoclonal platelet-antigen-specific T cells

In this decade, a pathophysiological role for T cells in ITP has been further established. Antiplatelet autoantibody production is controlled by platelet-specific helper T cells [14]. As shown in Fig. 1, T cell receptor (TCR) of Th cells could bind the peptide-MHC complex and then abberant signal activation occurs through CD154 and CD40 interaction in ITP. Binding of CD28 expressed on Th cells with the CD80 molecule overexpressed on the APC membrane of ITP patients could induce an additional co-stimulatory signal. And next, the activated Th cells could promote B cell differentiation and autoantibody production by secreting interleukin-2 and interferon-γ. Blocking the B7-CD28 interaction has been shown to induce platelet glycoprotein (GP)-specific T cell tolerance [15,16]. It has been reported that acute ITP had no significant T cell clone expansion, while chronic ITP usually demonstrates expanded abnormal T cell clones. Chronic ITP patients share certain common motifs for T cell receptor beta chain variable gene (TCRBV), which is possibly related to recognition of similar autoantigens [17]. Splenectomy may lead to reductions in T cell clonal expansions in responders, and the extent of T cell clonality impacts responsiveness to splenectomy in patients with ITP [18].

(2) Disequilibrium of T cell subsets

The balance of Th1 and Th2 subsets has been implicated in the regulation of many immune responses, and is known to be impaired in many autoimmune diseases. Several in vitro studies have found evidence supporting a T helper 0 (Th0)/Th1 polarization of the immune response in ITP [19]. Panitsas et al. observed that ITP patients with active disease exhibited significantly higher Th1/Th2 ratios than patients in remission or healthy controls, and lower peripheral platelet numbers correlated with higher Th1/Th2 ratios [20]. Correction of the Th1 polarization profile may provide a new idea for the management of ITP [21].

More recently, new subsets of T cells, such as Th17 or Th22 cells, have also been shown to be linked to the development of ITP. Th17 cells, and their effector cytokine IL-17, are significantly increased in patients with active ITP [22-25]. Genotype analysis further revealed that the IL17F 7488 T allele was significantly correlated with ITP [24]. Likewise, Th22 cells—another newly identified potent proinflammatory T cell subset—were significantly elevated in active ITP patients [26,27], and glucocorticoid therapy could downregulate expression of Th22 as well as plasma IL-22 [28].

CD4⁺CD25⁺ regulatory T cells (Treg cells) have immune-regulatory properties and are capable of inhibiting both CD4⁺ and CD8⁺ T cell responses. Cell-cell contact, IL-10, transforming growth factor (TGF)-beta or IL-35 have been suggested to mediate the suppressive function of Treg cells [29,30]. Several studies showed that both the numbers and immunoregulatory function of Treg cells are impaired in patients with active ITP, and therapies such as dexamethasone, IVIg, and Rituximab as well as TPO-agonists could correct the abnormalities of Tregs in ITP [31-36]. Furthermore, our group recently demonstrated that non-regulatory CD4⁺CD25^-CD45 RA⁺ T cells in ITP could successfully be induced into GP-specific Tregs, which could mediate tolerance-inducing effects on Th cells via modulating the antigen presenting function of DCs [37].

(3) Disturbed apoptosis of T-lymphocytes

FasL, TNFα and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) all belong to the TNF superfamily, each of which could induce apoptosis after binding with their respective receptors. The abnormal Fas and FasL expression in ITP patients suggests that altered Fas pathway signaling should be considered in the etiology of this disease [38]. In CD3⁺ lymphocytes of active ITP patients, anti-apoptotic genes, A20 and caspase-8, were upregulated, while the pro-apoptotic gene, Bax, was downregulated, suggesting an apoptotic resistant profile of T cells. Consequently, clearance of activated T-lymphocytes might be impaired, which could result in accumulation of potentially autoreactive T-lymphocytes and continued destruction of autologous platelets [39].

B cells and ITP

Besides differentiating into plasma cells and secreting pathogenic autoantibodies, abnormal B cells are involved in the development of ITP through other mechanisms. For example, B regulatory cells (Bregs)—a newly identified B cell subset characterized by CD19⁺CD24^hiCD38^hi—were reported to be deficient in active ITP [40]. In normal conditions, regulatory B cells (Bregs) could promote the differentiation and/or recruitment of Tregs to tissue, reduce Th-cell activation and monocyte proinflammatory production through an IL-10-dependent mechanism [41], thus playing an important role in maintaining immune tolerance. In ITP, both the frequency and immunosuppressive effect of Bregs were downregulated [40], suggesting that restoration of Bregs might shed new light on the reconstitution of immune balance.

Role of macrophages and DCs

IgG autoreactive responses against protein antigens are launched by activated autoreactive Th cells that recognize peptides presented by APCs such as MHC class II positive DCs or macrophages [42]. Cines et al. proposed a model in which APCs expressing novel cryptic epitopes from platelet GPs, along with co-stimulatory molecules, induce the activation of T cells that recognize these additional platelet antigens [43]. This acquired recognition of new self-determinants or epitope spreading played critical roles in the initiation and perpetuation of ITP. Kuwana showed that splenic macrophages were the major APCs responsible for presenting cryptic platelet peptides to autoreactive T cells and stimulating autoantibody production [44]. Moreover, several groups reported that the decreased inhibitory FcγRIIb on monocytes/macrophages might play a role in the pathogenesis of ITP [45-47]. Another study by Hamzeh-Cognasse revealed that by presenting the apoptotic platelets, DCs from ITP patients could efficiently stimulate autoreactive T cell proliferation [48]. Therefore, dysfunction of APCs plays important roles in the triggering of immune imbalance in ITP.

Abberant NK cell activity in ITP

It has been reported that numbers of NK cells and expansion of the CD56⁺CD3^- NK cells as well as CD56⁺CD3⁺ cytotoxic T cell subsets were increased in patients with active ITP [49,50]. Furthermore, elevated expression of MHC class II molecules was also found in CD56⁺CD3^- NK cells from refractory ITP patients, suggesting the in vivo activation of NK cells in ITP [49]. So it is reasonable to speculate that abberant NK cell activity might play a role in destruction of IgG-coated platelets.

Decreased platelet production

It has been well established that platelet clearance is accelerated in ITP. However, platelet turnover studies have shown that platelet production is decreased or normal in most ITP patients [51,52], and the absolute platelet reticulocyte counts are significantly reduced [53]. Therefore, ineffective platelet production in ITP has been proposed, and supported by a series of recently published data.

Autoantibody-mediated dysmegakariocytopoiesis

In vitro studies showing reduced megakaryocyte production and maturation in the presence of autoantibodies against platelet glycoproteins in ITP plasma provide evidence for autoantibody-induced suppression of megakaryocytopoiesis. Chang et al. described suppression of in vitro production of megakaryocytes from cord blood cells by plasma from ITP patients with detectable antiplatelet antibodies [54]. McMillan et al. similarly studied the effect of plasma from adult patients with chronic ITP on in vitro megakaryocytopoiesis. ITP plasma also inhibited megakaryocyte maturation, resulting in fewer 4N, 8N and 16N cells [55]. Moreover, our recently published data revealed that abnormal tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could impair apoptosis of megakaryocytes, which also contributes to dysmegakariocytopoiesis [56].

CTL-mediated dysmegakaryocytopoiesis

It has been suggested that CD8⁺ T cells may be involved in the pathogenesis of acquired amegakaryocytic thrombocytopenic purpura by exerting suppression on megakaryocyte differentiation [57]. Bone marrow CD8⁺ T cell proliferation in ITP was shown to be platelet specific, suggesting that the marked increase of activated CD8⁺ T cells in bone marrow is functional. Our previous published work demonstrated that CD8⁺ T cells from chronic ITP patients could induce downregulation of Fas and upregulation of Bcl-xl in megakaryocytes when cultured together, thus apoptosis of megakaryocytes was impaired, which would result in increased megakaryocyte counts and decreased platelet production [58].

Relative thrombopoietin (TPO) deficiency in ITP

Thrombopoiesis is regulated by cytokine named TPO, which is a ligand for the c-mpl receptor, a product of the cellular proto-oncogene c-MPL [59]. TPO is an acidic glycoprotein produced mainly in the liver, and is necessary for the expansion, differentiation and maturation of megakaryocytes, finally resulting in platelet production [60]. Levels of TPO may vary depending on disease status. A circumstantial evidence for decreased platelet production is that the serum TPO levels in patients with ITP were within the normal range or only slightly elevated in ITP patients compared with other thrombocytopenic disorders such as aplastic anemia with TPO levels up to 10 times higher [61]. Experimental evidence showed that TPO could improve colony formation of marrow megakaryocytes and increase peripheral platelets in ITP patients [62]. These studies suggested that relative endogenous TPO deficiency might play a role in the pathophysiology of thrombocytopenia in ITP, and exogenous TPO might be potential for the management of ITP. Recently, two major TPO-mimetic candidate drugs, romiplostim and eltrombopag, have been tested in several large multicenter clinical trials in ITP, and results are promising [63-66]. Bao recently reported that TPO-R treatment could improve Treg function by enhanced release of TGF-β1, suggesting that thrombopoietic agents have profound effects on the restoration of immune balance in ITP [36]. In 2008, both romiplostim and eltrombopag were approved by FDA for use in ITP patients who failed to response to corticosteroids, IVIg, or splenectomy. Additionally, another TPO agonist, recombinant human TPO, has been clinically used in Chinese ITP patients, and the therapeutic effects are also encouraging [67].

Summary

In conclusion, ITP is a complex and heterogeneous autoimmune disorder, and the low platelet count is the result of both increased platelet destruction and decreased platelet production. Abnormalities of multiple immune cells have been identified in ITP. The hypothesized pathophysiology of ITP is summarized in Fig. 1. Further exploration in immunologic derangement of cellular and humoral immunity will facilitate the design of new therapeutic approaches for the management of ITP.

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