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Egypt J Pediatr Immunol 2014;12(2):49-61.

Review article

Pathophysiology of immune thrombocytopenic purpura: a bird's-eye view.

Amira Abdel Moneam Adly Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt ABSTRACT and B (including T-helper, T- Immune thrombocytopenic purpura (ITP) is a cytotoxic, and T-regulatory lymphocytes) 6. common autoimmune disorder resulting in isolated The triggering event for ITP is unknown7, but thrombocytopenia. It is a disorder continued research is providing new insights into characterized by low platelet counts due to decreased the underlying immunopathogenic processes as well platelet production as well as increased platelet as the cellular and molecular mechanisms involved destruction by autoimmune mechanisms. ITP can in megakaryocytopoiesis and platelet turnover. present either alone (primary) or in the setting of other Although historically ITP-associated thrombo- conditions (secondary) such as infections or altered cytopenia was attributed solely to increased rates of immune states. ITP is associated with a loss of destruction of - coated platelets, it has tolerance to platelet and a phenotype of become evident that suboptimal platelet production accelerated platelet destruction and impaired platelet also plays a role 8. production. Although the etiology of ITP remains Bleeding is due to decreased platelet production unknown, complex dysregulation of the immune as well as accelerated platelet destruction mediated system is observed in ITP patients. Antiplatelet in part by -based destruction mediate accelerated clearance from the mechanisms9. Most in ITP are circulation in large part via the reticuloendothelial switched and harbor somatic mutations10, (monocytic phagocytic) system. In addition, cellular and as such a role for CD4+ helper T cells in is perturbed and T-cell and profiles pathogenesis has been invoked. Consistent are significantly shifted toward a type 1 and Th17 with this, ITP patients have activated platelet- proinflammatory with impaired autoreactive T cells with increasing cytokine regulatory compartment, including Tregs and Bregs, imbalance toward IL-2 and IFN-γ, indicating a shift have been reported, suggesting a generalized immune toward Th1 cells11. More recently, increased Th17 dysregulation in ITP. Understanding how cells or IL-17 cytokine were reported in ITP Th1/Th17/Treg differentiation and expansion are patients12, implicating a possible role for Th17 cells controlled is central to uncovering how autoimmunity in ITP immunopathology. may be sustained in ITP. Moreover, a role for cytotoxic T cells in direct lysis of platelets and megakaryocytes in the bone 13 INTRODUCTION marrow has been proposed . In addition to an Immune thrombocytopenia (ITP) is recognized as increase in the effector arm of the immune an immune mediated disorder in which platelets are response (Th1, Th17 and CD8 cells); a decrease in opsonized by autoantibodies and prematurely the regulatory immune compartment of patients destroyed by reticuloendothelial system1. It is a with ITP has been described. Specifically, a hematologic disorder affecting children with an deficiency in generation and/or defective functions 14 incidence of four to five cases per 100,000 children of ITP (Treg) and regulatory B 15 per year2.It is characterized by immune-mediated cells (Bregs) . accelerated platelet destruction and suppressed This brief review will highlights the platelet production. Although the etiology of ITP is mechanisms, and their elements, underlying the not yet known, and the diagnosis continues to be pathogenesis and cellular kinetics of ITP and one of exclusion.3 A number of studies have discusses the aspects of current understanding of provided evidence of disturbed innate and adaptive immune dysregulation. Also it addresses the recent immune responses in patients with ITP.4 The findings on the state of the Breg and Treg pathophysiology of ITP increasingly is understood compartments by which this information may guide better5. Not surprisingly, it is complex with in ITP patients in the future. involvement of many players in the human immune orchestra, including antibodies, , - presenting cells, costimulatory molecules, and T 49 Adly

Implicating agents in ITP of ITP often occurs seemingly rapidly and between ITP is a heterogeneous group of disorders with monitoring time points19. potentially differing etiologies, natural histories and Despite these limitations, data from numerous response to therapy3. Some ITP patients appear to studies in recent years are beginning to come have fundamental disturbances in their innate and together to form a picture of an unbalanced immune adaptive immunity, while in others a specific often response. Not surprisingly, the immune changes times self-liming inciting agent may be involved. observed during ITP are complex. The long-held Several potential inciting agents have been dogma of platelet-bound Abs leading to identified or proposed. Fc_receptor (Fc_R)–mediated clearance of platelets In the case of childhood ITP, many patients give by residing in the spleen (and liver) a history of a recent infection. In adults, infection continues to be a central theme in our current with human immune deficiency virus, Helicobacter understanding of ITP. In addition to this, the pylori and hepatitis C have been implicated in a evidence supports a wide array of immune shifts high percentage of cases of ITP in endemic areas; involving all components of the , the causal relationship is strongest in settings where resulting in both shortened platelet survival and microbial elimination leads to resolution of the inhibition of the production of platelets20. associated ITP16. Acute ITP can also occur after vaccination. The evidence for vaccine associated The role of the spleen ITP is most compelling in those immunized with In 1916, Kaznelson, while a medical student in measles-mumps-rubella vaccine (~1:40,000). There Vienna, prevailed upon the attending surgeon to have also been case reports of ITP in children perform a splenectomy in a patient with ITP. The following other vaccinations against hepatitis B, splenectomy was successful in normalizing the diphtheria-tetanus-pertussis, and hepatitis A, but the platelet count and, with other cases, first established relationship is less compelling17. the critical role of the spleen in ITP21. However, the It has been hypothesized that ITP results from cause of thrombocytopenia remained unclear. Was engrafted on a normal immune the spleen destroying the platelets or did it secrete a repertoire, such that the immune thrombocytopenia suppressive substance that inhibited platelet resolves once the inciting antigen has been production and/ or release into the circulation? eliminated. Although microbial antigens may play a Doan et al.22 examined a number of spleens from role in the induction of vaccine associated ITP, patients with ITP. They demonstrated sea blue many vaccines also contain adjuvants (e.g., alum) (lipid laden) histiocytes in the spleen, suggesting it that might potentiate the immune response. was the platelet ‘destroyer’. What directed the Recent work from Shoenfeld and coworkers spleen to prematurely destroy platelets, however, suggests that some autoimmune syndromes and remained unclear23. The spleen, with its unique inflammatory states might be induced by these microvascular architecture, is well suited for immune adjuvants themselves18. The name they immunologic surveillance of the body platelet mass have given to this activity is as it has the capacity to store a large number of Autoimmune/Inflammatory Syndrome Induced by platelets in an exchangeable pool in close proximity Adjuvants (ASIA). The role that ASIA might play to cells of the immune system23. The intrasplenic in the initiation of ITP requires further study, but platelet transit time has been estimated to be around the observation that ITP has been seen after the 10 min and is independent of spleen size. The administration of several different vaccines demonstration that splenic tissue from patients with provides an interest in this possibility. ITP produces antiplatelet IgG suggests that the Identification of factors that precipitate ITP is spleen has capacity of forming these autoantibodies extremely difficult due to the likely transient nature and that this mechanism is active in the disease24. In of the provoking event, the inherent difficulty in addition, the splenic have been shown diagnosing ITP early in its course, and the to avidly phagocytose platelets sensitized by serum prolonged time period over which monitoring from ITP patients25. The spleen therefore, both would need to occur to capture the onset of ITP. produces antiplatelet antibodies and sequesters Even in subjects known to be at high risk for ITP opsonized platelets. The central role of the spleen in due to a personal history of ITP, a strong family ITP is further illustrated by the fact that 70% of history of ITP (rare), or comorbidity with a chronic ITP patients enter a durable remission after condition predisposing to secondary ITP, the onset splenectomy26.

50 Pathophysiology of ITP

Figure 1. Model of relationship of contributing factors in ITP. (Johnsen J. Pathogenesis in immune thrombocytopenia: new insights. Hematology Am Soc Hematol Educ Program. 2012;2012:306-12).

Antigen presenting cells represents another mechanism by which platelet The role of antigen-presenting cells (APCs) for the autoantibodies may lead to immune-mediated loss of tolerance in ITP remains unclear, but a platelet destruction. Several studies have model has been advanced in which APCs are demonstrated increased platelet-associated C3 and crucial in generating a number of new or cryptic C4 on ITP platelets but these are thought to be from platelet glycoproteins. In this model, secondary in importance to platelet IgG and/or the APCs expressing these novel peptides, along with result of antiplatelet IgM29. Furthermore, there is an costimulatory molecules, induce the activation of T- association of (especially) C4 deficiency and ITP cells that recognize these additional platelet that has not been well studied and is of additional antigens. Thus, this acquired recognition of new interest because the C4 genes are in the midst of the self-determinants, or spreading, may play HLA region on chromosome 630. an important role in the initiation and perpetuation of ITP. T-cell clones that react with cryptic epitopes Role of T cells may escape the negative selection in the thymus T-cell abnormalities in patients with ITP when self-determinants are present at a sub- A number of T-cell abnormalities have been threshold concentration5. demonstrated in patients with ITP (summarized in Fig 2) and it is likely that there are three main mechanisms by which T cells could be involved in In general, Abs specifically bound to cell-surface the thrombocytopenia in patients with ITP. First, a antigens not only mediate clearance from number of studies suggest a Th1 bias, compared circulation by Fc_Rs, but also can serve to fix with Th2, in adults with chronic ITP. For example, complement on cells. Recently, plasma from ITP increased numbers of HLA-DR+ T cells, increased patients was shown by 2 groups to be capable of soluble IL-2 receptors, and a cytokine profile fixing complement to platelets in vitro27. suggesting the activation of precursor helper T and Furthermore, platelets from ITP patients also type 1 helper T cells have been described31. exhibit detectable complement, and the ability to fix Reduced levels of IL-10 have also been described complement was correlated with the presence of in patients with active disease when compared with detectable antiplatelet Abs28. Therefore, those in remission or healthy controls32 but complement-mediated immunity, either by conversely, raised IL-10 levels have been described enhanced clearance or direct cell destruction, in children with chronic ITP33. Further evidence of 51 Adly

Th1 involvement in the pathology of ITP is DNA microarray screening, Olsson et al.37 found illustrated by an increase in the Th1 cytokines, IL-2 increased expression of several cytotoxic genes, and IFN-c, in patients with ITP when compared such as granzyme A, and perforin, as with controls34. Interestingly, this increase was well as increased expression of genes involved in more marked in patients in remission than in those the Th1 cell response, such as INFc and IL-2 with active disease. -b in a small number of patients with ITP Overall, they describe significantly increased when compared with controls. As apparent Th1/Th2 ratios in patients with both active and compensation for this increased , they quiescent disease when compared with controls. also found increased expression of the killer cell These findings may be related to ongoing immune immunoglobulin-like receptor (KIR) family on activation as part of autoimmunity. The activity of CD3+ T cells in patients with ITP in remission regulatory T cells and the potential for in vivo T- when compared with controls and to those with cell exhaustion because of prolonged in vivo active ITP. KIRs downregulate cytotoxic T activation has not been well studied in ITP30. lymphocytes (CTL) and (NK) A second method of potential T-cell responses by binding to MHC class 1 molecules, involvement is the release of cytokines that preventing lysis of target cells. These findings interfere with megakaryocyte maturation and/or suggest that CTLs may be involved in ITP. In a platelet release. Transforming growth factor (TGF)- direct assay similar to that measuring NK cell b1 level has been inversely correlated with disease activity by using radiolabelled K562 targets, these activity32. The role of TGF-b1 in ITP is thought to investigators assessed platelet destruction in vitro be as a potent inhibitor of megakaryocyte by T cells. They found that six of eight patients maturation. Two studies have shown increased with active ITP showed platelet lysis by T cells - colony stimulating factor whereas none of the patients in remission did. The (GM-CSF) levels, and one increased macrophage effector cells were found to be CD3+CD8+ T (M) CSF levels, suggesting that cells37. /macrophage activation is associated with This expanded role of cytotoxic T cells may ITP35. Circulating cytokines may also alter the explain why not all of the patients originally response of HLA class II presentation, and/or described by Harrington et al.38 had a fall in their influence the interaction between B and T platelet count following the infusion of plasma from lymphocytes causing pre-existing B cells to patients with ITP, and may also explain a proliferate and produce high-affinity percentage of patients without measurable autoantibodies36. Finally, there is evidence to antiplatelet antibodies, and again points to the suggests a direct cytotoxic effect of T cells, by heterogeneity of this disease.

Figure 2. Summary of the variety of T-cell abnormalities found in adults with ITP. PBMC, peripheral blood mononuclear cells; MLR, mixed reaction; ITP, immune thrombocytopaenic purpura (Cooper N, Bussel J. The pathogenesis of immune thrombocytopaenic purpura. Br J Haematol. 2006 May;133(4):364-74.)

52 Pathophysiology of ITP

T-cell phenotypes in ITP (CD8+) cells42. Moreover, the increase in Tc17 cells From studies of the immune system in animals, is correlated with skewing of the CD4:CD8 ratio in immune homeostasis is thought to be maintained ITP42. via a balance of type 1 (IFNγ, IL-2, TNFα, and An additional discrete T-cell subset, Th22 TNFβ1 related) and type 2 (IL-4, IL-5, IL-6, IL-10, (CD4+IFNγ_IL-17_IL-22+), has recently been and IL-13 related) responses. Although the identified and found to be up-regulated in several distinctions between type 1 and type 2 T cells and autoimmune . Th22 cells are also cytokine profiles in humans are less clear, these significantly increased in ITP patients, and this categories are helpful in broadly placing common increase is correlated with the observed increased immune patterns into context. Type 1 reactions are numbers of Th1 and Th17 cells43. Another cytokine, classically thought to be involved in response to IL-21, is produced by some CD4+ T cells and intracellular , and a type 1 response is one natural killer T cells and is capable of up-regulating which generally promotes pro-inflammatory, cell- both Th17 cells and B cells. In ITP, IL-21 mediated, complement fixing phenotypes. On the expression on T cells is elevated in untreated newly other hand, type 2 immunity is thought to function diagnosed ITP patients, although circulating IL-21 in the fight against extracellular pathogens, and a is unchanged44. type 2 response typically elicits an immediate-type Simplistically, the evidence supports a type 1 T- response. During an inflammatory cell response with an up-regulated Th17 response in event, dominance of either a type 1 or 2 profile is ITP. Interestingly, similar patterns of immune characteristic, because the prevailing dominant type dysregulation can also be seen in other autoimmune both expands and engages in negative feedback disorders43. These types of responses, loops to suppress the “other” T-cell types. stereotypically proinflammatory, cell-mediated, Resolution of an immune inflammatory event is complement-fixing phenotypes, would be expected then characterized by suppression of the dominant to propagate and enhance the ongoing autoantibody type phenotype and restoration of the type 1/type 2 mediated platelet immune process. Moreover, the balance19. increase in Tc17 cells is correlated with skewing of Therefore, the shifting of the balance of type 1 the CD4:CD8 ratio in ITP44. and type 2 permits tailoring of the immune response to the perceived threat. In ITP, type 1/type 2 ratios are unbalanced (Figure 3). In primary ITP, adult chronic primary ITP patients have high Th1/Th2 (“helper” CD4+ cells) ratios and high Tc1/Tc2 (“cytotoxic” CD8+cells) ratios39. Furthermore, the Th1/Th2 ratio imbalance is inversely correlated with disease severity40, meaning the higher the Th1/Th2 ratio, the lower the platelet count. ITP patients also exhibit decreased numbers of CD4+CD25+ T-regulatory cells (Tregs), which 20 Figure 3. Schematic of shifts in the T-cell balance function to down-regulate T-cell responses . Not in ITP. surprisingly, the degree of decrease in numbers of (Cines DB, Bussel JB, Liebman HA, Luning Prak ET. Tregs is associated with more severe disease in ITP The ITP syndrome: pathogenic and clinical diversity. 41 . In addition to these type 1/2–specific changes, Blood. 2009; 113(26):6511-6521). the total CD4:CD8 ratio is also observed to be diminished in ITP42 and improves with disease T cell tolerance failure remission39. An autoantigenic stimulus may occur in the More recently, other subsets of T cells distinct extrathymic periphery that could derive from the from type 1 and type 2 have also been in implicated host’s tissues in the form of an altered self-antigen in autoimmune diseases, including ITP. Similar to or from a cross-reactive environmental stimulus type 1 and type 2 T cells, these T-cell subsets are that could mimic self-antigens. Alternatively high- defined by their cytokine secretion profiles. T cells, affinity atuoreactive CD4+ Th cells may have which secrete IL-17, are pro-inflammatory and of escaped thymic selection during T-cell ontogeny interest in ITP in part due to a large body of and may stimulate the anti-platelet autoantibody evidence implicating IL-17 in autoimmunity. response45. T cells recognizing peptides generated Within IL-17–secreting T-cell subsets, Th17 from native GPIIb/IIIa by normal processing (CD4+) cells are increased in ITP, as are Tc17 pathways are hypothesized to be deleted in the 53 Adly thymus (negative selection) since GPIIb/IIIa has Reduced Treg activity may also be due to been shown to be expressed abundantly as early as increased resistance of effector T cells to the 16th week of intrauterine life on epithelial cells suppression, although we specifically demonstrated of thymic stoma46. However, some autoreactive T that effector T cells from ITP patient and healthy cells directed against membrane antigens present on controls were equally inhibited by Tregs from bone marrow-derived cells and also expressed in healthy controls, arguing against the refractoriness the thymus may not be eliminated by intrathymic of ITP effector cells to suppression53. Indeed, Tregs deletion47. They can be detected in the peripheral, from ITP patients were less effective that Tregs even though they may not cause autoimmune from healthy controls in inhibiting effector T cell response. Some of these T cells to native GPIIb/IIIa proliferation from either patients or healthy that escape thymic deletion and exist in periphery, controls, suggesting that reduced Treg activity is are inactivated by a post-thymic mechanism of due to an intrinsic defect in ITP Tregs53. Failure to peripheral tolerance48 Thus, even though the maintain immune suppression by Tregs may be process of autoreactive T cells deletion or rendered responsible for the reported platelet autoantigen- anergic is not totally efficient, there are several specific T cell proliferative responses and the mechanisms that could proinflammatory phenotype in ITP patients53. potentially suppress autoreactive T and B cells from Nevertheless, the observed polyclonal Treg becoming activated (Fig. 4). These include dysregulation fails to explain why immune activation-induced cell death through expression of autoreactivity in ITP is directed toward platelets Fas and FasL, inhibitory action of membrane rather than other cell types. Detailed identification molecules like CTLA-4, presence of a group of and characterization of platelet antigen-specific suppressor T cells and some cytokines that help Tregs in ITP may help clarify why loss of tolerance inhibit the activation and proliferation of is toward platelets rather than other tissues54. autoreactive T cells, such as CD4+CD25+ T cells49. All these tolerance mechanisms contribute to the Role of B cells inhibition of autoreactive T cells that exist in Autoreactive B lymphocytes secrete antiplatelet peripheral tissue and it is their failure that in various antibodies ways leads the autoreactive T cells to mount an The most commonly occurring autoantibodies attack against self-antigen. In clinics, immuno- (~75%) in patients with ITP are directed against the suppressants such as azathioprine, cyclo- platelet surface glycoprotein (gp) complexes gpIIb– phosphamide and cyclosporine have been used to IIIa and gpIb–IX55. suppress T cells. Therefore, investigations of these Antibodies against other glycoproteins (Ia–IIa, aspects in the T cells tolerance not only help IV, and V) have been identified, and multiple understand the disorder or imbalance of immune platelet antigen specificities can be found in most system in ITP, but may also help development of patients Although antibodies are primarily of the novel clinical treatment of ITP by targeting these IgG subtype, IgM and IgA may be found56. mechanisms50. Platelets are targeted by the attachment of autoantibodies to their surface gp antigens, bound Defective Treg compartment in ITP to Fcγ receptors expressed on tissue macrophages Tregs, as characterized by high level expression of of the reticuloendothelial system and cleared from the CD25 surface marker and of the transcription the circulation. Complement- induced lysis factor forkhead box protein 3 (Foxp3) on CD4+ following antibody binding may also play a role57. cells, suppress proliferation of many immune cell After platelet internalization and degradation, types including T and B cells, either directly macrophages express platelet epitopes on their through cell contact or indirectly through secretion surface and secrete cytokines that stimulate of cytokines, thereby dampening inappropriate initiating CD4+ T-cell clones and clones with immune activation and autoreactivity51. Possible additional specificities5. reason for decreased Treg numbers can be due to Unique to patients with ITP, autoreactive CD4+ impaired development, proliferation, survival T cells recognize several distinct epitopes on gpIIb– and/or stability of Tregs whereas defective Treg IIIa, leading to autoimmune response expansion and function may be explained by failed cell contact accelerated platelet destruction. The trigger for the dependent suppression or reduced secretion of initiating autoantibody response is unknown, cytokines that mediate suppression including IL-10, although autoreactive T helper (Th) cells that TGF-β or IL-3552. interact with antibody-producing B cells are required7. Platelet-associated autoantibodies are 54 Pathophysiology of ITP detected in 50%– 70% of patients with ITP58, production. It is increasingly clear that cellular emphasizing the limitations of the currently immune mechanisms play a pivotal role in ITP5. available assays and/or suggesting that other or The production of antiplatelet antibodies by B additional mechanisms are involved. cells requires antigen-specific, CD4-positive, T-cell Assays for antibodies targeting gpIIb–IIIa, gpIb– help (Fig 5). It also is possible that in some ITP IX, and gpIIa–IIIa may be more specific59, but have cases, cytotoxic T cells play a role in the limited sensitivity, and the diagnosis remains destruction of platelets. A possible sequence of dependent on clinical presentation for the most events in ITP is as follows. A trigger, possibly an part.9 infection or toxin, leads to the formation of antibodies/ immune complexes that attach to Altered Bregs in ITP platelets. Antibody-coated platelets then bind to Similar to the T regulatory compartment, Bregs antigen-presenting cells (macrophages or dendritic inhibit T cell and monocyte activation and they do cells) through low-affinity Fcg receptors (Fcg so in part through secretion of anti-inflammatory RIIA/Fcg RIIIA) and are internalized and degraded. IL-1060, which in turn regulates Th polarization, Activated antigen-presenting cells then expose pro-inflammatory differentiation of other antigen novel peptides on the cell surface and with presenting cells (APCs) and autoimmune costimulatory help facilitate the proliferation of responses61. The alteration that we have detected in platelet antigen-specific, CD4- positive, T-cell ITP patients is both at the phenotypic and clones. These T-cell clones drive autoantibody functionality of B cells14. Specifically, frequency of production by platelet antigen-specific B-cell previously described as Breg population62, clones5. As part of the platelet destructive process characterized as CD19+CD24hiCD38hi cells, was in ITP, cryptic epitopes from platelet antigens are decreased in nonsplenectomized ITP patients off exposed, leading to the formation of secondary treatment. platelet antigen-specific T-cell clones, with ITP B cells have impaired IL-10 response after stimulation of new platelet antigen-specific B-cell stimulation and a reduced ability to dampen of clones and broadening of the immune response. The monocyte activation15. In mouse models, IL-10 autoantibody profile of individual patients who secreting regulatory B cells promote differentiation have ITP reflects activity of polyclonal autoreactive of Tregs63 or their recruitment64, indicating that B-cell clones derived by antigen-driven affinity these two immunoregulatory cell types interact with selection and somatic mutation6. each other. Although the ability of human Bregs to control tolerance failure Treg differentiation has not yet been demonstrated, Besides T cells, B cell tolerance are also important the possibility remains that altered Bregs in ITP in the pathogenesis of ITP, because they are the patients contributes to compromised Treg ultimate producer of autoantibodies. Genetic compartment14. analysis of autoantibodies in idiopathic This also brings up the question as to how these thrombocytopenic purpura reveals evidence of two immunoregulatory cell types interact with each clonal expansion and somatic mutation66. In the other and that there may be a hierarchy amongst germinal-center B cells, the degree of down these regulatory compartments with Bregs regulation is negatively correlated to the up controlling Tregs. Moreover, as with the data in regulation of autoantibody IgG response. Central Tregs, altered Breg activity identified in ITP and for tolerance suppresses the unwanted expansion of that matter in other disease states in humans62 does autoreactive B cell population. If some B cells not explain the antigen-specific autoreactivity, but escape this suppression or deletion, peripheral rather is consistent with a perturbed immune mechanism may also be launched to maintain reactive state. tolerance and one of the pivotal elements for maintaining peripheral tolerance is Fc_RIIB. Autoantibodies Suppress Megakaryopoiesis ITAM-containing FcRs, on the other hand can Chang and colleagues65 showed that plasma from prime autoimmune diseases and the impairment of patients with ITP containing autoantibodies against the functional balance between activating and gp1b and gpIIb–IIIa significantly suppressed inhibitory FcRs leads directly to - megakaryopoiesis in vitro. They proposed that mediated autoimmune diseases67. Possible platelet autoantibodies may affect megakaryocyte mechanisms that result in the activation of maturation or survival, leading to decreased platelet GPIIb/IIIa-reactive T cells are expression of cryptic determinants of GPIIb/IIIa and generation of cross- 55 Adly reactive B cells. The mechanism is thought to be mechanism. In ITP, these antibodies produced by B the generation of cross-reactive B cells, initially cells recognize self-antigens on platelet and cause primed by foreign protein serving as a molecular in platelet phagocytosis via the reticuloendothelial mimic that then bind, process, and resent self- system. In clinical therapy, patients may be protein. Then B cells would have the ability to responsive to splenectomy70, but treatment with an efficiently concentrate and present to T cells small immunosuppressant that inhibits T- and B-cell quantities of determinants that are typically function and cooperation, including azathioprine, sequestered. Cross-reactive B cells can cyclophosphamide, cyclosporine, mycophenolate subsequently prime naive autoreactive T cells if mofetil or anti-CD20, may be required. Monoclonal they express the appropriate costimulatory anti- CD20 antibody causes B-cell depletion, which molecules68. Autoantibodies can then be abundantly may inhibit T-cell–B-cell interactions50. produced by B cells after this T–B interactivation. In principle, autoimmune disorders arise because of Impaired Platelet Production the failure to eliminate or deactivate self-reactive Although increased platelet destruction clearly lymphocytes, which is reflected in a deficiency of plays a key role in the pathogenesis of ITP, it is central and/or peripheral tolerance induction now recognized that impaired platelet production mechanism69. selection in the also is important in many cases. In adults, as many thymus may be faulty and allows the release of high as 40% of ITP cases may have reduced platelet affinity autoreactive T cells. Alternatively, an turnover, reflecting the inhibitory effect of platelet environmental agent can mimic a self-antigen that autoantibodies on megakaryopoiesis71. leads to the breakdown of peripheral tolerance

Figure 4. Mechanisms of central and peripheral T cell tolerance. (1) Fas/FasL pathway induces the activated T cells to apoptosis, (2) inhibitory cell surface molecules help T cell anergy and (3) regulatory T cells secrete a profile of suppressive cytokines (Zhou B, Zhao H, Yang RC, Han ZC. Multi- dysfunctional pathophysiology in ITP. Crit Rev Oncol Hematol. 2005; 54(2):107-16)

Studies of platelet kinetics in children who have Summary of multi-dysfunctional ITP are limited but it is possible that a similar pathophysiology in ITP situation exists. There also is evidence that platelet The increase in serum cytokine levels and activated autoantibodies may induce thrombocytopenia by T cells, the alteration of cell communication and the inhibiting proplatelet formation72. Circulating impaired megakaryocytopoiesis in patients with thrombopoietin (TPO) levels in patients who have chronic ITP are related to a continual self-antigen- ITP typically are normal or increased only slightly, stimulated autoimmune response that is caused by reflecting the normal or only slightly reduced TPO tolerance failure. It is still unclear what causes the receptor mass in this acquired platelet disorder. In breakdown of central and/or peripheral tolerance to contrast, TPO levels are high in inherited platelet trigger autoreactive lymphocytes’ response, but production Disorders73. both environmental and genetic factors are thought 56 Pathophysiology of ITP to be crucial. It is apparent that dysfunction in step (e.g., induction of mimic antigen by foreign multi-steps, particularly in T of cellular immunity, antigen) is not necessarily accompanied by the play a central role in the final outcome of development of ITP because of the absence of megakaryocytopoietic suppression and platelet dysfunction of the latter steps. The immune system destruction. These steps are tightly connected and in human body is extremely delicate and should not be viewed in isolation or mutually complicated. An error in one step can sometimes be exclusive from each other. Dysfunction in one step compensated or be restored by other remedy may be a result of concurrent dysfunction of mechanism. ITP occurs only if multi-step another. For example, Th1 cells increase in the dysfunctions exist. Circulating antibodies to the circulation promotes an increase in cytokines IL-2, GPIIb/IIIa complex may represent only part of the IL-10 and INF-γ, which result in feedback relevant antibody pool therefore reducing platelet activation and proliferation of Th1 cells and APCs. antigen specific antibody is clearly downstream of Fig. 6 also shows that dysfunction of one item in a treatment and too insensitive to predict relapse50.

Figure 5. Pathogenesis of epitope spread in ITP. The factors that initiate autoantibody production are unknown. Most patients have antibodies against several platelet- surface glycoproteins at the time the disease becomes clinically evident. Here, glycoprotein IIb/IIIa is recognized by autoantibody (orange, inset), whereas antibodies that recognize the glycoprotein Ib/IX complex have not been generated at this stage (1). Antibody-coated platelets bind to antigen-presenting cells (macrophages or dendritic cells) through Fcg receptors and then are internalized and degraded (2). Antigen-presenting cells not only degrade glycoprotein IIb/IIIa (light blue oval), thereby amplifying the initial immune response, but also may generate cryptic epitopes from other platelet glycoproteins (light blue cylinder) (3). Activated antigen-presenting cells (4) express these novel peptides on the cell surface along with costimulatory help (represented in part by the interaction between CD154 and CD40) and the relevant cytokines that facilitate the proliferation of the initiating CD4-positive T-cell clones (T-cell clone 1) and those with additional specificities (T-cell clone 2) (5). B-cell immunoglobulin receptors that recognize additional platelet antigens (B-cell clone 2) thereby also are induced to proliferate and synthesize antiglycoprotein Ib/IX antibodies (green) in addition to amplifying the production of anti-glycoprotein IIb/IIIa antibodies (orange) by B-cell clone 1 (6) (Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002;346:995–1008)

57 Adly

Figure 6. Multi-step disorders in ITP. (1) Self-antigen generation, (2) T, B central and peripheral tolerance failure, (3) activation of T–B cells and (4) effector phase that lead to platelet clearance. (#) Single dysfunction does not always lead to clinical symptom, for the final stage of antibody production usually needs 1, 2 and 3 (Zhou B, Zhao H, Yang RC, Han ZC. Multi-dysfunctional pathophysiology in ITP. Crit Rev Oncol Hematol. 2005; 54(2):107-16).

Conclusion and future perspectives 5. Cines DB, Blanchette VS. Immune ITP is a complex, chronic, often cell-specific, thrombocytopenic purpura. N Engl J Med that is still not fully 2002;346:995–1008. understood. The improved understanding of the 6. Blanchette V, Bolton-Maggs P. Childhood innate and adaptive immune systems however is immune thrombocytopenic purpura: diagnosis and allowing us to understand and appreciate some of management. Hematol Oncol Clin North Am. 2010; the complex interactions between platelets, the 24(1):249-73. Kuwana M, Ikeda Y. immune system, and the development of ITP. 7. The role of autoreactive T-cells Immune-mediated platelet destruction in ITP in the pathogenesis of idiopathic thrombocytopenic occurs by a complex process involving multiple purpura. Int J Hematol 2005;81:106 –112. 8. Gernsheimer T, Stratton J, Ballem PJ, components of the immune system. The initiating Slichter SJ. event for the dysregulation remains unclear, Mechanisms of response to treatment although recent evidence helps to explain the in autoimmune thrombocytopenic purpura. N Engl J Med 1989; 320:974 –980. processes by which the disorder may perpetuate Gernsheimer T. itself. 9. Chronic idiopathic Rational therapy available for ITP awaits a more thrombocytopenic purpura: mechanisms of pathogenesis. Oncologist. 2009; 14:12–21 . thorough understanding of the relative contribution Roark JH, Bussel JB, Cines DB, Siegel DL. of each item in the different steps in the 10. maintenance of normal immune status and how the Genetic analysis of autoantibodies in idiopathic thrombocytopenic purpura reveals evidence of clonal relative contribution of dysfunction of each could expansion and somatic mutation. Blood. 2002; be accurately quantified in the treatment of ITP. 100:1388–1398 .

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