Immune pathophysiology of primary immune thrombocytopenia

2009Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) | 173 |
R. Stasi
Department of Haematology, St George’s Hospital, London, United Kingdom
Hematology Education: the education program for the annual congress of the European Hematology Association
2011;5:173-178
Introduction
Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder charac- terized by isolated thrombocytopenia in the absence of conditions known to cause thrombocytopenia, such as infections, other autoimmune disorders, drugs, and so on.1 In this review, we shall discuss the current
understanding of the pathophysiology of ITP.
Abnormalities of B and T cells Harrington’s seminal experiment provided
the first evidence that thrombocytopenia in ITP is caused by a plasma-derived factor,2 later identified as antiplatelet antibodies.3,4 The most commonly identified antigenic tar- get of these autoantibodies is platelet glyco- proteins (GP). Autoantibodies are often directed against a restricted number of “dominant” epitopes of GPIIbIIIa, or less fre- quently of GPIbIX or other platelet glycopro- teins.5 A number of ITP patients have anti- bodies directed to multiple platelet antigens.6 Antibodies against GP IIb/IIIa show clonal restriction in light-chain use,7 and antibodies derived from phage-display libraries show selective usage of a single Ig heavy-chain variable region gene (VH3-30).8 Sequencing of the antigen-combining regions of these anti- bodies suggests that they originate from a limited number of B-cell clones by antigen- driven affinity selection and somatic muta- tion.8 It should be noted, however, that
autoantibodies are not detectable in up to 50% of ITP patients,6,9 and that remission in ITP can occur despite the continued presence of platelet autoantibodies.10 Reasons for these findings may include technical factors (current monoclonal-based assays only detect antibodies with known specificity, typically GPIIb-IIIa and GPIb-IX; variable sensitivity of the assays), removal of autoan- tibodies by megakaryocytes, and the pres- ence of alternative mechanisms of the thrombocytopenia. As a matter of fact, several lines of evi-
dence also link T cells to the pathogenic process in ITP. Platelet-reactive T cells have been found in the blood of patients with this disorder, with the major target antigen being GP IIb/IIIa.11 In these patients, T cells stimu- late the synthesis of antibody after exposure to fragments of GP IIb/IIIa but not after exposure to native proteins.12 The derivation of these cryptic epitopes in vivo and the rea- son for sustained T-cell activation are unknown. It has been hypothesized that cryptic epitopes, normally not exposed in a self-antigen, may become exposed and rec- ognized by the immune system under cer- tain circumstances, for example, an infec- tion.13 The cytokine profile in the peripheral
blood of patients with ITP is consistent with a Th1 (proinflammatory) response,14 a pat- tern seen in most organ-specific autoim- mune diseases. These results were support- ed by flow cytometry studies, showing an
Immune thrombocytopenic purpura
Primary immune thrombocytopenia (ITP) is characterized by antibody mediated destruction of platelets and suppression of megakaryocyte and platelet development. The underlying defect leading to autoantibody production is unknown, and it is likely that both genetic and environmental factors are involved. Platelet-specific autoantibodies are often directed against a restricted number of “dom- inant” epitopes of GPIIbIIIa, or less frequently of GPIbIX or other platelet glycoproteins. The T cell compartment is now known to play a crucial role in ITP. Cytotoxic T cells may be involved
in platelet destruction and suppression of megakaryopoiesis. Many models of autoimmune disease have a Th1 bias, which is also seen in ITP, and which is reversed upon treatment. Furthermore, a reduc- tion in suppressor T-regulatory cells may predispose to the emergence of autoantibodies in response to exogenous antigens. Finally, an ITP-like presentation occurs in the setting of chronic infections, such as HIV, HCV, and
Helicobacter pylori. Antibodies that cross-react with platelets have been identified in patients who develop thrombocytopenia in association with these infections, suggesting that molecular mimicry and epitope spread may be a common pathway to antiplatelet antibody development in patients with ITP as well.
A B S T R A C T
increased Th1/Th2 ratio in ITP patients compared with controls.15 In keeping with these findings, investigation of whole blood gene expression profile using DNA microarrays identified an ITP-specific signature, which also included interferon (IFN)-induced genes, such as GBP2 and IFIT2.16 Pathway analysis demonstrated that IFN signaling, death receptor, and protein ubiquitination pathways were associated with ITP. Other studies have shown that patients with chronic
ITP often exhibit expansion of oligoclonal T-cells15,17 and the presence of cytotoxic T cells against autologous platelets.18 In fact, T cells from patients with ITP show increased expression of cytotoxic genes, such as tumor necrosis factor- alpha, perforin, and granzyme A and granzyme B.18,19 Interestingly, several members of the killer cell immunoglobulin-like receptor (KIR) family were upregulated in patients with active disease, and CD3+ lymphocytes expressing KIRs were greater in number in ITP patients in remission than in patients with active ITP or normal controls patients.18 Since KIRs downregulate cytotoxic T-lymphocyte (CTL) and natu- ral killer cell responses by binding to MHC class I mol- ecules, thereby preventing lysis of target cells, it may be speculated that cytotoxic T cells play a part in at least some patients with ITP. The emergence of antiplatelet autoantibodies and
antiplatelet cytotoxic T cells is a consequence of a loss of the immunological tolerance for self antigens. Filion et al. have shown that autoreactive T cells directed against GPIIb/IIIa are present in the peripheral blood of all healthy individuals,20 implying that peripheral toler- ance mechanisms are crucial to prevent autoreactive T cells from becoming activated. Several other T cell abnormalities have emerged from the investigation of immune regulation in ITP patients. Among these, CD4+CD25+ regulatory T cells have an impaired sup- pressive activity when compared with healthy sub- jects.21 Also, CD3+ T lymphocytes from patients with active ITP present an altered expression of genes associ- ated with apoptosis and are significantly more resistant to dexamethasone-induced suppression compared with normal lymphocytes.18,22 As far as B cells are concerned, the expansion of
autoreactive clones is suppressed in the bone marrow. If some B cells escape this suppression or deletion, periph- eral mechanisms, most importantly the functional bal- ance between activating and inhibitory Fc receptors (FcR), may also be launched to maintain tolerance.23
Antigen-presenting cells in ITP Like all immunoglobulin (Ig) isotype-switched IgG
antibody responses, autoreactive IgG against a protein antigen is initiated by activated T helper cells that rec- ognize specific peptide sequences on major histocom- patibility complex class II-positive antigen-presenting cells (APCs). The role of APCs for the loss of tolerance in ITP remains unclear, but dendritic cells from patients with ITP have upregulated costimulatory molecules enhancing autoreactive T- and B-cell responses against platelets.24 A model has been advanced in which APCs are crucial in generating a number of new or cryptic epi- topes from platelet glycoproteins.25 In this model, APCs expressing these novel peptides, and along with co- stimulatory molecules, induce the activation of T cells
that recognize these additional platelet antigens. Thus, this acquired recognition of new self-determinants, or epitope spreading, may play an important role in the ini- tiation and perpetuation of ITP. T-cell clones that react with cryptic epitopes may escape the negative selection in the thymus when self-determinants are present at a sub-threshold concentration.
Infection-associated ITP and the role of molecular mimicry Thrombocytopenia may accompany or follow a vari-
ety of infections from which ITP must be differentiated. In adults, the most prevalent infections associated with thrombocytopenia are those from hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Helicobacter pylori (H. pylori).26 In typical cases, the throm- bocytopenia presents with an insidious onset, has no tendency to remit spontaneously (although its severity may parallel the stage of the infectious disease), and may closely mimic chronic ITP. Response to infection may generate antibodies that cross-react with platelet antigens, most notably GP IIb-IIIa or immune complex- es that bind to platelet Fc receptors.27–30 Many patients with HIV-associated thrombocytope-
nia have autoantibodies that recognize a restricted pep- tide sequence (GPIIIa49-66) in platelet membrane GPIIIa, and can be recovered from patient plasma in the form of immune complexes consisting of autoantibody and platelet fragments.27,28 Recently, Zhang et al. found that sera from patients coinfected with HCV and HIV reacted with four peptides present in nonconserved regions of the HCV core envelope 1 protein.29 Antibodies raised against one of these peptides (PHC09) caused severe thrombocytopenia when injected into wild-type mice whose GPIIIa is more than 80% identical to that of humans. Immunization of wild-type mice with HCV core envelope protein 1 had no effect on platelet count. However, NZB/W F1 mice, a strain in which immune surveillance is defective, produced antibodies specific for PHC09 and became thrombocytopenic. The titer of PHC09-specific antibody in patients coinfected with HCV, and HIV correlated with both the incidence of thrombocytopenia and its severity. The authors con- clude that humans and immunodeficient mice immu- nized with HCV core envelope protein 1 often produce antibodies that recognize GPIIIa49-66 through molecu- lar mimicry and are capable of causing clinically signifi- cant thrombocytopenia. With regards to ITP and H. pylori infection, platelet-
cross-reactivity has been shown between platelet-asso- ciated immunoglobulins and bacterial components, in particular cytotoxin-associated gene (Cag) A protein30
and urease B.31 In support of the molecular mimicry theory, microbial
reduction/eradication leads to remission in a substantial fraction of infected patients. In the case of H. pylori, questions remain regarding why there appears to be marked variation in response rates among patients from different geographic areas.32
Genetic factors Little is known of the genetic factors that may con-
tribute to the predisposition to develop ITP or influence the natural history of ITP and response to treatment. A pilot study in 37 children with chronic ITP investigated
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common variants in the regulatory regions of cytokines (TNF, LTA, IL1RN, IL1A, IL1B, IL4, IL6, and IL10) and structural variants of the low affinity Fcg receptors (FCGR2A, FCGR3A, and FCGR3B).33 Two combinations of genotypes (TNF and FCGR3A; P = 0.0003, and LTA and FCGR3B; P = 0.011) were significantly associated with ITP compared with healthy controls. The NA1/NA1 genotype of the FCGR3B locus was observed in 30% of patients compared with 10% of controls and may be particularly relevant to ITP, as NA1 has a higher affinity than NA2 to IgG. The heterozygous V/F geno- type of the FCGR3A locus was also more frequent in patients than in controls (62% vs. 41%). These results suggest that immune complex handling may play a role in the pathophysiology of ITP, and that variant FcgR genes with decreased activity may provide partial pro- tection against ITP. With regards to cytokines, the transcriptionally more
active allele of TNF (allele 2 of −308) and the closely linked LTA allele 1 were both less common in children with ITP than in healthy controls. No clear hypotheses to account for these findings have been advanced. In an earlier study, deletions of Humhv3005, a devel-
opmentally regulated Ig variable (V) gene, and/or highly homologous VH genes were found more frequently in ITP patients (14 of 44, 31.8%) than in healthy controls (7/88, 8%, p = 0.002).34 Finally, associations with HLA-DRw235 and HLA-
DRB1*041036 have been reported, although the role played by these MHC molecules remains obscure.
Mechanisms leading to thrombocytopenia Ex vivo studies have shown that the spleen is the pri-
mary site of antibody production,37,38 whereas platelet kinetic studies have shown that the spleen is also the dominant organ for the clearance of IgG-coated platelets.39,40 In a minority of patients, hepatic clearance predominates. Human macrophages express several Fc receptors that
bind IgG specifically.41 Functionally, there are two differ- ent classes of Fc receptors: the activation and the inhibitory receptors, which transmit their signals via immunoreceptor tyrosine-based activation (ITAM) or inhibitory motifs (ITIM), respectively. Clinical data, along with information gained from animal models, suggest that the FcgRI, the high affinity receptor, does not play a relevant role in ITP.42,43 On the other hand, evi- dence has accumulated to indicate that the low-affinity receptors FcgRIIA and FcgRIIIA are primarily responsible for removal of opsonized platelets.44 Engagement of FcgRIIA on the surface of human macrophages by anti- GPIIb/IIIa-coated platelets triggers intracellular signaling through the tyrosine kinase Syk that leads to engulf- ment of the opsonized platelets. The presence of antibodies against GP Ib/IX has been
associated with resistance to intravenous immunoglob- ulin therapy both in a mouse model45 and in retrospec- tive series of ITP patients.46 These findings suggest the possibility of direct cytotoxicity or complement fixation as a mechanism of platelet destruction rather than anti- body-dependent, Fc receptor-mediated phagocytosis by macrophages. Interestingly, platelet kinetic studies using indium-111
(111In)-labeled autologous platelets have shown consider-
able heterogeneity in platelet turnover in patients with chronic ITP.39,40,47,48 While the platelet lifespan is often markedly decreased in most patients, in some it is only mildly reduced; furthermore, platelet turnover (a meas- ure of platelet production) is frequently subnormal. Overall, approximately 40% of patients with ITP were found to have a reduced platelet turnover.39,40 If platelet destruction were the only mechanism to
cause thrombocytopenia, then platelet production would be expected to increase and offset low platelet counts. It, therefore, was proposed that thrombocy- topenia may result not only from platelet destruction, but also from antibody-mediated damage to megakary- ocytes. Evidence to support this hypothesis has accu- mulated over time. McMillan et al. reported that IgG produced by cells
(grown in vitro) from the spleens of patients with ITP would bind to megakaryocytes, whereas IgG produced by cells from the spleens of healthy controls did not bind to megakaryocytes.49 A few years later, other inves- tigators demonstrated that antibodies against platelet antigens would bind to megakaryocytes as well.50,51 More recent in vitro experiments have further defined the role of autoantibodies in patients with ITP. Two studies in particular, by Chang et al.52 and McMillan et al.53 support the view that autoantibodies in ITP sup- press megakaryocyte production and maturation and platelet release. Electron microscopy studies have clarified some
aspects of the autoantibody-induced damage in bone marrow megakaryocytes from patients with ITP. Extensive megakaryocytic abnormalities were consis- tently present in a significant percentage of all stages of ITP megakaryocyte.54,55 In the most recent of these stud- ies, Houwerzijl et al. described the features characteris- tic of nonclassic apoptosis, including mitochondrial swelling with cytoplasmic vacuolization, distention of demarcation membranes, and condensation of nuclear chromatin.55 Para-apoptotic changes could be induced in megakaryocytes derived from CD34+ cells grown in ITP plasma, suggesting that autoantibodies may initiate the cascade of programmed cell death. In addition, megakaryocytes may be surrounded by neutrophils and macrophages, suggesting an inflammatory response against these cells. A role for direct T cell–mediated cytotoxicity against
platelets has been demonstrated in vitro.18 Whether this effect occurs in vivo and its relative importance in deter- mining platelet destruction has not been elucidated. There is also evidence that ITP is associated with accu- mulation and activation of T cells in the bone marrow that occurs through increased VLA-4 and CX3CR1 expression.56 It has been advanced that these activated T cells may mediate the destruction of platelets in the bone marrow.56 Thrombocytopenia associated with infectious dis-
eases is characterized by antibody-mediated platelet destruction. However, platelet production may be impaired by infection of megakaryocytes (HCV and HIV), decreased production of thrombopoietin (HCV), and splenic sequestration of platelets secondary to por- tal hypertension (HCV).26 A unique feature of antibodies specific for GP49-66,
frequently found in patients with HIV and HCV infec-
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tions, is their ability to induce reactive oxygen species through activation of 12-lipoxygenase and NADPH oxi- dase, leading to complement-independent platelet frag- mentation.57 This mechanism has not been described for antiplatelet antibodies with other epitope specificities.
Pathophysiology of secondary ITP Secondary forms of immune thrombocytopenia are
legion and include those associated with autoimmune and lymphoproliferative disorders, acute and chronic infec- tions, and certain drugs. Secondary ITP differs in specific aspects of pathobiology from primary ITP. We will con- fine our discussion to Systemic Lupus Erythematous (SLE) and lymphoproliferative disorders.
SLE is a multisystem disorder with wide-ranging clin- ical and laboratory manifestations. Of these, thrombo- cytopenia is a more common finding, with platelet counts less than 100 × 109/L found in 7–30% of patients.58 However, less than 3% of patients have counts below 20 × 109/L, which is associated with a sig- nificant risk of bleeding and usually requires treatment.59 Several genes correlated with ITP have been shown to be associated with expression signatures in systemic lupus erythematosis,60 indicating an overlap between the two autoimmune disorders. Purported mechanisms leading to thrombocytopenia in SLE include anti- GPIIb/IIIa antibody-mediated platelet destruction and inhibition of megakaryopoiesis by antibodies directed
| 176 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1)
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Figure 1. Simplified representation of the pathophysiology of ITP. The primary mechanism for the loss of tolerance in ITP remains unknown, although in some cases, infections may play a causative role. The emergence of antiplatelet autoan- tibodies remains the central pathogenetic mechanism. Platelet glycoproteins are cleaved to peptides by macrophages or another antigen-presenting cell (APC) and expressed on the APC cell surface via MHC class II molecules. APCs are crucial in generating a number of new or cryptic epitopes (“epitope spreading”). Genetic factors may be crucial in how the immune complexes are processed in APCs and expressed on MHC class II molecules. The T-cell receptor (TCR) of the Th cell can then bind the peptide-MHC complex and signal activation that upregulates CD154 (CD40 ligand) to interact with CD40 on the APC and cause additional costimulatory interactions to occur. An additional co-stimulatory signal can orig- inate from the binding of the CD80 molecule, overexpressed on the cell membrane of ITP platelets, with CD28 expressed on Th cells. The activated Th cell produces cytokines (interleukin-2 and interferon-g) that promote B-cell differentiation and autoantibody production. Tregs normally inhibit Th cell activity and proliferation, but their function in ITP is impaired. Autoantibodies opsonize platelets, which are taken up and destroyed by macrophages predominantly in the spleen. They also bind bone marrow megakaryocytes, thereby impairing megakaryocyte maturation and platelet production. An alter- native pathway of platelet destruction is by autoreactive cytotoxic T cells, although the relevance of this mechanism in vivo is not known. Tc cells in the bone marrow might also inhibit megakaryopoiesis and thrombopoiesis, although this has not been formally demonstrated. The role of infections such as H. pylori has not been completely elucidated, but cross-reactivity has been shown between bacterial antigens and platelet glycoproteins.
against the TPO receptor (cMpl).61 Predictably, the anti- body present dictates the clinical features: patients with anti-GPIIb/IIIa antibodies have normal or increased bone marrow megakaryocyte density and, similar to patients with immune thrombocytopenic purpura (ITP), show a good response to conventional immunosuppres- sion. Those with anti-TPO receptor antibodies demon- strate megakaryocytic hypoplasia and have a poor response to steroids and intravenous immunoglobulin.61 Patients with some lymphoproliferative disorders,
particularly those with chronic lymphocytic leukemia (CLL) and CD8 T-lymphocyte large granular lympho- cytic leukemia (LGL), appear to have an increased inci- dence of immune thrombocytopenia. ITP occurs in approximately 5% of patients with CLL when stringent diagnostic criteria are applied.62 The development of ITP is significantly associated with unmutated IgVH, a positive direct antiglobulin test, and the development of autoimmune hemolytic anemia. Patients with CLL and IT have poorer survival than other patients with CLL, and this effect appears to be independent from common clinical…