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Clearance of autoantibody-sensitized platelets through Fcγ receptors on phagocytic cells is one of the main mechanisms of thrombocytopenia in idiopathic thrombocytopenic purpura (ITP). We examined the FcγRIIA-131R/H and FcγRIIIA-158V/F polymorphisms in 104 adult chronic ITP patients, and in 59 healthy control subjects using polymerase chain reaction-based allele-specific restriction analysis. The frequency of FcγRIIA genotypes (131H/H, H/R, R/R) was not significantly different between patients and controls, and did not correlate with the responsiveness to treatment. In contrast, among FcγRIIIA genotypes, frequency of 158F/F homotype was smaller in ITP (P < 0·05). Furthermore, in FcγRIIIA-158V/V homotype, the complete remission (CR) rate with medication (treatment with corticosteroid or other immunosuppressive agents) was significantly higher (60%) than that in 158V/F (10%) or 158V/F plus 158F/F, (P < 0·01, P < 0·05). Conversely, the CR rate after splenectomy in 158F/F and 158V/F types (64·3% and 54·6%) was higher than in 158V/V (25%). Our results indicate that the polymorphism of FcγRIIIA, but not FcγRIIA, influences the response to treatment in ITP.
In idiopathic thrombocytopenic purpura (ITP), an immunological mechanism has been under consideration (Fujimura et al, 1996; Shimomura et al, 1996; Karpatkin, 1997; George & Raskob, 1998) since the autoantibodies against platelet membrane glycoprotein (GP)Ib, GPIIb/IIIa or GPVI were detected using radio or enzyme immunoassay. Although several immunological platelet destruction mechanisms have been proposed, the clearance of sensitized platelets by phagocytic cells is thought to be one of the main mechanisms of thrombocytopenia. That is, the Fab portions of the autoantibodies bind to platelet-specific membrane antigens, and these sensitized platelets are phagocytosed through the Fc portion of these antibodies by monocytes and/or macrophages in spleen or liver which express the Fc receptors. The effectiveness of splenectomy to remove the platelet destruction organ and intravenous immunoglobulin treatment (IVIG) to block the Fc receptor for IgG in the phagocytic cells support the clinical importance of the clearance mechanisms carried out by Fc receptors.
The Fcγ receptors are expressed on the effector cells that have a phagocytic function of autoantibody-sensitized platelets and antibody-dependent cell-mediated cytotoxity function (Newland & Macey, 1994). Three different families of Fc γR exist: FcγRI, FcγRII and FcγRIII, which are quite diverse in both their structure and function (Schreiber et al, 1992; van de Winkel & Capel, 1993; Indik et al, 1995; van der Pol & van de Winkel, 1998). Fc receptors are members of the immunoglobulin superfamily found on many different cells (neutrophils, macrophages, lymphocytes and platelets), and form a critical link between the humoral and cellular immune responses (Voura et al, 1997). FcγRI has a very strong affinity for monomeric IgG, whereas FcγRII and FcγRIII only bind effectively to IgG in the form of immune complexes. FcγRII class is encoded by three genes (IIA, IIB and IIC) and FcγRIII class is encoded by two genes (IIIA and IIIB). Several genetic polymorphisms of FcγR were reported and, in some of them, amino acid differences may alter the affinity of the receptors to bind immunoglobulins. The polymorphism of FcγRIIA is caused by a single base substitution at nucleotide position 494. The variant allele of 131 histidine (494 A) is characterized by a high affinity for human IgG2 and a low affinity for murine IgG1, whereas the other allele of 131 arginine (494G) has the opposite binding properties (Salmon et al, 1992). In FcγRIIIA, which is mainly expressed on mononuclear phagocytes, polymorphism of T to G substitution at nucleotide 559 changes phenylalanine (F) into valine (V) at position 176 in the membrane proximal EC2 domain (De Haas et al, 1996; Tamm & Schmidt, 1996). This domain influences ligand binding because FcγRIIIA-V/V homozygotes bind more IgG1 and IgG3 than F/F type. These FcγR polymorphisms suggest the influence of antibody-mediated phagocytic activity or antigen presentation activity.
In this study, we examined the involvement of polymorphism of FcγRIIA and FcγRIIIA in the effectiveness of treatments, retrospectively, and the presence of platelet antibodies in ITP.
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The human genome project suggests that a polymorphism occurs approximately once every 800–1200 bp, and the frequency of selected variant alleles of the genes are different among races (Lehrnbecher et al, 1999). When our results were compared with a previous report (Lehrnbecher et al, 1999), the frequency of the variant genotype of FcγRIIA was different from the results analysed in Caucasians and African-American (H/H 22–26%, H/R 50–51% and R/R 24–23%), but was close to the results in the Far East populations (H/H 49%, H/R 47% and R/R 8%). Our data therefore support that the R/R homotype of FcγRIIA is found in a relatively small population, characteristically in people from the Far East, including the Japanese. On the other hand, the frequency of the FcγRIIIA polymorphism analysed in this study is the first to be reported because there are no previous publications about the FcγRIIIA polymorphism in Japanese or Far East populations. However, it was not too different from the reported frequency in Americans except for the V/V genotype (Koene et al, 1997). The V/V type is also a minor allele in American (11%) but was found in a smaller population (6·8%) in this study.
Recently, a number of groups have investigated the clinical significance of variant alleles in FcγRIIA and FcγRIIIA in several disease populations (Lehrnbecher et al, 1999). FcγRIIA polymorphism has been associated with various clinical conditions, including heparin-induced thrombocytopenia (HIT), systemic lupus erythematosus (SLE) and bacterial infections (Brandt et al, 1995; Burgess et al, 1995; Salmon et al, 1996; Wu et al, 1997; Carisson et al, 1998). Although these observations showed the correlation of these disease predispositions with FcγR polymorphisms there is still some controversy because of differences in race or number of subjects investigated. In this study, we could not determine the difference between the frequency of FcγRIIA genotypes in healthy control subjects and ITP patients. A report from Ireland (Williams et al, 1998) showed that their group of refractory ITP patients showed an increased frequency of R/R genotype (48%) compared with the healthy control group (18%), and suggested that the FcγRIIA polymorphism may be implicated in the pathophysiology of ITP or may be responsible for modulating the immune response in ITP. In contrast, a report from Canada (Horsewood et al, 1998) presented data that indicated no correlation, and suggested that the FcγRIIA polymorphism did not produce susceptibility to and severity of ITP, as demonstrated here. There have been no reports of FcγRIIIA in ITP patients. We have now shown that the frequency of 158F/F genotype in ITP was significantly lower than in control subjects.
We further analysed the relationship between the Fcγ receptor polymorphism and the response to treatment. Our data did not provide evidence that the FcγRIIA polymorphism has a relationship with the effectiveness of ITP treatment. In contrast, the 158V/V genotype of FcγRIIIA illustrates a trend that medication is more effective than splenectomy compared with other genotypes. Conversely, the 158F/F and V/F types predispose to ineffective response by medication, but not by splenectomy. These results suggest that the polymorphism of FcγRIIIA, rather than FcγRIIA, influences the effectiveness of ITP treatment. It is known that most of the platelet autoantibodies in ITP belong to the subclass of IgG1 or IgG3, but not to IgG2 (Tijhuis et al, 1991). Regarding the affinity of the receptors, it was reported that the FcγRIIA polymorphism influences the binding affinity mainly for IgG2 (Warmerdam et al, 1991), and that the FcγRIIIA-158V/V homotype binds to IgG1 and IgG3 with a higher affinity than the F/F homotype (Koene et al, 1997; Wu et al, 1997). Taken together, FcγRIIIA polymorphism, but not FcγRIIA, may contribute to the different levels of clearance of antibody-sensitized platelets and further supports the role of the FcγRIIIA polymorphism on the response to treatment in ITP. It is not clear why the reverse effect was observed between medication and splenectomy among the different polymorphisms of the FcγRIIIA. One possible explanation is that in the V/V type, which has a higher affinity for antibody-sensitized platelets, medications such as prednisolone are more effective because of suppression of the function of phagocytes carrying this receptor. In contrast, splenectomy is less effective because the other reticuloendothelial organs such as the liver could still destroy platelets with high affinity even after the spleen was removed. In the F/F or the F/V type, however, the effectiveness of the elimination of the main platelet destruction site (the spleen) is more obvious because phagocytes of the other organs have only lower affinity for the platelets, which may induce the compensatory state for the platelet destruction by platelet production.
It is known that the Fcγ receptors regulate not only phagocytic activity but also the antibody production indirectly through the phagocytosis followed by the antigen presentation (Gessner et al, 1998). In this study, no correlation was observed between the presence of platelet antibodies and the polymorphisms of the Fcγ receptors. These results might indicate that the polymorphisms of the Fcγ receptors did not regulate the production of autoantibody against platelets.
In summary, our results suggest the possibility that the analysis of FcγRIIIA polymorphism could indicate the effectiveness of medication and splenectomy, which would be clinically useful information for the selection of treatment in ITP.