SEARCH

SEARCH BY CITATION

Keywords:

  • immune thrombocytopenic purpura;
  • single nucleotide polymorphism;
  • inflammatory cytokine;
  • tumour necrosis factor-β;
  • anti-platelet autoantibody

Summary

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

Single nucleotide polymorphisms (SNPs) of inflammatory cytokine genes were examined in 84 adult Japanese patients with chronic immune thrombocytopenic purpura (ITP) and 56 race-matched healthy controls. The SNPs examined were within the genes encoding tumour necrosis factor (TNF)-α (−238 G/A and −308 G/A), TNF-β (+252 G/A), and interleukin (IL)-1β (−511 C/T and +3953 T/C). Of these SNPs, the frequency of the TNF-β (+252) G/G phenotype was significantly higher in ITP patients than in healthy controls (21% vs. 7%, P = 0·04, odds ratio = 3·6, 95% confidence interval 1·1–11·1), while no significant association was detected for the other SNPs. The distribution of the TNF-β (+252) phenotype was not associated with human leucocyte antigen class II alleles or the therapeutic response in ITP patients. The frequency of circulating anti-glycoprotein IIb/IIIa antibody-producing B cells was significantly higher in ITP patients with the TNF-β (+252) G/G phenotype than in those with the G/A or A/A phenotype (11·9 ± 4·9 vs. 6·8 ± 4·9 and 3·7 ± 2·8 per 105 peripheral blood mononuclear cells; P = 0·02 and P < 0·001, respectively). These findings suggest that the SNP located at TNF-β (+252) contributes to susceptibility to chronic ITP by controlling the autoreactive B-cell responses to platelet membrane glycoproteins.

Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by the presence of autoantibodies against platelet membrane glycoproteins (GPs), such as GPIIb/IIIa and GPIb/IX (Karpatkin, 1997; Cines & Blanchette, 2002). The pathogenic process of ITP primarily involves an accelerated clearance of the antibody-coated platelets by phagocytes in the reticuloendothelial system. The aetiology of ITP remains unclear, but both genetic and environmental factors are thought to play a role in the development of the disease. Several immune-related genes have been examined as candidates for the genes susceptible to ITP. Associations between ITP and polymorphisms within the genes encoding human leucocyte antigen (HLA)-DRB1 (Nomura et al, 1998), DPB1 (Kuwana et al, 2000), and Fcγ receptors (Foster et al, 2001; Fujimoto et al, 2001; Carcao et al, 2003) have been reported, although these associations are generally weak.

On the other hand, infectious agents may influence the onset and course of several autoimmune diseases, including ITP (Benoist & Mathis, 1998). Childhood acute ITP is often preceded by infection of the respiratory or gastrointestinal tract (Lusher & Iyer, 1977). In addition, several groups have reported that an increased platelet count was observed in some patients with chronic ITP after the successful eradication of Helicobacter pylori (Gasbarrini et al, 1998; Emilia et al, 2002). The influence of H. pylori infection on susceptibility to ITP and platelet response to the eradication treatment varies among countries (Franchini & Veneri, 2003). However, only a small number of individuals develop ITP after exposure to these infectious agents. Such variability could be explained by differences in genetic factors associated with the host defence to pathogens, such as genes encoding inflammatory cytokines.

In this study, we examined a total of five single nucleotide polymorphisms (SNPs) within the genes for three inflammatory cytokines, including tumour necrosis factor (TNF)-α, TNF-β, and interleukin (IL)-1β, in adult Japanese patients with chronic ITP. These cytokines were selected from seven inflammatory cytokines (TNF-α, β, IFN-α, β, γ, IL-1α, and β) based on the presence of well-defined SNPs associated with autoimmune diseases. The role of these SNPs in the development of the disease, therapeutic response, and anti-GPIIb/IIIa autoantibody response in these patients was also examined.

Patients and controls

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

We studied 84 unrelated adult Japanese patients diagnosed as having chronic ITP (22 men and 62 women). All the patients who were seen at Keio University Hospital, Tokyo, Japan between January 1997 and December 1998 were included unless the patients did not agree to participate in this particular study. The diagnosis of chronic ITP was based on thrombocytopenia (platelet count <100 × 109/l) for at least 6 months, normal or increased bone marrow megakaryocytes without morphological evidence of dysplasia, and no secondary diseases that could account for the thrombocytopenic state (Karpatkin, 1997; Cines & Blanchette, 2002). Clinical information on all the ITP patients was retrospectively obtained by reviewing their clinical charts. The demographic and clinical information recorded were sex, age at onset, and responses to individual treatment regimens. Refractory ITP was defined as having a platelet count <50 × 109/l despite treatment with a standard dose of corticosteroids and splenectomy (Figueroa et al, 1993; Karpatkin, 1997). Fifty-six unrelated healthy adult Japanese volunteers living in the Tokyo area (21 men and 35 women) were used as controls. All samples from ITP patients and healthy controls were collected during the same period. Written informed consent was obtained from all participants in accordance with the Keio University Institutional Review Board guidelines.

Detection of TNF-β in plasma and PBMC culture supernatants

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

PBMCs were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum, 2 mmol/l l-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin at 2 × 106/ml, then stimulated with 25 μg/ml of phytohemagglutinin (PHA) for 72 h at 37°C (Messer et al, 1991; Whichelow et al, 1996; Lee et al, 1997). The culture supernatants were collected and stored at −80°C until analysis. TNF-β levels in platelet-poor plasma and PHA-stimulated PBMC culture supernatants were measured using an enzyme-linked immunosorbent assay kit (Quantikine; R & D Systems, Minneapolis, MN, USA), according to the manufacturer's protocol. The detection limit of this kit ranged from 31·2 to 2000 pg/ml.

Detection of circulating anti-GPIIb/IIIa antibody-producing B cells

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

Peripheral blood B cells producing IgG anti-GPIIb/IIIa antibodies were detected and quantified using an enzyme-linked immunospot (ELISPOT) assay as described previously (Kuwana et al, 2003). Briefly, polyvinylidene difluoride-bottomed 96-well multi-titre plates (Millipore-Amicon, Bedford, MA, USA) were coated with 30 μg/ml of purified human GPIIb/IIIa (Enzyme Research Laboratories, South Bend, IN, USA) and subsequently blocked with 1% bovine serum albumin. PBMCs (105 cells/well) in complete medium were incubated in the GPIIb/IIIa-coated plates at 37°C in a humidified atmosphere of 5% CO2 for 4 h. After washing away the cells, the membranes were incubated with alkaline phosphatase-conjugated goat anti-human IgG (ICN/Cappel, Aurora, OH, USA), and antibodies bound to the membrane were visualized as spots by incubation with nitro blue tetrazolium/5-bromo-4-chloro-indolyl phosphate. Each experiment was carried out in five independent wells, and the results represent the mean of the five values and are expressed as the number per 105 PBMCs. The maximum frequency during follow-up (mostly at presentation) in individual patients was recorded. The cut-off level, based on the results of healthy individuals, was 2/105 PBMCs (Kuwana et al, 2003).

Statistical analysis

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

The continuous variables were calculated as the mean ± SD. Phenotypic frequencies were tested for statistical significance using the chi-square test. The odds ratio (OR) with 95% confidence interval (CI) was calculated for statistically significant differences. Differences in continuous variables were examined by the Kruskal–Wallis test, followed by the non-parametric Mann–Whitney U-test between two patient groups. Interactive effects of HLA class II alleles and inflammatory cytokine SNPs on susceptibility to ITP were assessed using contingency tables by fitting log-linear models according to the hierarchical principle, as described previously (Kameda et al, 1998). Differences were considered significant if P < 0·05.

Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

The phenotypic frequencies of the five SNPs studied within the TNF-α, TNF-β, and IL-1β loci in ITP patients and healthy controls are shown in Table I. There was no difference in the distribution of SNPs present at TNF-α (−238), TNF-α (−308), IL-1β (−511), and IL-1β (+3953) between ITP patients and healthy controls, but the frequency of TNF-β (+252) G/G was significantly higher in ITP patients than in healthy controls (21% vs. 7%, P = 0·04, OR = 3·6, 95% CI 1·1–11·1). When the allelic frequencies of individual SNPs were also compared, the A allele at TNF-β (+252) was less frequently detected in ITP patients than in healthy controls (79% vs. 93%, P = 0·04).

Table I.  Phenotypic frequencies of five SNPs within the TNF-α, TNF-β, and IL-1β genes in Japanese patients with ITP and race-matched healthy controls.
Cytokine SNPsPhenotypeITP patients (n = 84) (%)Healthy controls (n = 56) (%) P-value
  1. P-values were calculated by the chi-square test.

  2. *OR = 3·6 (95% CI 1·1–11·1).

TNF-α (−238)G/G83 (99)53 (95)0·4
G/A1 (1)3 (5)0·4
A/A001·0
TNF-α (−308)G/G80 (95)56 (100)0·3
G/A4 (5)00·3
A/A001·0
TNF-β (+252)G/G18 (21)4 (7)0·04*
G/A35 (42)23 (41)0·9
A/A31 (37)29 (52)0·1
IL-1β (−511)C/C22 (26)18 (32)0·6
C/T34 (41)25 (45)0·8
T/T28 (33)13 (23)0·3
IL-1β (+3953)C/C79 (94)52 (93)0·9
C/T5 (6)4 (7)0·9
T/T001·0

Clinical characteristics and the TNF-β (+252) phenotype

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

The ITP patients were divided into three groups according to the TNF-β (+252) phenotype (G/G, G/A, and A/A). As shown in Table II, there was no difference in the sex distribution or age at onset among the groups. Of the 84 ITP patients, 66 were treated with corticosteroids, 31 of which subsequently received splenectomy, and nine patients were defined as having refractory ITP. Refractory ITP was not associated with the TNF-β (+252) phenotype.

Table II.  Clinical characteristics, HLA class II alleles, and anti-GPIIb/IIIa antibody-producing B-cell frequency in ITP patients according to the TNF-β (+252) phenotype.
Demographic and clinical findingsTNF-β (+252) G/G (n = 18)TNF-β (+252) G/A (n = 35)TNF-β (+252) A/A (n = 31)P-value
  1. P-values were calculated by the chi-square test or the Kruskal–Wallis test.

Sex (male:female)5:1311:246:250·5
Age (years) at onset42·9 ± 17·744·1 ± 12·443·9 ± 17·10·9
Refractory ITP2 (11%)2 (6%)5 (16%)0·4
HLA-DRB1*045 (28%)10 (29%)14 (45%)0·3
HLA-DPB1*020113 (72%)17 (49%)14 (45%)0·2
Anti-GPIIb/IIIa antibody- producing B cells (per 105 PBMCs)11·9 ± 4·9 (n = 11)6·8 ± 4·9 (n = 20)3·7 ± 2·8 (n = 15)<0·001

HLA class II alleles and the TNF-β (+252) phenotype

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

The HLA-DRB1, DQB1, and DPB1 alleles were determined in all 84 ITP patients and 56 healthy controls. There was no difference in frequencies of individual HLA class II alleles except DPB1**0201, which was more frequently detected in ITP patients than in healthy controls (52% vs. 23%, P = 0·001, OR = 3·6, 95% CI 1·7–7·9). Among the three ITP patient groups classified according to the TNF-β (+252) phenotype, there was no difference in the frequencies of individual DRB1, DQB1, and DPB1 alleles, including DRB1**04 and DPB1**0201, which were previously reported to be associated with chronic ITP in the Japanese population (Nomura et al, 1998; Kuwana et al, 2000) (Table II). In addition, we failed to detect any interactive effect of TNF-β (+252) and HLA class II alleles on the development of ITP.

Capacity for TNF-β production and the TNF-β (+252) phenotype

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

Since the SNP at TNF-β (+252) is reported to influence TNF-β production (Messer et al, 1991), TNF-β secretion was evaluated in ITP patients. No TNF-β could be detected in plasma samples from 36 ITP patients with the assay we used. Therefore, the PBMCs from 24 ITP patients were maximally stimulated with an excessive concentration of PHA, and the TNF-β level was measured in the culture supernatants. The mean TNF-β level produced was 1148 ± 392 pg/ml from the PBMCs of seven patients with the G/G phenotype, 1008 ± 376 pg/ml from those of 11 patients with G/A, and 870 ± 391 pg/ml from those of six patients with A/A. ITP patients with the G/G phenotype tended to produce a greater amount of TNF-β upon PHA stimulation, but these differences were not statistically significant, probably because of the small number of patients examined.

Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

The frequency of circulating B cells producing anti-GPIIb/IIIa antibodies was investigated for 46 ITP patients by ELISPOT assay. This ex vivo assay system quantitatively detects circulating memory B cells spontaneously producing anti-GPIIb/IIIa antibodies, and the frequency of these B cells reflects magnitude of the ongoing antoantibody response in ITP patients (Kuwana et al, 2002). An increase in anti-GPIIb/IIIa antibody-producing B cells was found in 40 patients (87%). When the ITP patients were grouped according to TNF-β (+252) phenotype, the increase in circulating anti-GPIIb/IIIa antibody-producing B cells was detected in all 11 patients with the G/G phenotype, 18 (90%) of 20 patients with G/A, and 11 (73%) of 15 patients with A/A. These differences were not statistically significant (P = 0·1), but the absolute number of B cells producing anti-GPIIb/IIIa antibodies in 105 PBMCs was significantly different among these three groups (P < 0·001) (Table II). The specific B-cell frequency was the highest in patients with the G/G phenotype, and the lowest in patients with the A/A phenotype (Fig 1).

image

Figure 1. Frequency of IgG anti-GPIIb/IIIa antibody-producing B cells in ITP patients according to the TNF-β (+252) phenotype. The cut-off value of 2/105 PBMCs, determined from healthy controls, is shown by a broken line. Statistically significant differences between two patient groups were calculated by the Mann–Whitney U-test.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References

We have examined potential associations between the development of chronic ITP and the SNPs within the genes for several inflammatory cytokines, and found that the TNF-β (+252) G/G phenotype was marginally more frequent in adult patients with chronic ITP than in healthy controls. The SNP at TNF-β (+252) has been reported to be associated with various autoimmune diseases, including the G allele with systemic lupus erythematosus (Bettinotti et al, 1993; Tomita et al, 1993), the G/A phenotype with Graves’ disease (Badenhoop et al, 1992), and the A/A phenotype with systemic sclerosis (Pandey & Takeuchi, 1999), whereas a lack of significant association was reported for patients with myasthenia gravis (Zelano et al, 1998), multiple sclerosis, rheumatoid arthritis (Vandevyver et al, 1994), and Behcet's disease (Lee et al, 2003). Taken together, it is likely that the SNP at TNF-β (+252) is a genetic factor that influences the onset of several distinct autoimmune diseases, although these observed associations may merely reflect linkage disequilibrium with other susceptibility genes within the HLA region of chromosome 6. Recently, Foster et al (2001) reported that the TNF-β (+252) A/A phenotype was higher in Caucasian patients with childhood chronic ITP than in healthy controls. The reason for the difference between the report by Foster et al (2001) and our finding is unclear, but possible explanations include differences in the patients’ ethnic background (Japanese versus Caucasian) or in the pathogenic process of chronic ITP between adults and children.

The TNF-β (+252) phenotype was not associated with HLA class II alleles or the therapeutic response in ITP patients, but the frequency of circulating anti-GPIIb/IIIa antibody-producing B cells was strongly influenced by the TNF-β (+252) phenotype. The finding that the highest frequency was seen in patients with the G/G phenotype and the lowest in patients with the A/A phenotype suggests the presence of a gene dosage effect of the TNF-β (+252) G allele on the activation of GPIIb/IIIa-specific B cells.

The SNP at TNF-β (+252) is located in the first intron, but in linkage disequilibrium with an additional SNP that results in a variant amino acid residue at position 26, asparagine in the G allele and threonine in the A allele (Messer et al, 1991). This haplotype is reported to be functional and to influence the capacity of lymphocytes to produce TNF-β. Namely, individuals possessing the TNF-β (+252) G allele are high producers of TNF-β, while those possessing the A allele are low producers (Messer et al, 1991; Lee et al, 1997). This tendency was also observed in the ITP patients in this study. TNF-β is mainly produced by activated T cells and is involved in the maturation and activation of B cells (Kehrl et al, 1987; Steffen et al, 1988). In addition, TNF-β is reported to act as an autocrine growth factor for B cells (Seregina et al, 1989). A recent study using mice deficient in TNF-β has shown that TNF-β is essential for the generation of the germinal centre in the spleen, where T and B cell interaction for priming and maintenance of the humoral immune response occurs (De Togni et al, 1994). Anti-GPIIb/IIIa antibody-producing B cells in the peripheral blood of ITP patients are mostly memory B cells that are released from the spleen after activation through the antigen-specific interaction with CD4+ T cells, and the frequency of these autoreactive B cells reflects the magnitude of ongoing autoantibody response in the spleen (Kuwana et al, 2002). Therefore, it is plausible that the SNP at TNF-β (+252) influences the capacity of T and B cells to produce TNF-β, and controls the threshold of the autoreactive T- and B-cell responses to platelet membrane antigens, such as GPIIb/IIIa, in secondary lymphoid organs. Other genetic and environmental factors are definitely necessary for the development of ITP as well, but the SNP at TNF-β (+252) may play a role in an individual's susceptibility to this disease by promoting the specific autoantibody response.

References

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients and controls
  5. Cell and plasma preparation
  6. HLA class II allele genotyping
  7. Cytokine SNP genotyping
  8. Detection of TNF-β in plasma and PBMC culture supernatants
  9. Detection of circulating anti-GPIIb/IIIa antibody-producing B cells
  10. Statistical analysis
  11. Results
  12. Distribution of inflammatory cytokine SNPs in ITP patients and healthy controls
  13. Clinical characteristics and the TNF-β (+252) phenotype
  14. HLA class II alleles and the TNF-β (+252) phenotype
  15. Capacity for TNF-β production and the TNF-β (+252) phenotype
  16. Anti-GPIIb/IIIa antibody-producing B-cell frequency and the TNF-β (+252) phenotype
  17. Discussion
  18. Acknowledgments
  19. References
  • Badenhoop, K., Schwarz, G., Schleusener, H., Weetman, A.P., Recks, S., Peters, H., Bottazzo, G.F. & Usadel, K.H. (1992) Tumor necrosis factor beta gene polymorphisms in Graves’ disease. Journal of Clinical Endocrinology and Metabolism, 74, 287291.
  • Benoist, C. & Mathis, D. (1998) Autoimmunity. The pathogen connection. Nature, 394, 227228.
  • Bettinotti, M.P., Hartung, K., Deicher, H., Messer, G., Keller, E., Weiss, E.H. & Albert, E.D. (1993) Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes. Immunogenetics, 37, 449454.
  • Carcao, M.D., Blanchette, V.S., Wakefield, C.D., Stephens, D., Ellis, J., Matheson, K. & Denomme, G.A. (2003) Fcgamma receptor IIa and IIIa polymorphisms in childhood immune thrombocytopenic purpura. British Journal of Haematology, 120, 135141.
  • Cines, D.B. & Blanchette, V.S. (2002) Immune thrombocytopenic purpura. New England Journal of Medicine, 346, 9951008.
  • De Togni, P., Goellner, J., Ruddle, N.H., Streeter, P.R., Fick, A., Mariathasan, S., Smith, S.C., Carlson, R., Shornick, L.P., Strauss-Schoenberger, J., Russell, J.H., Karr, R. & Chaplin, D.D. (1994) Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science, 264, 703707.
  • Emilia, G., Luppi, M., Morselli, M., Potenza, L., D'Apollo, N. & Torelli, G. (2002) Helicobacter pylori infection and idiopathic thrombocytopenic purpura. British Journal of Haematology, 118, 11981199.
  • Figueroa, M., Gehlsen, J., Hammond, D., Ondreyco, S., Piro, L., Pomeroy, T., Williams, F. & McMillan, R. (1993) Combination chemotherapy in refractory immune thrombocytopenic purpura. New England Journal of Medicine, 328, 12261229.
  • Foster, C.B., Zhu, S., Erichsen, H.C., Lehrnbecher, T., Hart, E.S., Choi, E., Stein, S., Smith, M.W., Steinberg, S.M., Imbach, P., Kuhne, T. & Chanock, S.J. (2001) Polymorphisms in inflammatory cytokines and Fcgamma receptors in childhood chronic immune thrombocytopenic purpura: a pilot study. British Journal of Haematology, 113, 596599.
  • Franchini, M. & Veneri, D. (2003) Helicobacter pylori infection and immune thrombocytopenic purpura. Haematologica, 88, 10871091.
  • Fujimoto, T.T., Inoue, M., Shimomura, T. & Fujimura, K. (2001) Involvement of Fc gamma receptor polymorphism in the therapeutic response of idiopathic thrombocytopenic purpura. British Journal of Haematology, 115, 125130.
  • Galbraith, G.M. & Pandey, J.P. (1995) Tumor necrosis factor alpha (TNF-alpha) gene polymorphism in alopecia areata. Human Genetics, 96, 433436.
  • Gasbarrini, A., Franceschi, F., Tartaglione, R., Landolfi, R., Pola, P. & Gasbarrini, G. (1998) Regression of autoimmune thrombocytopenia after eradication of Helicobacter pylori. Lancet, 352, 878.
  • di Giovine, F.S., Takhsh, E., Blakemore, A.I. & Duff, G.W. (1992) Single base polymorphism at -511 in the human interleukin-1 beta gene (IL1 beta). Human Molecular Genetics, 1, 450.
  • Inoko, H. & Ota, M. (1993) PCR-RFLP. In: Handbook of HLA Typing Techniques. (ed by K.M.Hui & J.Bidwell), pp. 970. CRC Press, Boca Raton.
  • Kameda, H., Pandey, J.P., Kaburaki, J., Inoko, H. & Kuwana, M. (1998) Immunoglobulin allotype gene polymorphisms in systemic sclerosis: interactive effect of MHC class II and KM genes on anticentromere antibody production. Annals of the Rheumatic Diseases, 57, 366370.
  • Karpatkin, S. (1997) Autoimmune (idiopathic) thrombocytopenic purpura. Lancet, 349, 15311536.
  • Kehrl, J.H., Alvarez-Mon, M., Delsing, G.A. & Fauci, A.S. (1987) Lymphotoxin is an important T cell-derived growth factor for human B cells. Science, 238, 11441146.
  • Kornman, K.S., Crane, A., Wang, H.Y., di Giovine, F.S., Newman, M.G., Pirk, F.W., Wilson, T.G., Higginbottom, F.L. & Duff, G.W. (1997) The interleukin-1 genotype as a severity factor in adult periodontal disease. Journal of Clinical Periodontology, 24, 7277.
  • Kuwana, M., Kaburaki, J., Pandey, J.P., Murata, M., Kawakami, Y., Inoko, H. & Ikeda, Y. (2000) HLA class II alleles in Japanese patients with immune thrombocytopenic purpura. Associations with anti-platelet glycoprotein autoantibodies and responses to splenectomy. Tissue Antigens, 56, 337343.
  • Kuwana, M., Okazaki, Y., Kaburaki, J., Kawakami, Y. & Ikeda, Y. (2002) Spleen is a primary site for activation of platelet-reactive T and B cells in patients with immune thrombocytopenic purpura. Journal of Immunology, 168, 36753682.
  • Kuwana, M., Okazaki, Y., Kaburaki, J. & Ikeda, Y. (2003) Detection of circulating B cells secreting platelet-specific autoantibody is useful in the diagnosis of autoimmune thrombocytopenia. American Journal of Medicine, 114, 322325.
  • Lee, S.H., Park, S.H., Min, J.K., Kim, S.I., Yoo, W.H., Hong, Y.S., Park, J.H., Cho, C.S., Kim, T.G., Han, H. & Kim, H.Y. (1997) Decreased tumour necrosis factor-beta production in TNFB**2 homozygote: an important predisposing factor of lupus nephritis in Koreans. Lupus, 6, 603609.
  • Lee, E.B., Kim, J.Y., Lee, Y.J., Park, M.H. & Song, Y.W. (2003) TNF and TNF receptor polymorphisms in Korean Behcet's disease patients. Human Immunology, 64, 614620.
  • Lusher, J.M. & Iyer, R. (1977) Idiopathic thrombocytopenic purpura in children. Seminars in Thrombosis Hemostasis, 3, 175199.
  • Messer, G., Spengler, U., Jung, M.C., Honold, G., Blomer, K., Pape, G.R., Riethmuller, G. & Weiss, E.H. (1991) Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production. Journal of Experimental Medicine, 173, 209219.
  • Nomura, S., Matsuzaki, T., Ozaki, Y., Yamaoka, M., Yoshimura, C., Katsura, K., Xie, G.L., Kagawa, H., Ishida, T. & Fukuhara, S. (1998) Clinical significance of HLA-DRB1*0410 in Japanese patients with idiopathic thrombocytopenic purpura. Blood, 91, 36163622.
  • Pandey, J.P. & Takeuchi, F. (1999) TNF-alpha and TNF-beta gene polymorphisms in systemic sclerosis. Human Immunology, 60, 11281130.
  • Sambrook, J., Fritsh, E.F. & Maniatis, T. (1989) Analysis and cloning of eukaryotic genomic DNA. In: Molecular Cloning: A Laboratory Manual. pp. 9.169.19. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • Seregina, T.M., Mekshenkov, M.I., Turetskaya, R.L. & Nedospasov, S.A. (1989) An autocrine growth factor constitutively produced by a human lymphoblastoid B-cell line is serologically related to lymphotoxin (TNF-beta). Molecular Immunology, 26, 339342.
  • Steffen, M., Ottmann, O.G. & Moore, M.A. (1988) Simultaneous production of tumor necrosis factor-alpha and lymphotoxin by normal T cells after induction with IL-2 and anti-T3. Journal of Immunology, 140, 26212624.
  • Tomita, Y., Hashimoto, S., Yamagami, K., Sawada, S. & Horie, T. (1993) Restriction fragment length polymorphism (RFLP) analysis in the TNF genes of patients with systemic lupus erythematosus (SLE). Clinical and Experimental Rheumatology, 11, 533536.
  • Vandevyver, C., Raus, P., Stinissen, P., Philippaerts, L., Cassiman, J.J. & Raus, J. (1994) Polymorphism of the tumour necrosis factor beta gene in multiple sclerosis and rheumatoid arthritis. European Journal of Immunogenetics, 21, 377382.
  • Whichelow, C.E., Hitman, G.A., Raafat, I., Bottazzo, G.F. & Sachs, J.A. (1996) The effect of TNF**B gene polymorphism on TNF-alpha and -beta secretion levels in patients with insulin-dependent diabetes mellitus and healthy controls. European Journal of Immunogenetics, 23, 425435.
  • Wilson, A.G., di Giovine, F.S., Blakemore, A.I. & Duff, G.W. (1992) Single base polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene detectable by NcoI restriction of PCR product. Human Molecular Genetics, 1, 353.
  • Zelano, G., Lino, M.M., Evoli, A., Settesoldi, D., Batocchi, A.P., Torrente, I. & Tonali, PA. (1998) Tumour necrosis factor beta gene polymorphisms in myasthenia gravis. European Journal of Immunogenetics, 25, 403408.