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Keywords:

  • TGF-β1;
  • chronic ITP;
  • Th3 cytokine;
  • PBMC;
  • active disease

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Summary. Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which activated T-helper (Th) cells and different Th-cell cytokines might play an important role. We have recently reported that chronic ITP patients in remission had elevated plasma levels of the Th3 cytokine transforming growth factor-β1 (TGF-β1), possibly as a part of a bystander immune suppression. In the present study we found that, in ITP patients with active disease [platelet count (plc) < 50 × 109/l], mitogen-stimulated peripheral blood mononuclear cells (PBMC) had a significantly reduced production of TGF-β1 (444 ± 178 pg/ml; n = 6) compared with patients with plc 50–150 × 109/l (1293 ± 374 pg/ml; n = 9; P < 0·05), patients with plc > 150 × 109/l (1894 ± 244 pg/ml; n = 12; P < 0·005) and healthy controls (1698 ± 241 pg/ml; n = 10; P < 0·01). Nineteen per cent of ITP patients expressed a platelet-induced PBMC proliferation. Surprisingly, 22% of the ITP patients had a PBMC proliferation below the normal range, i.e. a suppressed proliferation in the presence of platelets; five of these six patients had active disease. In summary, this study demonstrated that chronic ITP patients with active disease had reduced PBMC production of the Th3 cytokine TGF-β1. This result gives further support to the theory that chronic ITP in active phase is associated with a downregulated Th3-response.

Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which the antibodies produced cause platelet destruction and enhanced bleeds (McMillan, 1981). Although the humoral immune response in chronic ITP has been extensively studied, the cellular immunology is less well known. Nevertheless, previous studies have shown an increased T-lymphocyte activation (Mizutani et al, 1987; Semple & Freedman, 1991; Garcia-Suarez et al, 1993b) and T helper (Th)-cell cytokine patterns, indicating that Th cells may play an important role in this disorder (Garcia-Suarez et al, 1993a; Haznedaroglu et al, 1995; Nomura et al, 1995; Crossley et al, 1996; Semple et al, 1996; Erduran et al, 1998). Furthermore, a platelet-induced Th cell (CD4+) proliferation with a concomitant interleukin 2 (IL-2) production in the majority of children with chronic ITP has been reported (Semple & Freedman, 1991; Semple et al, 1996). Recently, Kuwana et al (1998) described chronic ITP patients that had autologous T-cell reactivity against platelet membrane glycoprotein (GP) IIb/IIIa and a concomitant in vitro anti-GPIIb/IIIa IgG production. Taken together, these findings indicate that, in ITP T-lymphocytes, probably Th cells, are activated by antigen-presenting cells, thereby initiating the B-cell autoantibody production.

A bystander immune suppression has been demonstrated in experimental models of oral immune tolerance induction, a phenomenon associated with expression of the Th3 cytokine transforming growth factor-β1 (TGF-β1) (Miller et al, 1991). We have recently reported (Andersson et al, 2000) that chronic ITP patients in remission had elevated plasma levels of TGF-β1, possibly as a part of a bystander immune suppression. In the present work we studied the platelet-induced reactivity and the Th-cytokine profile in mononuclear cell cultures from chronic ITP patients.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients. Twenty-seven patients with chronic ITP were examined. The diagnosis of ITP was based on thrombocytopenia lasting more than 6 months, normal or increased number of megakaryocytes in a bone marrow biopsy, absence of splenomegaly and exclusion of other known causes of thrombocytopenia. The patients were divided into three groups based on their platelet count (plc): (i) ‘active disease’ < 50 × 109/l (range 10–40; mean ± SD 25 ± 13) (n = 6), (ii) ‘stable disease’ 50–150 × 109/l (range 60–143; mean ± SD 101 ± 33) (n = 9), and (iii) ‘in remission’ > 150 × 109/l (range 166–389; mean ± SD 251 ± 79) (n = 12). Patients in remission were stable, with platelet counts persisting above 150 × 109/l for at least 3 months. The control material consisted of 10 healthy volunteers, aged 22–60 years (six women and four men) with normal plc (range 151–263; mean ± SD 226 ± 32). Further patient characteristics are given in Table I.

Table I.  Clinical characteristics, treatment at the time of analysis and previous therapy of idiopathic thrombocytopenic purpura (ITP) patients with ‘active disease’[platelet count (plc) < 50 × 109/l], ‘stable disease’ (plc 50–150 × 109/l) and ‘remission’ (plc > 150 × 109/l).
  Age (years) (mean ± SD) Sex (male/female) Splenectomized (number of patients) Corticosteroid therapy at time of study (number of patients) Other immunosuppressive therapy at time of study (number of patients)Previous therapy, i.e. corticosteroids or other immunosuppressive drugs (number of patients) Antiplatelet antibodies detected in MAIPA (number of patients)
  1. MAIPA, monoclonal antibody-specific immobilization of platelet antigens.

Active (n = 6)42 ± 172/424None 63
Stable (n = 9)49 ± 182/731None 43
Remission (n = 12)50 ± 236/642None124

Preparation of peripheral blood mononuclear cells (PBMC).  PBMC were isolated from heparinized blood using 1·077 g/ml Ficoll-Hypaque (Lymphoprep, Nycomed Pharma AS, Norway) gradient centrifugation (900 g for 30 min, 20°C). The isolated PBMC consisted of 86 ± 7 (SD)% lymphocytes, 6 ± 5 (SD)% monocytes and 7 ± 6 (SD)% neutrophils. The isolated PBMC were washed twice with 0·9% NaCl then resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco BRL Life Technologies, USA) containing 1% l-glutamine and supplemented with 5% heat-inactivated human AB + serum, 5 × 10−5 mol/l mercaptoethanol, 0·5 µg/ml amphotericin B and 50 µg/ml gentamicin.

Preparation of platelets.  Autologous platelets were separated from EDTA-anticoagulated blood by centrifugation at 200 g for 15 min at 20°C. The platelet-rich plasma (PRP) was then centrifuged at 800 g for 10 min and the platelet pellet was washed once in 0·9% NaCl and resuspended in 1 ml of 0·9% NaCl. For patients with platelets < 100 × 109/l, 5 ml of 0·9% NaCl was added to the packed red cells and additional platelets were isolated by differential centrifugation; this procedure was repeated and the harvested platelets were pooled, pelleted and washed as described above. Allogeneic donor platelets were used in PBMC cultures in two patients with active disease in whom an insufficient number of autologous platelets was collected.

In order to estimate the platelet content of TGF-β1, washed platelets were lysed with 1% Triton X-100 (Pierce, USA) for 30 min. Insoluble material was removed by centrifugation at 26 000 g for 30 min, and the supernatant was harvested and analysed for TGF-β1, using a commercially available enzyme-linked immunosorbent assay (ELISA).

Anti-platelet reactivity – proliferation assay.  PBMC were adjusted to 2 × 106/ml and 150 µl of the cell suspension (total 3 × 105 PBMC) was added to each well of a U-bottomed 96-well tissue culture plate (Nunclon, Nalge Nunc Int., Denmark). Thereafter, 50 µl of a platelet suspension (200 × 109/l) were added. All samples were cultured at least in triplicate. The plate was then incubated for 6 d at 37°C and 5% CO2. On d six, 37 kbq [3H]-thymidine was added to each well and the plate was incubated for an additional 24 h. The cells were then harvested with a cell harvester (Harvester 96, Tomtec, USA) onto filter paper and incorporated [3H]-thymidine was determined by a scintillation counter (1450 Microbeta plus, Wallac, Finland). The results are presented as stimulation index (SI), defined as mean cpm for triplicate wells with platelets divided by the mean cpm for the corresponding triplicate wells not holding platelets. The normal range for SI was defined as the mean SI ± 2 SD recorded for the 10 healthy controls.

Anti-platelet reactivity – cytokine assay.  PBMC were adjusted to 1 × 106/ml and 2 ml of the cell suspension was added in triplicate to the wells of a flat-bottomed 24-well tissue culture plate (Nunclon, Nalge Nunc Int., Denmark). Thereafter, 500 µl of a platelet suspension in RPMI-1640 medium, containing 50 × 106 platelets, was added to each well. A corresponding volume of RPMI-1640 medium without platelets was used as negative control. The PBMC, in presence or absence of platelets, were then incubated for 7 d at 37°C and 5% CO2. The supernatants were then removed, filtered through 0·22 µm filters (Millipore Co., USA) and stored at −20°C until assay.

Mitogen response – cytokine assay.  PBMC were adjusted to 1 × 106/ml and 2 ml of the cell suspension was added in triplicate to the wells of a flat-bottomed 24-well tissue culture plate. Cultures were then incubated in presence or absence of phytohaemagglutinin M (PHA-M; final concentration 10 µg/ml). After 72 h of incubation at 37°C and 5% CO2, the supernatants were removed, filtered through 0·22 µm filters and stored at −20°C until assay.

Cytokine analysis.  The levels of interleukin 2 (IL-2), interferon γ (IFN-γ), IL-4, IL-10 and TGF-β1 were determined using commercially available ELISA kits (R & D Systems, USA) according to the manufacturer's recommendations.

Modified monoclonal antibody-specific immobilization of platelet antigens (MAIPA) This assay has been described in detail previously (Stockelberg et al, 1995).

Statistics.  Unless otherwise stated, the mean values ± SEM are reported. The differences were evaluated using the Mann–Whitney U-test. For paired data, i.e. the antiplatelet reactivity cytokine assay, the Wilcoxon signed rank test was used. A P-value < 0·05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Anti-platelet reactivity – proliferation assay

Five out of the 26 (19%) ITP patients expressed a platelet-induced PBMC proliferation above the normal range (reactive patients). Out of these five patients, four had stable disease and one was in remission. It was also found that six patients had a PBMC proliferation below the normal range, i.e. a suppressed proliferation in the presence of platelets; five out of these six patients had active disease and one patient had a stable disease. None of the patients in remission displayed a suppressed PBMC proliferation. Patients with active disease had a significantly reduced mean SI (0·21 ± 0·16) compared with patients with stable disease (2·17 ± 0·45; P < 0·01), patients in remission (1·66 ± 0·30; P < 0·005) and healthy controls (1·70 ± 0·17; P < 0·005) (Fig 1). Basal PBMC proliferation, i.e. without platelets, were similar in all groups (active 5229 ± 792 cpm, stable 3907 ± 995 cpm, in remission 4654 ± 647 cpm and controls 4963 ± 677 cpm).

image

Figure 1. Results of the antiplatelet reactivity proliferation assay. The results are given as stimulation index (SI), i.e. mean cpm for triplicate peripheral blood mononuclear cell (PBMC) cultures in the presence of platelets divided by the mean cpm for the corresponding cultures in the absence of platelets. The study population comprised idiopathic thrombocytopenic purpura (ITP) patients with active disease [platelet count (plc) < 50 × 109/l], stable disease (plc 50–150 × 109/l), in remission (plc > 150 × 109/l) and healthy controls. The area within the dotted lines denotes the normal range and the solid horizontal lines indicate the mean values.

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Anti-platelet reactivity – cytokine assay

Both in the presence and absence of platelets, the IFN-γ, IL-2 and IL-4 concentrations in the culture supernatants were below the detection limit for the assay; < 8 pg/ml, < 7 pg/ml and < 10 pg/ml respectively. This was observed both for ITP patients and healthy controls. Patients in remission had statistically higher IL-10 production in the presence of platelets than in the absence of platelets (6·6 ± 1·5 and 4·1 ± 0·9 pg/ml; P = 0·007). A similar difference in IL-10 production, however, not statistically significant, was also seen in patients with active disease (4·6 ± 1·0 and 3·3 ± 1·1 pg/ml; P = 0·14), stable disease (5·2 ± 1·4 and 3·7 ± 1·0 pg/ml; P = 0·07) and healthy controls (7·4 ± 1·1 and 5·2 ± 0·9 pg/ml; P = 0·06). Regarding the Th3 cytokine TGF-β1, all patients and the healthy controls had significantly higher concentrations in the culture supernatant in the presence of platelets than in the absence of platelets; ‘active disease’ (3955 ± 346 versus 746 ± 518 pg/ml; P < 0·04), ‘stable disease’ (3628 ± 229 versus 1184 ± 384 pg/ml; P < 0·01), ‘in remission’ (3628 ± 256 versus 1695 ± 222 pg/ml; P < 0·005) and the healthy controls (3386 ± 177 versus 1468 ± 255 pg/ml; P < 0·01). Detergent lysis of platelets released approximately 250 ± 3 pg TGF-β1/106 platelets into the supernatant (mean of five different experiments).

Mitogen response – cytokine assay

Th1 cytokines.  There were no statistically significant differences, regarding the PHA-induced IL-2 production by PBMC, between any of the patient subgroups and controls (‘active disease’ 157 ± 91 pg/ml, ‘stable disease’ 37 ± 8 pg/ml, ‘remission’ 11 ± 3 and controls 20 ± 8 pg/ml). Similarly, no statistical differences were seen regarding IFN-γ production (‘active disease’ 12718 ± 3904 pg/ml, ‘stable disease’ 9074 ± 2091 pg/ml, ‘remission’ 7374 ± 2206 and controls 7540 ± 854 pg/ml).

Th2 cytokines.  The cytokine IL-4 was below the detection limit for the assay (< 10 pg/ml). IL-10 was within the assay range but no statistically significant differences between patient subgroups and controls were noticed (‘active disease’ 160 ± 52 pg/ml, ‘stable disease’ 253 ± 55 pg/ml, ‘remission’ 196 ± 47 pg/ml and controls 272 ± 45 pg/ml).

Th3 cytokine.  In patients with active disease, PHA-stimulated PBMC had a significantly reduced production of the Th3 cytokine TGF-β1 (444 ± 178 pg/ml) compared with both patients with ‘stable disease’ (1293 ± 374 pg/ml; P < 0·05), ‘patients in remission’ (1894 ± 244 pg/ml; P < 0·005) and healthy controls (1698 ± 241 pg/ml; P < 0·01). Also, patients with ‘stable disease’ had a significantly lower TGF-β1 production compared with patients ‘in remission’ (P < 0·05) and controls (P < 0·05) (Fig 2). The contamination of platelets in the cultures was measured and no statistically significant difference was seen between the patient groups and healthy controls; ‘active disease’ 3·2 ± 0·96 (SD) × 106 platelets/106 PBMC, ‘stable disease’ 0·8 ± 0·82 (SD), ‘in remission’ 1·13 ± 0·96 (SD) and healthy controls 1·6 ± 1·6 (SD).

image

Figure 2. Transforming growth factor β1 (TGF-β1) production in phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cell (PBMC) cultures from idiopathic thrombocytopenic purpura (ITP) patients with active disease (plc < 50 × 109/l), stable disease (plc 50–150 × 109/l), in remission (plc > 150 × 109/l) and controls. The horizontal lines denote the mean values.

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MAIPA assay

Sera from all the patients were tested for presence of IgG antibodies against GPIa/IIa, GPIb/IX, GPIIb/IIIa and GPIV. Antibodies against one or more of these GP were detected in 10 (37%) of the 27 patients. No difference in antibody frequency was seen between any of the patient groups (Table I).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In the present study PHA-stimulated PBMC from chronic ITP patients with ‘active’ disease had a significantly lower production of TGF-β1 than patients with stable disease, patients ‘in remission’ and healthy controls. Similarly, patients with ‘stable disease’ had a lower TGF-β1 production than patients ‘in remission’ and healthy controls.

Chronic ITP is an autoimmune disorder in which activated Th cells and different Th-cell cytokines might play an important role. Th cells are divided into three main subsets, Th1 , Th2 and Th3 cells. Th1 cells produce IL-2 and IFN-γ whereas Th2-cells release IL-4 and IL-10 (Romagnani et al, 1997). A Th1 cytokine profile has been suggested in chronic ITP. However, the results reported have not been concordant (Ware & Howard, 1993; Haznedaroglu et al, 1995; Crossley et al, 1996; Semple et al, 1996; Erduran et al, 1998). A characteristic of Th3 cells are their production of the immune-modulating cytokine TGF-β1 (Chen et al, 1994; Fukaura et al, 1996). TGF-β1 has been found to be an important inhibitor of both B-cell proliferation and immunoglobulin secretion (Kehrl et al, 1986a, 1989; Cross & Cambier, 1990). Also, it inhibits T-cell proliferation (Kehrl et al, 1986b) and suppresses some Th1 and Th2 cell-mediated autoimmune diseases (Kehrl et al, 1986a; Holter et al, 1994; Mosmann & Sad, 1996; Bridoux et al, 1997). Most recently it was shown that TGF-β1 is one of the key negative regulators of immune homeostasis and its absence leads to activation of a self-targeted immune response (Gorelik & Flavell, 2000).

We have previously reported that patients with chronic ITP in remission had significantly higher plasma levels of TGF-β1 than patients with active disease and healthy controls (Andersson et al, 2000). This finding is further supported by the results from the present study, demonstrating a reduced TGF-β1 production by PBMC from ‘active’ ITP patients. Thus, chronic ITP in active phase appears to be associated with a downregulated Th3 response. Presumably, a remission might be induced by upregulation of the Th3 response. It is known that platelets are a rich source of TGF-β1, stored in the α-granules (Fava et al, 1990). The differences we observed for the TGF-β1 production by PBMC cultures could not, however, be explained by in vitro platelet degranulation, as the mean platelet count in the PBMC suspensions was similar in all groups. Hence, our data indicates that the severity of the disease is reflected by an inability to produce the Th3 cytokine TGF-β1, i.e. high disease activity seems to be associated with a downregulated Th3 response. In addition, it has previously been reported that systemic lupus erythematosus (SLE), a disorder of generalized autoimmunity and T-cell dysfunction, is associated with a decreased production of TGF-β by lymphocytes (Ohtsuka et al, 1998). Taken together, these findings suggest that decreased production of this cytokine could be important in the maintenance of B-cell hyper-reactivity in autoimmune disorders.

Furthermore, others have previously demonstrated that 21–68% of ITP patients display a platelet-induced Th-cell proliferation (Semple & Freedman, 1991; Ware & Howard, 1993; Kuwana et al, 1998). Only 19% of our ITP patients expressed a platelet-induced PBMC proliferation; the majority of our ‘reactive’ patients were in stable disease and none was in active stage. This finding seems to be contradictory to the results of Kuwana et al (1998); all their ‘reactive’ patients had an active disease. Also, Semple and Freedman (1991) and Ware and Howard (1993) reported a platelet count between 50 and 70 × 109/l in their study population; however, the platelet count was not specifically given for ‘reactive’ patients. Conversely, we found that most patients with plc < 50 × 109/l had a suppressed PBMC proliferation in the presence of platelets. A larger proportion of our patients with active disease were on oral corticosteroids at time of study. Basal PBMC proliferation were, however, similar in all groups, indicating that corticosteroid treatment did not interfere with the unstimulated PBMC proliferation in vitro. The explanation for the platelet-induced suppression of PBMC proliferation is unclear. However, the PBMC cultures that contained platelets and which were used for cytokine analysis, had significantly higher TGF-β1 concentrations than the control cultures without platelets. This difference in TGF-β1 concentrations is at least partly explained by TGF-β1 being released from the platelets added to the cultures. TGF-β1 is known to inhibit proliferation of both B and T cells and to induce apoptosis in B cells (Inman & Allday, 2000). Furthermore, Kehrl et al (1986b) has reported that activated T cells have increased number of TGF-β receptors. Indeed, several investigators have found an elevated percentage of activated T cells in ITP patients with active disease (Mizutani et al, 1987; Semple & Freedman, 1991; Garcia-Suarez et al, 1993b; Andersson et al, 2000). Thus, it could be hypothesized that PBMC from patients with active ITP might be more sensitive to TGF-β1 and the suppression of PBMC proliferation might be mediated by this immune modulating cytokine.

Furthermore, recent reports have shown that the effect of certain immunosuppressive agents such as cyclosporin A, tacrolimus and rapamycin is mediated, at least in part, by TGF-β production (Khanna et al, 1994, 1999a,b; Dodge et al, 2000). Even though not commonly used, cyclosporin A has been reported to be effective in some patients with refractory chronic ITP (Kelsey et al, 1985; Emilia et al, 1996; Blanchette et al, 1998; Kappers-Klunne & van't Veer, 2001). In the view of our data, one possible explanation for the cyclosporine effect in ITP could be a TGF-β1-mediated immune suppression.

In conclusion, the present study demonstrated that chronic ITP patients with active disease had a reduced PBMC production of the Th3 cytokine TGF-β1. This finding gives further support to the hypothesis that chronic ITP in active phase is associated with a downregulated Th3 response. Thus, TGF-β1 might be an important regulator of both B- and T-cell proliferation in chronic ITP. Further studies should focus on the effect of different therapeutic measures in ITP and their effect on the Th3 response.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by grants from the Swedish Medical Research Council (project K2001–71X-11630–06A), the Göteborg Medical Society, ‘Assar Gabrielssons Foundation’, ‘Stiftelsen Jubileumsklinikens Forskningsfond mot Cancer’ and ‘FoU Västra Götaland’. The authors thank Ms Iréne Andersson and Ms Karin Staffansson for expert technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • Andersson, P.O., Stockelberg, D., Jacobsson, S. & Wadenvik, H. (2000) A transforming growth factor-beta1-mediated bystander immune suppression could be associated with remission of chronic idiopathic thrombocytopenic purpura. Annals of Hematology, 79, 507513.
  • Blanchette, V., Freedman, J. & Garvey, B. (1998) Management of chronic immune thrombocytopenic purpura in children and adults. Seminars in Hematology, 35, 3651.
  • Bridoux, F., Badou, A., Saoudi, A., Bernard, I., Druet, E., Pasquier, R., Druet, P. & Pelletier, L. (1997) Transforming growth factor beta (TGF-beta)-dependent inhibition of T helper cell 2 (Th2)-induced autoimmunity by self-major histocompatibility complex (MHC) class II-specific, regulatory CD4 (+) T cell lines. Journal of Experimental Medicine, 185, 17691775.
  • Chen, Y., Kuchroo, V.K., Inobe, J., Hafler, D.A. & Weiner, H.L. (1994) Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science, 265, 12371240.
  • Cross, D. & Cambier, J.C. (1990) Transforming growth factor beta 1 has differential effects on B cell proliferation and activation antigen expression. Journal of Immunology, 144, 432439.
  • Crossley, A.R., Dickinson, A.M., Proctor, S.J. & Calvert, J.E. (1996) Effects of interferon-alpha therapy on immune parameters in immune thrombocytopenic purpura. Autoimmunity, 24, 81100.
  • Dodge, I.L., Demirci, G., Strom, T.B. & Li, X.C. (2000) Rapamycin induces transforming growth factor-beta production by lymphocytes. Transplantation, 70, 11041106.
  • Emilia, G., Messora, C., Longo, G. & Bertesi, M. (1996) Long-term salvage treatment by cyclosporin in refractory autoimmune haematological disorders. British Journal of Haematology, 93, 341344.
  • Erduran, E., Aslan, Y., Aliyazicioglu, Y., Mocan, H. & Gedik, Y. (1998) Plasma soluble interleukin-2 receptor levels in patients with idiopathic thrombocytopenic purpura. American Journal of Hematology, 57, 119123.DOI: 10.1002/(SICI)1096-8652(199802)57:2<119::AID-AJH5>3.3.CO;2-S
  • Fava, R.A., Casey, T.T., Wilcox, J., Pelton, R.W., Moses, H.L. & Nanney, L.B. (1990) Synthesis of transforming growth factor-beta 1 by megakaryocytes and its localization to megakaryocyte and platelet alpha-granules. Blood, 76, 19461955.
  • Fukaura, H., Kent, S.C., Pietrusewicz, M.J., Khoury, S.J., Weiner, H.L. & Hafler, D.A. (1996) Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-beta1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. Journal of Clinical Investigation, 98, 7077.
  • Garcia-Suarez, J., Prieto, A., Reyes, E., Manzano, L., Merino, J.L. & Alvarez-Mon, M. (1993a) The clinical outcome of autoimmune thrombocytopenic purpura patients is related to their T cell immunodeficiency. British Journal of Haematology, 84, 464470.
  • Garcia-Suarez, J., Prieto, A., Reyes, E., Manzano, L., Merino, J.L. & Alvarez-Mon, M. (1993b) Severe chronic autoimmune thrombocytopenic purpura is associated with an expansion of CD56+ CD3- natural killer cells subset. Blood, 82, 15381545.
  • Gorelik, L. & Flavell, R.A. (2000) Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity, 12, 171181.
  • Haznedaroglu, I.C., Sayinalp, N.M., Ozcebe, O.I., Ozdemir, O., Dundar, S.V. & Kirazli, S. (1995) Megakaryocytopoietic cytokines in autoimmune thrombocytopenic purpura [letter]. American Journal of Hematology, 49, 265.
  • Holter, W., Kalthoff, F.S., Pickl, W.F., Ebner, C., Majdic, O., Kraft, D. & Knapp, W. (1994) Transforming growth factor-beta inhibits IL-4 and IFN-gamma production by stimulated human T cells. International Immunology, 6, 469475.
  • Inman, G.J. & Allday, M.J. (2000) Apoptosis induced by TGF-beta 1 in Burkitt's lymphoma cells is caspase 8 dependent but is death receptor independent. Journal of Immunology, 165, 25002510.
  • Kappers-Klunne, M.C. & Van't Veer, M.B. (2001) Cyclosporin A for the treatment of patients with chronic idiopathic thrombocytopenic purpura refractory to corticosteroids or splenectomy. British Journal of Haematology, 114, 121125.DOI: 10.1046/j.1365-2141.2001.02893.x
  • Kehrl, J.H., Roberts, A.B., Wakefield, L.M., Jakowlew, S., Sporn, M.B. & Fauci, A.S. (1986a) Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes. Journal of Immunology, 137, 38553860.
  • Kehrl, J.H., Wakefield, L.M., Roberts, A.B., Jakowlew, S., Alvarez-Mon, M., Derynck, R., Sporn, M.B. & Fauci, A.S. (1986b) Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. Journal of Experimental Medicine, 163, 10371050.
  • Kehrl, J.H., Taylor, A.S., Delsing, G.A., Roberts, A.B., Sporn, M.B. & Fauci, A.S. (1989) Further studies of the role of transforming growth factor-beta in human B cell function. Journal of Immunology, 143, 18681874.
  • Kelsey, P.R., Schofield, K.P. & Geary, C.G. (1985) Refractory idiopathic thrombocytopenic purpura (ITP) treated with cyclosporine. British Journal of Haematology, 60, 197198.
  • Khanna, A., Li, B., Stenzel, K.H. & Suthanthiran, M. (1994) Regulation of new DNA synthesis in mammalian cells by cyclosporine. Demonstration of a transforming growth factor beta-dependent mechanism of inhibition of cell growth. Transplantation, 57, 577582.
  • Khanna, A., Cairns, V. & Hosenpud, J.D. (1999a) Tacrolimus induces increased expression of transforming growth factor-beta1 in mammalian lymphoid as well as nonlymphoid cells. Transplantation, 67, 614619.
  • Khanna, A.K., Cairns, V.R., Becker, C.G. & Hosenpud, J.D. (1999b) Transforming growth factor (TGF)-beta mimics and anti-TGF-beta antibody abrogates the in vivo effects of cyclosporine: demonstration of a direct role of TGF-beta in immunosuppression and nephrotoxicity of cyclosporine. Transplantation, 67, 882889.
  • Kuwana, M., Kaburaki, J. & Ikeda, Y. (1998) Autoreactive T cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura. Role in production of anti-platelet autoantibody. Journal of Clinical Investigation, 102, 13931402.
  • McMillan, R. (1981) Chronic idiopathic thrombocytopenic purpura. New England Journal of Medicine, 304, 11351147.
  • Miller, A., Lider, O. & Weiner, H.L. (1991) Antigen-driven bystander suppression after oral administration of antigens. Journal of Experimental Medicine, 174, 791798.
  • Mizutani, H., Tsubakio, T., Tomiyama, Y., Katagiri, S., Tamaki, T., Kurata, Y., Yonezawa, T. & Tarui, S. (1987) Increased circulating Ia-positive T cells in patients with idiopathic thrombocytopenic purpura. Clinical and Experimental Immunology, 67, 191197.
  • Mosmann, T.R. & Sad, S. (1996) The expanding universe of T-cell subsets: Th1, Th2 and more. Immunology Today, 17, 138146.DOI: 10.1016/0167-5699(96)80606-2
  • Nomura, S., Yanabu, M., Kido, H., Lan, X.G., Ichiyoshi, H., Katsura, K., Miyake, T., Miyazaki, Y., Kagawa, H. & Fukuhara, S. (1995) Significance of cytokines and CD68-positive microparticles in immune thrombocytopenic purpura. European Journal of Haematology, 55, 4956.
  • Ohtsuka, K., Gray, J.D., Stimmler, M.M., Toro, B. & Horwitz, D.A. (1998) Decreased production of TGF-beta by lymphocytes from patients with systemic lupus erythematosus. Journal of Immunology, 160, 25392545.
  • Romagnani, S., Parronchi, P., D'Elios, M.M., Romagnani, P., Annunziato, F., Piccinni, M.P., Manetti, R., Sampognaro, S., Mavilia, C., De Carli, M., Maggi, E. & Del Prete, G.F. (1997) An update on human Th1 and Th2 cells. International Archives of Allergy and Immunology, 113, 153156.
  • Semple, J.W. & Freedman, J. (1991) Increased antiplatelet T helper lymphocyte reactivity in patients with autoimmune thrombocytopenia. Blood, 78, 26192625.
  • Semple, J.W., Milev, Y., Cosgrave, D., Mody, M., Hornstein, A., Blanchette, V. & Freedman, J. (1996) Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity. Blood, 87, 42454254.
  • Stockelberg, D., Hou, M., Jacobsson, S., Kutti, J. & Wadenvik, H. (1995) Evidence for a light chain restriction of glycoprotein Ib/IX and IIb/IIIa reactive antibodies in chronic idiopathic thrombocytopenic purpura (ITP). British Journal of Haematology, 90, 175179.
  • Ware, R.E. & Howard, T.A. (1993) Phenotypic and clonal analysis of T lymphocytes in childhood immune thrombocytopenic purpura. Blood, 82, 21372142.