The Hematology Journal (2001) 2, 242 249 2001 The European Haematology Association All rights reserved 1466

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Release of cytokines and soluble cytokine receptors after intravenous anti-D treatment in children with chronic thrombocytopenic purpura
Iwona Malinowska*,1, Agnieszka Obitko-Pludowska1, E Stephen Buescher2, Maria Wa sik3 and 1 Roma Rokicka-Milewska
1

Department of Pediatrics, Hematology and Oncology, Medical University of Warsaw, Warsaw, Poland; 2Center for Pediatric Research, Eastern Virginia Medical School, Children's Hospital of The King's Daughters, 855 West Brambleton Avenue, Norfolk, Virginia, USA; 3Department of Laboratory Diagnostics and Immunology, Medical University of Warsaw, Marszalkowska 24, 00-576 Warsaw, Poland

Introduction: Immunoglobulin anti-D administration is one of several methods used in treating children with chronic immune thrombocytopenic purpura. Fc receptor blockade of the reticuloendothelial cell system and of mononuclear phagocytes is an important mechanism of the action of anti-D in ITP. Recently other possible mechanisms by which anti-D works in ITP have been considered. Methods: The aim of this study was to obtain a better understanding of the eect of anti-D administration on cytokine, soluble cytokine receptors and platelet count in children with chronic ITP and to determine the pre-treatment plasma cytokine prole in this group of patients. Eighteen children with chronic ITP were examined. In our study the impact of antiD on the cytokine network was evaluated by analysing serum levels of IL-6, IL-8, tumor necrosis alpha and soluble TNF receptors I and II by the EASIA method before and 1, 3, 20 and 40 h then seven days and one month after anti-D infusion. Results: Anti-D caused a signicant increase in platelet count 20 h postinfusion in 10 out of 18 children, 96 h postinfusion in three children and 168 h postinfusion in one child. The mean duration of the response was four weeks. A signicant and rapid increase in plasma levels of IL-6, IL-8 and TNF-a was seen within 1 to 20 h after anti-D infusion. This increase was accompanied by a prolonged elevation of soluble TNF receptors. There was a signicant correlation between TNF-a and IL-8, IL-8 and IL-6, TNF-a and sTNFRI, and sTNF receptors I and II. Conclusion: These data demonstrate that anti-D infusion caused changes in the cytokine network and raises the question of whether the therapeutic eectiveness of anti-D is related to its immunomodulating properties. The Hematology Journal (2001) 2, 242 249 Keywords: IL-6; IL-8; TNF-a; sTNFRI; sTNFRII

Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disease that results from peripheral blood platelet destruction due to binding of IgG autoantibodies against Gp Iib/IIIa or GP Ib/IX platelet glycoproteins.2 Due to the lack of fully eective methods of treatment, numerous side eects of current treatments and the high costs of currently used methods, new ITP therapies are under investigation. Treatment of ITP has included conventional modalities such as
*Correspondence: I Malinowska, Department of Pediatrics, Hematology and Oncology, Medical University of Warsaw, Marszalkowska 24, 00-576 Warsaw, Poland; Tel/fax: +48(22) 621-53-62; E-mail: iwonamal@interia.pl Received 24 November 2000; accepted 18 December 2000

steroids, intravenous immunoglobulin IgG (IVIG), splenectomy, and recently immunoglobulin anti-D (anti-D). Infusions of IVIG result in substantial platelet increases in 470% of ITP patients.1,3 No single dominant mechanism appears responsible for the rapid eect of IVIG in ITP, but mechanisms including Fc receptor blockade of phagocytic cells,1,4 modulation of Fc receptor expression or anity and suppression of autoantibodies with anti-idiotypes4 have been proposed. However, high-dose IVIG has been reported to interfere with cytokine cascades in Kawasaki disease.4,5 Increases in interleukin-6 (IL-6), IL-8, tumor necrosis alpha (TNF-a) and IL-1 accompanied by prolonged elevation in levels of soluble TNF receptors I and II (sTNFRI and sTNFRII) as well as IL-1 receptor antagonist

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(IL-1RA) after IVIG infusion in patients with hypogammaglobulinemia. The eects on cytokine networks, especially on anti-inammatory cytokines, might be important contributors to the therapeutic eect of IVIG in immune-mediated disorders.5 The mechanism by which anti-D exerts its action in ITP is not entirely clear. FcR blockade by the substitution of antibody-coated RBCs for antibodycoated platelets has been postulated as one of mechanisms. An immunomodulatory eect of antiD infusion has also been postulated because increases in FcR expression on monocytes and in immunoglobulin production have been observed. Our study was undertaken to examine whether immuno/cytokine modulatory eects occur with antiD treatment. Furthermore, we sought to determine the pre-treatment plasma cytokine prole of children with chronic ITP.

5 min) to obtain platelet-free plasma. These samples were frozen in aliquots at 7708C until used. Samples were frozen and thawed only once. Urine samples Urine was collected from ve examined children. Samples were collected before and after anti-D administration at time points corresponding to plasma collection whenever possible. EASIAs for IL-6, IL-8, TNF-a and sTNFRI and sTNFRII Enzyme Amplied Sensitivity Immunoassay (EASIAs) for IL-6, IL-8, TNF-a and sTNFRI and sTNFRII, based upon multiple MoAbs directed against distinct epitopes on the target antigen, were commercially available assays (Medgenix Diagnostics, Fleurus, Belgium). According to the manufacturer's instructions, minimum detectable concentration are as follows: IL-6 EASIA, 2 pg/ml; IL-8 EASIA, 0.7 pg/ml; TNF EASIA, 3 pg/ml; TNFRI EASIA, 50 pg/ml and TNFRII EASIA, 0.1 ng/ml.

Materials and methods Patients

From January 1997 through December 1998, 18 nonsplenectomized children (11 males) aged two to 12 years with chronic ITP were treated at the Department of Pediatrics, Hematology and Oncology, Medical University of Warsaw. Entry criteria included a clinical diagnosis of chronic ITP and a platelet count of 5506109/L. Bone marrow examination showed a normal or elevated number of megakaryocytes. All patients were Rh+(D+) and HIV negative. The study has been approved by an ethics committee.

Cytokine levels in anti-D preparation

Using the EASIA assays described above, we could not detect IL-6, IL-8, TNF-a, sTNFRI or sTNFRII in anti-D products used in this study.

Statistical analysis
Data are presented as mean+s.d. values. Statistical comparisons are based on Student t-test and paired sample t-test. Coecients of correlation (r) were calculated by the Spearman rank test using Statistica software. A probability of P50.05 was taken as statistically signicant.

Treatment
Anti-D (WinRho SD, Winnipeg, Manitoba, Canada) was administered intravenously in single doses of 40 mg/kg. Six patients required repeat infusions with anti-D. Criteria for repeat infusions were the same as those at the start of therapy, i.e. a platelet count below 506109/L 21 days after treatment.

Results Platelet counts

The initial platelet count was 226109+146109/L (range 1 486109/L). Fourteen of 18 patients (78%) responded to anti-D with an increase in their platelet count to above 506109/L. Seven (39%) achieved a very good response (platelet increase to greater than 1006109/L and seven (39%) a good response, with platelet increase greater than 506109/L. Four of 18 cases (22%) failed to respond (5506109/L). Anti-D treatment increased platelet counts to above 506109/L by 20 h postinfusion in 11 of the 18 examined children (61%), by 96 h in two children (11%) and by 168 h in one child (6%). The platelet count achieved maximal value of 1096109/L+866109/L at 168 h (Figure 1). The maximal platelet count after infusion of anti-D was observed at 20 h in two children, 96 h in three children and at 169 h in nine children. The mean duration of the response was four weeks with a range of one to six weeks.
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Laboratory evaluation
A complete blood count was done before treatment and repeated at 20 and 96 h, and seven and 30 days after anti-D administration. Response to treatment was regarded as good when platelet count increased to above 506109/L and very good when platelet count increased to above 1006109/L. Plasma samples Plasma samples prepared from heparinized whole blood were obtained from the patients before and 1, 3, 20 and 40 h then seven days and one month after anti-D infusion. Blood was centrifuged within 20 min of collection (400 g, 10 min). Plasma fractions were transferred to sterile microfuge tubes and further sedimented (10 000 g,

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Figure 1 Plasma platelets count before and after anti-D infusion. Data are shown as scatter of points on log scale. The mean is expressed as geometric mean. Value of 50 000 represents a reference for responding patients.

Plasma cytokine concentration before and after anti-D infusion

IL-6, IL-8 and TNF-a levels pre and post anti-D treatment were examined in 18 patients. Trace levels of IL-6 and IL-8 were present before anti-D administration. While the mean IL-6 concentration before anti-D administration was 3+7 pg/ml (14 patients had nondetectable levels, four patients had levels of 2, 5, 18 and 26 pg/ml respectively), the mean IL-8 concentration before anti-D was 0.1+0.27 pg/ml with a range of 0 1 pg/ml and the mean TNF-a concentration was 18+8 ml (range 6 34.2/pg/ml). As shown in Figures 2 4, there was a signicant increase in mean plasma levels of IL-6, IL-8 and TNFa after anti-D infusion. TNF-a peaked 1 h postinfusion while IL-8 and IL-6 both peaked at 20 h post anti-D treatment. IL-6 and IL-8 levels remained signicantly elevated through 168 h then returned to pre-anti-D values. TNF-a levels remained signicantly elevated over 720 h of observation.

tions of sTNFR1 and sTNFR2 before treatment were 1.56+0.36 ng/ml (range 0.8 2.1) and 3.08+2.3 ng/ml (range 2.0 6.6) respectively. After anti-D infusion, a signicant and rapid increase in levels of both circulating TNF-a receptors was observed with peak values of R1 at 20 h and R2 at 3 h.

Correlation between maximal changes in plasma levels of cytokines, their soluble receptors and platelets after anti-D infusion
Maximal changes in plasma levels of cytokines, their soluble receptors and platelets were calculated as the dierence between post-treatment maximal values and pretreatment values. Correlations between IL-8 and IL-6 (r=0.65, P=0.003), IL-8 and TNF-a (r=0.55, P=0.02), TNFa and sTNFR1 (r=0.53, P=0.03), TNFR1 and TNFRII (r=0.75, P=0.0002) maximal changes were present. The maximal changes in platelet count were not signicantly correlated with sTNFR1 and TNFR2.

Content of plasma soluble cytokine receptors

Two soluble receptors for TNF-a (sTNFR1 and sTNFR2) were examined. Figures 5 and 6 show EASIA results from plasma before and after anti-D administration. The concentraThe Hematology Journal

Correlation between plasma levels of cytokines before treatment and maximal changes in platelets count after anti-D infusion
Pre-treatment values of cytokines and soluble receptors were correlated with maximal change in platelet count

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Figure 2 Plasma levels of IL-6 before and after anti-D infusion. Data are shown as scatter points on log scale. Non-detectable cytokine levels are converted to 0.001 to allow their presentation on the log scale.

Figure 3 Plasma levels of IL-8 before and after anti-D infusion. Data are shown as scatter points on log scale. Non-detectable cytokine levels are converted to 0.001 to allow their presentation on the log scale.
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Figure 4 Plasma levels of TNF-a before and after anti-D infusion. Data are shown as scatter points on log scale. Non-detectable cytokine levels are converted to 0.001 to allow their presentation on the log scale.

Figure 5 Plasma levels of sTNFR1 before and after anti-D infusion. Data are shown as scatter points on log scale. Non-detectable cytokine levels are converted to 0.001 to allow their presentation on the log scale.
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Figure 6 Plasma levels of sTNFR2 before and after anti-D infusion. Data are shown as scatter points on log scale. Non-detectable cytokine levels are converted to 0.001 to allow their presentation on the log scale.

after treatment to see whether there is any predictive value for responses to anti-D treatment. Correlation between TNF-a (P50.01) and maximal platelet rise was present. Pre-treatment values of IL-6, IL-8 and soluble receptors were not signicantly correlated with maximal platelet change after anti-D treatment.

Concentration of cytokines and soluble receptors in urine

Urine samples for cytokine and soluble receptor measurements were obtained from ve children. Mean concentrations of IL-6, IL-8, TNF-a and soluble receptors for TNF-a were signicantly higher in urine than in plasma before anti-D administration, and there was no correlation between plasma and urine concentrations after anti-D administration.

Discussion
The purpose of this study was to examine whether the hypothetical mechanism of anti-D immunomodulatory eect previously proposed6 can actually be demonstrated. Our results show that anti-D infusion caused a signicant increase in the concentration of cytokines (IL-6, IL-8 and TNF-a) and soluble cytokine receptors (sTNFR1 and sTNFR2), as well as the platelet count in children with chronic ITP. Previous observations1,2

indicate that there is no correlation of parameters of hemolysis with platelet increase, suggesting that anti-D eect is not limited to FcR blockade. The actual mode by which anti-D exerts its eect is not clear. In addition to direct saturation of macrophage Fc receptors with antibody coated RBCs, infusion of anti-D may also work to increase the platelet count by a variety of immunomodulatory eects.1,6 These studies show a correlation of platelet number with an increase of monocyte Fc receptor I expression and increased in vitro production of antibody to sheep RBCs following anti-D infusion. We suspect that Fc receptor blockade explains the immediate eects of anti-D on the rapid rise in platelet count, while the longer eects are related to a cytokinemodulatory eect. A rapid increase of TNF-a within 1 h postinfusion may represent the activation of macrophages in a process of phagocytosis. Subsequently TNF-a induces release of IL-6, which is a potent stimulator of megakaryopoiesis and thrombopoiesis. IL-6 belongs to the category of proinflammatory cytokines, which can both suppress TNF-a synthesis and induce production of TNF antagonists like soluble receptors.7,8 The mechanisms controlling the shedding of TNF receptors have not been fully elucidated; both TNF-a and stimuli causing TNF-a release can induce shedding of soluble TNFR which neutralizes TNF-a and the degree of biologic activity may depend on the
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balance between cytokine and soluble receptor.9,10 In our study there was a signicant positive correlation between the maximum change in TNF-a and the levels of sTNFRI, which possibly reects the compensation of TNF-a activity by its soluble receptor. The trend we observed between the decreasing level of sTNFRI and increasing platelet count may indicate that inhibition of TNF-a activity promotes platelet production in ITP patients. There is also evidence that anti-D and IVIG do not share an identical mechanism of eects. The most striking dierence is that IVIG is equally eective in splenectomized patients and this is irrespective of the patient's blood type.1,6 The time lapse until the peak response is seen and therapeutic doses of both immunoglobulins diered. Infusion of anti-D at a dose of 40 mg/kg gives a very small circulating concentration of anti-D and leads to platelet increase with peak at 168 h. In comparison the platelet increase seen after 2 g/kg of IVIg in ITP (50 000 times higher than anti-D) is usually more dramatic and occurs within 48 h.7 Our study also indicates that there may be dierences in eects of both immunoglobulins on cytokine cascade. Anti-D caused a remarkably larger increase in IL-6 concentration and only small increases of IL-8 concentrations in comparison to IVIG. Comparable eects were observed for TNF-a and its soluble receptors. Several studies have shown in vitro cellular immune defects in patients with chronic ITP,11,12,14,15 but little is known regarding plasma cytokines in ITP and their relationship to autoimmune pathogenesis. Trace levels of IL-6 and IL-8 were present in our ITP patients

before anti-D infusion. While increased levels of IL-2, interferon-g and IL-10 are present in patients with chronic ITP,11 patients did not have detectable serum levels of IL-4 and IL-6.11 TNF-a which is absent in the serum of healthy individuals was detected at physiologically signicant levels before anti-D infusion in our patients, suggesting a possible primary role for this cytokine in the etiology of ITP or a reection of inammation/immune response to the causative agent.16,17 The signicant correlation between TNF-a concentration before anti-D treatment and maximal platelet rise after treatment might indicate a specic role for this cytokine in ITP and its predictive value for responses to the treatment. TNF-a has an exceedingly short half-life of less than 20 min in the circulation, and is totally cleared within hours.8 However, we observed a very slow disappearance of TNF-a from the circulation of our patients, indicating the changes in metabolism or continuous production. These observations may account for the lack of correlation between plasma and urine levels in our study. These eects of anti-D may also be explained by the interaction of immune complexes or immunoglobulin, which are slowly cleared from the circulation, with Fc receptors on monocytes, as well as other cell types.7,18 In summary, these ndings highlight the complexity of response to immunoglobulin therapy and can be attributed to the immunomodulatory eects of anti-D on cytokine synthesis combined with a blockade of the Fc receptor on macrophages of the reticuloendothelial system.

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6 Bussel JB, Graziano JN, Kimberly RP, Pahwa S, Aledort L. Intravenous anti-D treatment of immune thrombocytopenic purpura: analysis of ecacy, toxicity and mechanism of action. Blood 77: 1884, 1991. 7 Durum SK, Oppenheim JJ. Proinammatory cytokines and immunity. In: William EP (ed) Fundamental immunology, Raven Press, New York, pp 801, 1993. 8 Tilg H, Trehu E, Atkins MB, Dinarello CA, Mier J. Interleukin-6 as an anti-inammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood 83: 113, 1994. 9 Botran RF. Soluble cytokine receptors: their role in immunoregulation. FASEB J 5: 2567, 1991. 10 Heaney ML, Golde DW. Soluble cytokine receptors. Blood 87: 847, 1996. 11 Semple JW, Milev Y, Cosgrave D, Mody M, Hornstein A, Blanchette V, Freedman J. Dierences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity. Blood 87: 4245, 1996.

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12 Chang M, Suen Y, Meng G, Buzby JS, Bussel J, Shen V, van de Ven C, Cairo M. Dierential mechanisms in the regulation of endogenous levels of thrombopoietin and interleukin-11 during thrombocytopenia: insight into the regulation of platelet production. Blood 88: 3354, 1996. 13 Kimura F, Nakamura Y, Sato K, Wakimoto N, Kato T, Tahara T, Yamada M, Nagata N, Motoyoshi K. Cyclic changes of cytokines in patients with cyclic thrombocytopenia. British Journal of Haematology 94: 171, 1996. 14 Nomura S, Yanabu M, Kido H, Gui Lan X, Ichiyoshi H, Katsura K, Miyake Y, Miyazaki Y, Kagawa H, Fukuhara S. Signicance of cytokines and CD68 positive microparticles in immune thrombocytopenic purpura. European Journal of Haematology 55: 49, 1995.

15 Je drzejczak WW, Dawidziak-Podolak M. Cytokines, VOLUMED, Wroclaw, 103 pp, 1997. 16 Hale KK, Smith CG, Baker lice RW, Squires CH, Gleason TM, Tucker KK, Kohno T, Russell D Vander. Multifunctional regulation of the biological eects of TNF alpha by the soluble type I and type II TNF receptors. Cytokine 7: 26, 1995. 17 Schimozato T, Iwata M, Tamura N. Suppression of tumor necrosis factor alpha production by human immunoglobulin preparation for intravenous use. Infection and Immunology 58: 1384, 1990. 18 Andersson JP, Andersson UG. Human intravenous immunoglobulin modulates monokine production in vitro. Immunology 71: 372, 1990.

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