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Blood, 1 January 2007, Vol. 109, No. 1, pp. 155-158. Prepublished online as a Blood First Edition Paper on September 5, 2006; DOI 10.1182/blood-2006-05-023796. This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: blood-2006-05-023796v1 109/1/155    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Crow, A. R. Articles by Lazarus, A. H. Search for Related Content PubMed PubMed Citation Articles by Crow, A. R. Articles by Lazarus, A. H. Related Collections Immunotherapy Brief Reports Hemostasis, Thrombosis, and Vascular Biology Immunobiology Transfusion Medicine Related Article in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Brief report A role for IL-1 receptor antagonist or other cytokines in the acute therapeutic effects of IVIg? Andrew R. Crow, Seng Song, John W. Semple, John Freedman, and Alan H. Lazarus Canadian Blood Services, Department of Laboratory Medicine, St Michael's Hospital, Toronto, ON, Canada; Departments of Pharmacology, Medicine, and Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada; Toronto Platelet Immunobiology Group, ON, Canada    Abstract Top Abstract Introduction Materials and methods Results and discussion References   The exact mechanism of action of IVIg in the amelioration of immune thrombocytopenic purpura (ITP) is still unclear. Studies have suggested that IVIg may function through the regulation of cytokines, including interleukin-1 receptor antagonist (IL-1Ra), an inhibitor of phagocytosis. Using a mouse model relevant to ITP, we confirm an increase in mouse serum levels of IL-1Ra after exposure to IVIg, yet a recombinant IL-1Ra did not ameliorate thrombocytopenia. IVIg has also been shown to affect the expression of other regulatory cytokines. We have also recently established that IVIg specifically targets activating FcRs on CD11c+ dendritic cells (DCs) as its primary mechanism of action in the amelioration of murine ITP. Herein, we show that IVIg functions therapeutically in mice lacking specific cytokines or their receptors that can potentially affect DC/macrophage function (IL-1 receptor, IL-4, IL-10, IL-12ß, TNF-, IFN- receptor, MIP-1). This suggests that while IVIg may mediate the release of a variety of cytokines, the cytokines tested do not directly participate in the mechanism of IVIg action.    Introduction Top Abstract Introduction Materials and methods Results and discussion References   The exact mechanism of action of IVIg in the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP) remains unresolved. Proposed modes of action include Fc-receptor blockade, inhibitory effects mediated by FcRIIB, immunomodulation (reviewed in Bayry et al1 and Crow and Lazarus2), and the recent finding that IVIg specifically targets activating FcRs on CD11c+ dendritic cells (DCs) in a mouse model of ITP.3 Modified cytokine production may play a role in the pathogenesis of autoimmune diseases such as ITP (reviewed in Zhou et al4), and thus cytokine modulation by IVIg might contribute to the reversal of thrombocytopenia. Indeed, studies have demonstrated modulation of numerous proinflammatory and anti-inflammatory cytokines both in vivo and in vitro after exposure to IVIg.5-7 Work by Aukrust et al7 has demonstrated an increase in IL-6, -8, and TNF- within 1 hour of IVIg administration. Both IVIg7,8 and anti-D9 have been shown to induce the production of IL-1 receptor antagonist (IL-1Ra), a potent anti-inflammatory cytokine that can counteract the inflammatory effects of IL-1.10 IVIg can also induce secretion of IL-1Ra in human monocytes,11 and administration of IL-1Ra can successfully inhibit the phagocytosis of anti-D–opsonized red blood cells (RBCs).12 In addition, IL-1Ra can also regulate the activated state of DCs.13 IVIg can also inhibit the differentiation/maturation of human DCs and abrogate their production of IL-12 while increasing production of the anti-inflammatory cytokine IL-10.14 The exact role that cytokines play in the therapeutic action of IVIg, however, remains unresolved. Using a mouse model of ITP,15 we have analyzed the therapeutic effect of IVIg (Gamimune) in mice lacking specific cytokines/receptors that can potentially affect DC and/or macrophage function (IL-1R, IL-4, IL-10, IL-12ß, TNF-, IFN-R, MIP-1), or in mice lacking the common cytokine receptor chain (required for signal transduction through the receptors for IL-2, -4, -7, -9, -15, and -21). We present data that suggest that while cytokine modulation may play some role clinically in the long-term effects of IVIg in humans with true ITP, the key cytokines tested were not required for the acute protective effect of IVIg in murine ITP.    Materials and methods Top Abstract Introduction Materials and methods Results and discussion References   Reagents The IVIg used was from Bayer (Elkhart, IN). The anti–integrin IIb antibody (clone MWReg30) was from PharMingen (Mississauga, ON, Canada). Recombinant IL-1Ra (anakinra) was from Amgen Canada (Mississauga, ON, Canada). The murine IL-1Ra enzyme-linked immunosorbent assay (ELISA) kit was from R&D Systems (Minneapolis, MN). Mice Mice deficient in IL-1R (B6.129S7-Il1r1tm1Imx), IL-4 (B6.129P2-Il4tm1Cgn), IL-10 (B10.129P2(B6)-Il10tm1Cgn), IL-12ß (B6.129S1-Il12btm1Jm), TNF- (B6;129S6-Tnftm1Gkl), IFN-R (B6.129S7-Ifngrtm1Agt), MIP-1 (B6.129P2-Ccl3tm1Unc), or common cytokine receptor chain (B6.129S4-Il2rgtm1Wjl/J) and appropriate control mice were all purchased from The Jackson Laboratory (Bar Harbor, ME). CD1 mice were purchased from Charles River Laboratories (St Constant, PQ, Canada). Mice were housed in the St Michael's Hospital Research Vivarium. Induction and reversal of murine ITP ITP was induced and treated as previously described.15 Briefly, mice were injected daily (days 0-3) with 2 µg antiplatelet antibody to induce thrombocytopenia; platelets were enumerated by flow cytometry as described.15 Mice received 50 mg IVIg (generally equivalent to 2 g/kg body weight) intraperitoneally on day 2. Detection of serum IL-1Ra C57BL/6 mice were bled for serum at the indicated times after injection of IVIg. Murine IL-1Ra was detected by ELISA, according to the manufacturer's directions. Statistics Statistics were calculated according to Student t test and are presented as mean ± SEM. Note, in some cases where the error bars are not apparent, this is due to a minimal SEM.    Results and discussion Top Abstract Introduction Materials and methods Results and discussion References   To determine the potential role that IL-1Ra may play in the amelioration of murine ITP by IVIg, we first questioned if IVIg could increase the levels of IL-1Ra in mice. Analysis of mouse serum taken at 0, 1, 6, 24, and 48 hours after administration of IVIg revealed that IVIg did indeed induce the production of IL-1Ra, reaching peak levels at 24 hours, the time in which platelet recovery is readily observed in our model (Figure 1A) This finding is consistent with the possibility that IVIg-induced IL-1Ra might play a role in the amelioration of thrombocytopenia. View larger version (17K): [in this window] [in a new window]   Figure 1. IL-1Ra does not play a role in the acute effects of IVIg. (A) C57BL/6 mice were injected with 50 mg IVIg and each mouse was bled at the times indicated on the x-axis. Sera were assessed for IL-1Ra by ELISA. Values are expressed in pg IL-1Ra/mL on the y-axis. Data are expressed as mean ± SEM; n = 4 mice from 2 separate experiments. (B) C57BL/6 mice and IL-1R–deficient mice were injected on days 0 to 3 with antiplatelet antibody to induce thrombocytopenia. On day 2, mice received 50 mg IVIg (arrow). Mice were bled daily for platelet enumeration by flow cytometry just prior to antiplatelet antibody injection. C57BL/6 mice (), IL-1R–deficient mice (); n = 6 mice per each group. (C) CD1 mice were injected with antiplatelet antibody and assessed as in panel B, and mice received either 50 mg IVIg on day 2 (; arrow) or 50 µg rIL-1Ra on day 2 (); n = 6 mice per each group. (D) Mice were injected with antiplatelet antibody and received either 50 mg IVIg on day 2 (; down arrow) or 1 mg rIL-1Ra on days 2 and 3 (; up arrows); n = 6 mice per each group. (E) CD1 mice were pretreated with 50 mg IVIg (column 2), nothing (column 3), or 1 mg rIL-1Ra (column 4) on day 0. On day 1, antiplatelet antibody was given as indicated. On day 2, all mice were bled for platelet counts; n = 6 mice per each group.   Due to the fact that deletion of the IL-1 receptor (IL-1R) gene is epistatic to IL-1Ra gene deletion,16 we next questioned if IVIg would function in mice lacking the IL-1R, a ligand for IL-1Ra, by using IL-1R–deficient mice. IL-1R–deficient mice and control mice were rendered thrombocytopenic and treated with IVIg (Figure 1B). Platelet enumeration revealed that both wild-type mice and IL-1R–deficient mice responded to IVIg treatment, suggesting that IVIg function is not dependent on the presence of IL-1Ra. This result also implies that IVIg function is not reliant on the presence of IL-1, a potent proinflammatory cytokine that can also promote DC maturation.17 In addition, we found that a monoclonal antibody that reacts with RBCs (TER-119) and mimics the action of anti-D in the amelioration of murine ITP18,19 also induced the in vivo production of IL-1Ra in mice (Figure S1, available at the Blood website; see the Supplemental Figures link at the top of the online article). These data correlate with the IL-1Ra production observed in children with chronic ITP infused with anti-D.9 However, similar to our findings with IVIg, this anti-RBC antibody also protected against thrombocytopenia in IL-1R–deficient mice (Figure S1). Thus, these findings do not support a role for IL-1Ra in the amelioration of murine ITP by either IVIg or TER-119. In spite of these results, we nonetheless examined the ability of a recombinant IL-1Ra to reverse ITP or inhibit its induction. Initial experiments using a standard dose of IL-1Ra demonstrated that the administration of 50 µg of recombinant IL-1Ra to thrombocytopenic mice had no effect on thrombocytopenia (Figure 1C). Mice injected with a supraphysiologic dose (1 mg) of IL-1Ra on 2 consecutive days also showed no increase in the platelet count (Figure 1D). In addition, pretreatment of mice with 1 mg IL-1Ra did not prevent the induction of thrombocytopenia (Figure 1E). Thus there does not appear to be any direct role for IL-1Ra in the amelioration of murine thrombocytopenia. However, since IL-1Ra is produced during inflammatory responses,10 the elevated IL-1Ra seen in mice after IVIg administration is hypothesized to be more of a marker of the inflammatory process rather than having a role in reversing thrombocytopenia. Modulation of serum cytokines has been reported in patients exposed to IVIg.7,20-22 Pertinent to ITP, a recent study has demonstrated increases in IL-10 and MCP-1 in ITP patients after IVIg treatment.5 IVIg can also inhibit the differentiation/maturation of human DCs and abrogate their production of IL-12.14 IVIg has also demonstrated the ability to inhibit TNF- production by immature DCs.14 To address the potential role that selected cytokines may play in the action of IVIg, we analyzed the treatment of thrombocytopenia in mice lacking key proinflammatory regulators that could potentially affect DC or macrophage function (Figure 2A) Mice lacking TNF-, IL-12ß, MIP-1, or IFN-R were rendered thrombocytopenic as in Figure 1B, and all mice fully responded to IVIg treatment despite the lack of individual expression of these cytokines. View larger version (9K): [in this window] [in a new window]   Figure 2. Mice lacking key inflammatory or anti-inflammatory cytokines respond normally to IVIg treatment of ITP. Control mice (wild-type) and mice lacking the indicated specific proinflammatory cytokines (A), anti-inflammatory cytokines (B), or mice genetically lacking the common cytokine receptor chain (C) were rendered thrombocytopenic by daily injection (days 0-3) of antiplatelet antibody as in Figure 1B. On day 2, all mice received 50 mg IVIg (arrow). Mice were bled daily just prior to antiplatelet antibody injection and platelets enumerated as in Figure 1B. The y-axis represents platelet counts; the x-axis, the length of the experiment in days; n = 6 mice per each strain.   The anti-inflammatory cytokine IL-10 can play an important role in dampening macrophage activation,23 and its production by DCs is up-regulated in response to IVIg in vitro.14 The anti-inflammatory cytokine IL-4 can induce the production of IL-1Ra24 and up-regulate expression of the inhibitory IgG receptor FcRIIB,25 which is absolutely required for IVIg to ameliorate murine models of ITP.26,27 We found, however, that IVIg did function therapeutically in IL-10–deficient mice as well as in IL-4–deficient mice (Figure 2B). Mice deficient for the common cytokine receptor chain, which is required for signal transduction through the receptors for IL-2, -4, -7, -9, -15, and -21, responded to IVIg treatment (Figure 2C), which suggests that IVIg function is not reliant upon the expression of these cytokines. The immunoregulatory compound nitric oxide (NO) can be induced by IL-1,28 and NO's production in murine macrophages by IFN- can be inhibited by IL-10.29 NO can also be positively and negatively regulated by IVIg.30,31 Based upon this, we also analyzed IVIg-mediated amelioration of thrombocytopenia in mice that were subjected to aminoguanidine, a specific inhibitor of iNOS, or L-NAME, a multi-spectrum NOS inhibitor (Figure S2). We found that after inhibition of iNOS (aminoguanidine), or of iNOS, eNOS, and nNOS (L-NAME), IVIg successfully ameliorated murine ITP, suggesting that NO is not required for IVIg to reverse murine ITP. IVIg does indeed induce/suppress the production of multiple cytokines, and these cytokines may, for example, affect the synthesis of the antiplatelet antibody in humans with true ITP, an event not captured in our acute murine model. Thus while we demonstrate that the acute effects of IVIg do not require the presence of the individual cytokines tested, some of these cytokines may nevertheless play a role in the long-term effects of IVIg in humans with ITP.    Acknowledgments   We thank Mr Davor Brinc, Mr Hoang Le-Tien, and Dr Vinayakumar Siragam for assistance and helpful discussion, and the St Michael's Hospital Research Vivarium staff. This work was supported by the Bayer–Canadian Blood Services–Héma Québec Partnership Fund.    Footnotes   Submitted May 22, 2006; accepted August 14, 2006. Prepublished online as Blood First Edition Paper, September 5, 2006 DOI: 10.1182/blood-2006-05-023796 Contribution: A.R.C. designed research, performed research, analyzed data, and wrote the paper; S.S. performed research and analyzed data; J.W.S. designed research and edited the manuscript; J.F. contributed analytical tools and edited the manuscript; and A.H.L. designed research, analyzed data, and wrote the paper. The online version of this article contains a data supplement. An Inside Blood analysis of this article appears at the front of this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Alan H. Lazarus,Transfusion Medicine Research, St Michael's Hospital, 30 Bond St, Toronto, ON, Canada M5B 1W8; e-mail: lazarusa{at}smh.toronto.on.ca.    References Top Abstract Introduction Materials and methods Results and discussion References   Bayry J, Misra N, Latry V, et al. Mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases. Transfus Clin Biol 2003; 10:165–169.[CrossRef][Medline] [Order article via Infotrieve] Crow AR and Lazarus AH. Role of Fc receptors in the pathogenesis and treatment of idiopathic thrombocytopenic purpura. J Pediatr Hematol Oncol 2003; 25:S14–S18. Siragam V, Crow AR, Brinc D, Song S, Freedman J, Lazarus AH. Intravenous immunoglobulin ameliorates ITP via activating Fcgamma receptors on dendritic cells. Nat Med 2006; 12:688–692.[CrossRef][Medline] [Order article via Infotrieve] Zhou B, Zhao H, Yang RC, Han ZC. Multi-dysfunctional pathophysiology in ITP. Crit Rev Oncol Hematol 2005; 54:107–116.[Medline] [Order article via Infotrieve] Cooper N, Heddle NM, Haas M, et al. Intravenous (IV) anti-D and IV immunoglobulin achieve acute platelet increases by different mechanisms: modulation of cytokine and platelet responses to IV anti-D by FcgammaRIIa and FcgammaRIIIa polymorphisms. Br J Haematol 2004; 124:511–518.[CrossRef][Medline] [Order article via Infotrieve] Sewell WA, North ME, Cambronero R, Webster AD, Farrant J. In vivo modulation of cytokine synthesis by intravenous immunoglobulin. Clin Exp Immunol 1999; 116:509–515.[CrossRef][Medline] [Order article via Infotrieve] Aukrust P, Froland SS, Liabakk NB, et al. Release of cytokines, soluble cytokine receptors, and interleukin-1 receptor antagonist after intravenous immunoglobulin administration in vivo. Blood 1994; 84:2136–2143.[Abstract/Free Full Text] Arend WP and Leung DY. IgG induction of IL-1 receptor antagonist production by human monocytes. Immunol Rev 1994; 139:71–78.[CrossRef][Medline] [Order article via Infotrieve] Semple JW, Allen D, Rutherford M, et al. Anti-D (WinRho SD) treatment of children with chronic autoimmune thrombocytopenic purpura stimulates transient cytokine/chemokine production. Am J Hematol 2002; 69:225–227.[CrossRef][Medline] [Order article via Infotrieve] Arend WP, Malyak M, Guthridge CJ, Gabay C. Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol 1998; 16:27–55.[CrossRef][Medline] [Order article via Infotrieve] Ruiz de Souza V, Carreno MP, Kaveri SV, et al. Selective induction of interleukin-1 receptor antagonist and interleukin-8 in human monocytes by normal polyspecific IgG (intravenous immunoglobulin). Eur J Immunol 1995; 25:1267–1273.[Medline] [Order article via Infotrieve] Coopamah MD, Freedman J, Semple JW. Anti-D initially stimulates an Fc-dependent leukocyte oxidative burst and subsequently suppresses erythrophagocytosis via interleukin-1 receptor antagonist. Blood 2003; 102:2862–2867.[Abstract/Free Full Text] Iizasa H, Yoneyama H, Mukaida N, et al. Exacerbation of granuloma formation in IL-1 receptor antagonist-deficient mice with impaired dendritic cell maturation associated with Th2 cytokine production. J Immunol 2005; 174:3273–3280.[Abstract/Free Full Text] Bayry J, Lacroix-Desmazes S, Carbonneil C, et al. Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin. Blood 2003; 101:758–765.[Abstract/Free Full Text] Crow AR, Song S, Semple JW, Freedman J, Lazarus AH. IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity. Br J Haematol 2001; 115:679–686.[CrossRef][Medline] [Order article via Infotrieve] Irikura VM, Lagraoui M, Hirsh D. The epistatic interrelationships of IL-1, IL-1 receptor antagonist, and the type I IL-1 receptor. J Immunol 2002; 169:393–398.[Abstract/Free Full Text] Yamada N and Katz SI. Generation of mature dendritic cells from a CD14+ cell line (XS52) by IL-4, TNF-alpha, IL-1 beta, and agonistic anti-CD40 monoclonal antibody. J Immunol 1999; 163:5331–5337.[Abstract/Free Full Text] Song S, Crow AR, Freedman J, Lazarus AH. Monoclonal IgG can ameliorate immune thrombocytopenia in a murine model of ITP: an alternative to IVIG. Blood 2003; 101:3708–3713.[Abstract/Free Full Text] Song S, Crow AR, Siragam V, Freedman J, Lazarus AH. Monoclonal antibodies that mimic the action of anti-D in the amelioration of murine ITP act by a mechanism distinct from that of IVIg. Blood 2005; 105:1546–1548.[Abstract/Free Full Text] Ochi K, Kohriyama T, Higaki M, Ikeda J, Harada A, Nakamura S. Changes in serum macrophage-related factors in patients with chronic inflammatory demyelinating polyneuropathy caused by intravenous immunoglobulin therapy. J Neurol Sci 2003; 208:43–50.[CrossRef][Medline] [Order article via Infotrieve] Sewell WA and Jolles S. Immunomodulatory action of intravenous immunoglobulin. Immunology 2002; 107:387–393.[CrossRef][Medline] [Order article via Infotrieve] Noh GW, Lee WG, Lee W, Lee K. Effects of intravenous immunoglobulin on plasma interleukin-10 levels in Kawasaki disease. Immunol Lett 1998; 62:19–24.[CrossRef][Medline] [Order article via Infotrieve] Bogdan C, Vodovotz Y, Nathan C. Macrophage deactivation by interleukin 10. J Exp Med 1991; 174:1549–1555.[Abstract/Free Full Text] Marie C, Pitton C, Fitting C, Cavaillon JM. IL-10 and IL-4 synergize with TNF-alpha to induce IL-1ra production by human neutrophils. Cytokine 1996; 8:147–151.[CrossRef][Medline] [Order article via Infotrieve] Tridandapani S, Siefker K, Teillaud JL, Carter JE, Wewers MD, Anderson CL. Regulated expression and inhibitory function of Fcgamma RIIb in human monocytic cells. J Biol Chem 2002; 277:5082–5089.[Abstract/Free Full Text] Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatory activity of IVIG mediated through the inhibitory Fc receptor. Science 2001; 291:484–486.[Abstract/Free Full Text] Crow AR, Song S, Freedman J, et al. IVIg-mediated amelioration of murine ITP via Fc{gamma}RIIB is independent of SHIP1, SHP-1, and Btk activity. Blood 2003; 102:558–560.[Abstract/Free Full Text] Dinarello CA. The IL-1 family and inflammatory diseases. Clin Exp Rheumatol 2002; 20:S1–S13.[Medline] [Order article via Infotrieve] Cunha FQ, Moncada S, Liew FY. Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages. Biochem Biophys Res Commun 1992; 182:1155–1159.[CrossRef][Medline] [Order article via Infotrieve] Wang CL, Wu YT, Lee CJ, Liu HC, Huang LT, Yang KD. Decreased nitric oxide production after intravenous immunoglobulin treatment in patients with Kawasaki disease. J Pediatr 2002; 141:560–565.[CrossRef][Medline] [Order article via Infotrieve] Stangel M and Compston A. Polyclonal immunoglobulins (IVIg) modulate nitric oxide production and microglial functions in vitro via Fc receptors. J Neuroimmunol 2001; 112:63–71.[CrossRef][Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? Related Article in Blood Online: IVIg in ITP: no role for cytokines? Bethan Psaila and James B. Bussel Blood 2007 109: 4-5. [Full Text] [PDF] This article has been cited by other articles: J. Chang, P. A. Shi, E. Y. Chiang, and P. S. Frenette Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion Blood, January 15, 2008; 111(2): 915 - 923. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: blood-2006-05-023796v1 109/1/155    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Crow, A. R. Articles by Lazarus, A. H. Search for Related Content PubMed PubMed Citation Articles by Crow, A. R. 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