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Prepublished online as a Blood First Edition Paper on March 20, 2003; DOI 10.1182/blood-2003-01-0023. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-01-0023v1 102/2/558    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 Hemostasis, Thrombosis, and Vascular Biology Immunobiology Phagocytes Apoptosis Signal Transduction Immunotherapy Brief Reports Related Letter in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 July 2003, Vol. 102, No. 2, pp. 558-560 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Brief report IVIg-mediated amelioration of murine ITP via FcRIIB is independent of SHIP1, SHP-1, and Btk activity Andrew R. Crow, Seng Song, John Freedman, Cheryl D. Helgason, R. Keith Humphries, Katherine A. Siminovitch, and Alan H. Lazarus From the Transfusion Medicine Research, St Michael's Hospital, Toronto, ON, Canada; Canadian Blood Services, Toronto, ON, Canada; Department of Cancer Endocrinology and Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada; Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, ON, Canada; and Toronto Platelet Immunobiology Group, Toronto, ON, Canada.    Abstract Top Abstract Introduction Study design Results and discussion References   It has been established that amelioration of murine immune thrombocytopenia purpura (ITP) by IVIg is dependent on the inhibitory receptor FcRIIB. Co-cross-linking of the FcRIIB with the B-cell receptor complex or with FcRI in mast cells results in cell inhibition, which is mediated by recruitment of the inositol phosphatase SHIP1 to the cytoplasmic tail of the FcR. The FcRIIB can also associate with protein tyrosine phosphatase SHP-1 as a potential secondary target of the receptor. Alternatively, homoaggregation of FcRIIB can induce a proapoptotic state in B cells that is dependent on the presence of Bruton tyrosine kinase (Btk), a kinase also expressed in monocytes. We sought to determine if these signaling pathways may direct IVIg-mediated FcRIIB-dependent regulation of in vivo monocyte function in a murine model of ITP in which IVIg functions in an FcRIIB-dependent manner. We demonstrate that mice deficient in SHIP1, SHP-1, and Btk respond to the ameliorating effects of IVIg with the same kinetics as control mice. We conclude that IVIgmediated inhibitory pathways operating via monocyte FcRIIB may involve a transmembrane signaling pathway different from that of B cells.    Introduction Top Abstract Introduction Study design Results and discussion References   The inhibitory low-affinity receptor for immunoglobulin G (IgG), FcRIIB, is widely expressed by hematopoietic cells such as B cells, mast cells, and monocytes. Two main FcRIIB-mediated inhibitory signaling pathways in B cells have been identified. The first pathway is initiated by co-cross-linking of the FcRIIB with the B-cell receptor complex, which leads to a dominant-negative signal, resulting in cell inactivation. This event delivers a signal through a motif in the cytoplasmic tail of FcRIIB, the immunoreceptor tyrosine-based inhibitory motif (ITIM; for a review, see Ravetch and Lanier1). SHIP1 binds directly, via its SH2 domain, to the phosphorylated receptor ITIM of FcRIIB. This same inhibitory pathway also blocks antigen-induced mast cell degranulation by recruitment of SHIP1 to the ITIM via co-crosslinking of FcRIIB with the activating high-affinity IgE receptor, FcRI.2-4 Evidence also indicates that ITIM-dependent FcRIIB-mediated immune suppression could involve the tyrosine phosphatase SHP-1.5-7 The second major inhibitory pathway mediated by FcRIIB in B cells is ITIM independent and involves immune complex-mediated homoaggregation of FcRIIB.1,8 This event leads to delivery of a proapoptotic signal to the B cell. It has been shown that signaling via FcRIIB homoaggregation is dependent on the presence of Bruton tyrosine kinase (Btk), a B-cell kinase also expressed in monocytes.9 We10 and others11,12 have shown that intravenous immunoglobulin (IVIg) can successfully ameliorate thrombocytopenia in a murine model of immune thrombocytopenia purpura (ITP), a disease state that is mediated by monocyte phagocytosis of opsonized platelets.13,14 Samuelsson et al11 recently demonstrated that the acute activity of IVIg in preventing murine ITP requires the expression of FcRIIB, and we have shown that the activity of IVIg in treating murine ITP is not dependent on the presence of B or T cells, nor anti-idiotype reactivity of IVIg.10 To determine if the FcRIIB-dependent activity of IVIg uses the ITIM-dependent inhibitory pathway (SHIP1/SHP-1) versus the homoaggregation (Btk)-dependent signaling pathway, we used mice genetically deficient for these key signaling molecules. We found that FcRIIB knockout (KO) mice rendered thrombocytopenic were refractory to IVIg treatment. Mice lacking FcRIIB-dependent signaling mediators including SHIP1, SHP-1, and Btk, however, all responded to IVIg treatment, indicating that either the known FcRIIB signaling pathways are not used by IVIg for its effect or that monocytes use another inhibitory pathway via FcRIIB.    Study design Top Abstract Introduction Study design Results and discussion References   Mice Wild-type, heterozygous, and homozygous SHIP1 KO mice were as previously described15 and bred at the British Columbia Cancer Agency (Vancouver, BC, Canada). SHP-1-deficient motheaten (me/me) mice and control littermates were bred at the Mt Sinai Hospital Samuel Lunenfeld Research Institute (Toronto, ON, Canada). Btk KO mice, FcRIIB KO mice, and C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Induction and reversal of ITP Thrombocytopenia was induced by intraperitoneal injection of 2 µg rat antimouse integrin IIb antibody (PharMingen, Mississauga, ON, Canada) as previously described.10 The following day, mice were injected with 2 g/kg human serum albumin (HSA) control protein, followed by 2 g/kg IVIg (Bayer, Elkhart, IN) on day 2. Platelet counts were assessed as previously described.10    Results and discussion Top Abstract Introduction Study design Results and discussion References   It is well accepted that inhibition of cell function through the inhibitory receptor FcRIIB requires recruitment of the inositol phosphatase SHIP1 to the receptor ITIM in both B cells1,3,16-19 and mast cells.2-4 The expression of FcRIIB on monocytes can lead to the down-regulation of phagocytosis,20 although the pathway by which FcRIIB down-modulates monocyte function remains unclear. To better understand this inhibitory event in vivo, we examined IVIg-mediated amelioration of ITP, which uses FcRIIB to alleviate thrombocytopenia.11 ITP was the first autoimmune disease successfully treated with IVIg, and IVIg is currently used to treat many autoimmune diseases.21-23 We have shown that IVIg can successfully ameliorate a murine model of antibody-induced thrombocytopenia with similar doses and kinetics as in humans with ITP.10 We investigated the signaling mediators used by IVIg through analyzing IVIg treatment of ITP in mice genetically deficient for primary signaling molecules juxtaposed to FcRIIB. Mice injected with antiplatelet antibody became thrombocytopenic by day 1 after injection (Figure 1A). Treatment of mice with 2 g/kg HSA (control protein) did not affect ITP. However, treatment of mice with 2 g/kg IVIg successfully reversed the disease in wild-type mice (Figure 1A). In separate experiments, thrombocytopenic wild-type mice treated with HSA alone for the duration of the experiment displayed no increase in platelet count (data not shown). In contrast to wild-type mice, IVIg had no effect in FcRIIB KO mice (Figure 1A); platelet counts in these mice remained low throughout the course of the experiment. These findings confirm that in our model system, the activity of IVIg in treating ITP is completely dependent on FcRIIB expression. In addition, the complete lack of any elevation in platelet counts in FcRIIB KO mice following IVIg treatment (Figure 1A, day 2 versus days 3 and 4) suggests that "competitive" reticuloendothelial system (RES) blockade per se does not significantly contribute to IVIg-mediated amelioration of thrombocytopenia. View larger version (13K): [in this window] [in a new window]   Figure 1.. IVIg requires expression of FcRIIB but not SHIP1 to ameliorate murine ITP. (A) FcRIIB KO () or control wild-type () mice (C57BL/6) were injected with 2 µg anti-integrin IIb antibody on days 0, 1, 2, and 3. The arrow () denotes injection of anti-integrin IIb antibody. All mice received an injection of 2 g/kg HSA control protein on day 1, followed by 2 g/kg IVIg on day 2 (, denotes injection of HSA or IVIg). Mice were bled daily for platelet enumeration. (B) SHIP1 heterozygous KO () and homozygous KO () mice and littermate control () mice were treated as described in panel A. Data are expressed as mean ± SEM; n = 10 mice/data point. *P < .001 versus control.   The inositol phosphatase SHIP1 preferentially binds the FcRIIB ITIM and it has been established that SHIP1 is the primary negative signaling pathway used by FcRIIB in B cells1,3,16-18 and mast cells.2-4 SHIP1 can negatively regulate FcR-mediated phagocytosis24 and overexpression of SHIP1 in murine macrophages can inhibit phagocytosis.25 Thus, we surmised that SHIP1 expression might play a key role in monocyte FcRIIB-dependent inhibition by IVIg and hypothesized that SHIP1 KO mice would be unresponsive to IVIg treatment. We found, however, that IVIg was able to exert its effects in both homozygous and heterozygous SHIP1 KO mice to the same extent as littermate controls (Figure 1B). Thus the FcRIIB-dependent activity of IVIg in immune thrombocytopenia is not dependent on the expression or activity of SHIP1. The tyrosine phosphatase SHP-1 can dephosphorylate multiple immunoreceptor-regulated substrates, leading to cell inactivation.5,6 SHP-1 has been shown to bind FcRIIB under extreme conditions such as receptor superclustering6 or hyperphosphorylation.7 We examined whether this enzyme is a possible mediator in the pathway by which IVIg exerts its protective effect. Administration of IVIg to thrombocytopenic SHP-1-deficient mice resulted in an increase in platelet counts, indistinguishable from that of control littermates (Figure 2A). This suggests that like SHIP1, SHP-1 is not required for the FcRIIB-mediated activity of IVIg. The FcRIIB ITIM contains 2 distinct, albeit overlapping, binding sites for SHIPs and SHPs17; thus there exists the possibility that a redundancy may exist in the SHIP/SHP families. However, evidence suggests that FcRIIB ITIM binds to SHIP proteins, but not SHP-1 or SHP-2 in vivo.26 View larger version (12K): [in this window] [in a new window]   Figure 2.. Mice deficient for SHP-1 or Btk respond to IVIg treatment of ITP. Mice were treated as described in Figure 1A. (A) Littermate control (), SHP-1-deficient (me/me; ) mice; n = 6 except for day 4, where n = 3 for SHP-1-deficient mice. (B) Btk KO () and control wild-type () mice (C57BL/6) were treated as in Figure 1A; n = 10 mice/data point. Data are expressed as mean ± SEM.   Because these major known ITIM-dependent signaling intermediates did not appear to be required for activity of IVIg, we questioned whether IVIg therapy resulted in ITIM-independent FcRIIB-mediated inhibition. This inhibitory pathway focuses on delivery of a proapoptotic signal generated through FcRIIB homoaggregation27 and is dependent on the presence and expression of the Tec family kinase Btk,8 a B-cell kinase also expressed in monocytes.9 IVIg has been shown to induce apoptosis in multiple cell types, including monocytes.28 IVIg ameliorated thrombocytopenia in both Btk KO mice and control mice (Figure 2B), indicating that the activity of IVIg is not dependent on a Btk-dependent proapoptotic event. We demonstrate that FcRIIB-dependent IVIg activity is not reliant on the established signaling pathways downstream of FcRIIB. Mice lacking the normal FcRIIB downstream signaling mediators, SHIP1, SHP-1, or Btk, all responded to IVIg treatment of ITP as successfully as control mice. This suggests that inhibitory signaling through FcRIIB on monocytes may use a different pathway than B cells. Indeed, a recent report has demonstrated that SHIP-dependent inhibition of monocyte function can occur independently of FcRIIB, a mechanism distinct from that used by B-cell expressed FcRIIB.24 It may be that inhibition of monocyte function by IVIg occurs via an FcRIIB signaling pathway unique to monocytes.    Acknowledgements   We thank Ms Alison F Starkey, Mr Hoang Le-Tien, Mr Davor Brinc, and Dr Vinayakumar Siragam for assistance and helpful discussion; Dr John W Semple for critical review of the manuscript; and Ms Carolyn Bateman of the British Columbia Cancer Agency Joint Animal Facility, Ms Michele Deverill of the Samuel Lunenfeld Research Institute, and the St Michael's Hospital Research Vivarium staff.    Footnotes   Submitted January 6, 2003; accepted March 12, 2003. Prepublished online as Blood First Edition Paper, March 20, 2003; DOI 10.1182/blood-2003-01-0023. Supported by The Bayer-Canadian Blood Services-Héma Québec Partnership Fund and the National Cancer Institute of Canada with funds from the Terry Fox Foundation (R.K.H.). C.D.H. was a recipient of a BC Health Research Foundation Joint Scholarship and holds a Canadian Institutes of Health Research (CIHR) New Investigator Scholarship. K.A.S. is a CIHR Senior Scientist. 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 U.S.C. section 1734. Reprints: 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 Study design Results and discussion References   Ravetch JV, Lanier LL. Immune inhibitory receptors. Science. 2000;290: 84-89.[Abstract/Free Full Text] Daeron M, Malbec O, Latour S, Arock M, Fridman WH. 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Lazarus IVIg-mediated amelioration of murine ITP via Fc{gamma}RIIb is not necessarily independent of SHIP-1 and SHP-1 activity Blood, March 1, 2004; 103(5): 1973 - 1974. [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2003-01-0023v1 102/2/558    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. 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