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Author Query Form Journal: SJI Article:
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Dear Author, During the copyediting of your manuscript the following queries arose. Please refer to the query reference callout numbers in the page proofs and respond to each by marking the necessary comments using the PDF annotation tools. Please remember illegible or unclear comments and corrections may delay publication. Many thanks for your assistance.
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Funding Info Query Form Please confirm that the funding sponsor list below was correctly extracted from your article: that it includes all funders and that the text has been matched to the correct FundRef Registry organization names. If a name was not found in the FundRef registry, it may not be the canonical name form, it may be a program name rather than an organization name, or it may be an organization not yet included in FundRef Registry. If you know of another name form or a parent organization name for a “not found” item on this list below, please share that information. FundRef name
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Lunds Universitet
Lunds Universitet
Canadian Blood Services
Canadian Blood Services
Health Canada
Health Canada
Swiss National Science Foundation
Schweizerischer Nationalfonds zur F€orderung der Wissenschaftlichen Forschung
Received: 4 April 2018
Accepted: 29 May 2018
DOI: 10.1111/sji.12678
EXPERIMENTAL IMMUNOLOGY
1
Antiplatelet antibody-induced thrombocytopenia does not correlate with megakaryocyte abnormalities in murine immune thrombocytopenia L. Guo1,2,3,4 | R. Kapur1,2,3,5,6 | R. Aslam1,2 | K. Hunt1,2 | Y. Hou1,2 | A. Zufferey1,2 | E. R. Speck1,2 | M. T. Rondina4 | A. H. Lazarus1,2,3,7,8 | H. Ni1,2,3,7,8 |
Institute of Medical Science, University of Toronto, Toronto, ON, Canada 4 University of Utah, Salt Lake City, UT, USA 5
Canadian Blood Services, Lund University, Lund, Sweden 6
Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden 7
Department of Medicine, University of Toronto, Toronto, ON, Canada 8
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada 9
4
Department of Pharmacology, University of Toronto, Toronto, ON, Canada Correspondence J. W. Semple, Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden. Email:
[email protected]
in the disease. However, the exact interplay between platelet destruction, megakaryocyte dysfunction and the elements of both humoral and cell-mediated immunity in ITP remains incompletely defined. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the antiplatelet antibodies can also destroy bone marrow megakaryocytes. To address this, we negated the effects of T cells by utilizing an in vivo passive ITP model where BALB/c mice were administered various anti-aIIb, anti-b3 or anti-GPIb antibodies or antisera and platelet counts and bone marrow megakaryocytes were enumerated. Our results show that after 24 hours, all the different antiplatelet antibodies/sera induced variable degrees of thrombocytopenia in recipient mice. Compared with na€ıve control mice, however, histological examination of the bone marrow revealed that only 2 antibody preparations (mouse-anti-mouse b3 sera and an anti- aIIb monoclonal antibody (MWReg30) could affect bone marrow megakaryocyte counts. Our study shows that while most antiplatelet antibodies induce acute thrombocytopenia, the majority of them do not affect the number of megakaryocytes in the bone marrow. This suggests that other mechanisms may be responsible for megakaryocyte abnormalities seen during immune thrombocytopenia.
Funding information Lunds Universitet; Canadian Blood Services, Grant/Award Number: 340668; Health Canada; Swiss National Science Foundation, Grant/Award Number: P2GEP3_151966
L. Guo and R. Kapur are contributed equally to this work. Scand J Immunol. 2018;e12678. https://doi.org/10.1111/sji.12678
PE: Premalatha S.
the bone marrow. Although ITP was originally thought to be primarily due to antibody-mediated autoimmunity, it is now clear that T cells also play a significant role
No. of pages: 8
3
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in
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2 Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, Toronto, ON, Canada
Abstract
Manuscript No.
The Toronto Platelet Immunobiology Group, Toronto, ON, Canada
Dispatch: 6.6.18
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CE: Sathiyaseelan R
J. W. Semple1,2,3,5,6,7,8
S J I
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© 2018 The Foundation for the Scandinavian Journal of Immunology
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| INTRODUCTION
Immune thrombocytopenia (ITP) is an autoimmune bleeding disease characterized by an isolated thrombocytopenia without any other haematological abnormalities except anaemia due to blood loss.1 The pathogenesis of ITP is complex both at the level of immune system abnormalities and how the thrombocytopenia is induced in the disease.2-4 For example, the thrombocytopenia of ITP is due to at least 2 major mechanisms, either increased peripheral platelet destruction in the spleen or reduced platelet production from the bone marrow.4-7 The mechanism of peripheral platelet destruction in ITP has been studied extensively and is thought to be primarily due to increased FcR-dependent phagocytosis of opsonized platelets by splenic macrophages.2-4 This immune platelet destruction is associated with a vast array of peripheral and splenic B cell and T cell defects involving, for example, cytokine defects, Th1-skewness, Treg abnormalities and loss of tolerance.2-4 Less is known, however, about the immune mechanism(s) of reduced platelet production in ITP although there are some early and recent clues. In 1915, Frank first proposed that there was a platelet production abnormality in patients with ITP.8 Subsequently, Dameshek and Miller examined the bone marrow of patients with ITP and found increased numbers of megakaryoblasts along with elevated numbers of degenerated megakaryocytes and decreases in the number of mature megakaryocytes shedding platelets.5 Houwerzijl et al6 confirmed these findings by studying the fine ultrastructural characteristics of bone marrow megakaryocytes from patients with ITP using electron microscopy. Of interest, they observed apoptotic morphological abnormalities of megakaryocytes and this was confirmed by showing increased caspase-3 expression in the megakaryocytes.6 The immune nature of the megakaryocyte abnormalities was first shown by 2 groups that both demonstrated reduced in vitro megakaryocyte growth upon addition of antiplatelet autoantibodies with various specificities, for example anti-aIIb, vs anti-b3. More recently, it has become evident from murine models that cytotoxic T cells may also play a significant role in megakaryocyte destruction in ITP;9,10 however, the exact interplay between megakaryocyte dysfunction, platelet production and the elements of both humoral and cell-mediated immune systems remains incompletely understood. To better address how antiplatelet antibodies can solely affect megakaryocyte numbers and morphology in vivo, we utilized a well-characterized murine model of passive ITP and demonstrate that while most antibodies mediate thrombocytopenia, the majority are not associated with in vivo megakaryocyte abnormalities. The results show that while antiplatelet antibodies can mediate peripheral destruction of platelets, they have little effect in
GUO
ET AL.
the bone marrow suggesting that other immune mechanisms may be responsible for the megakaryocyte abnormalities seen in ITP.
2 2.1
| MATERIALS AND METHODS | Mice
BALB/c mice (H-2d; Charles River Laboratories, Montreal, QC, Canada) were used as antibody recipients. BALB/c CD61 (b3 integrin) knockout (KO) mice were supplied by Dr. Heyu Ni and used as a source of mouse-anti-mouse CD61 sera. All mice were 8-12 weeks of age, and all studies were approved by the St. Michael’s Hospital Animal Care Committee.
2.2
| Antiplatelet antibodies and Passive ITP
Rabbit-anti-mouse platelet serum (RAMS; Cedarlane Laboratories, Mississauga, ON) and monoclonal antibodies (mAbs) including MWReg30, 2C9.G2, NIT-B, NIT-E, NITG and NIT-H1 obtained for Drs. Alan Lazarus and Heyu Ni (St. Michael’s Hospital) were examined in this study. Their antigen specificities, isotypes as well as the doses used for ITP induction, are listed in Table 1. The antigen expression on megakaryocytes including aIIb, b3 and GPV was measured by flow cytometry as shown in Figure S1. Mouseanti-mouse CD61 sera (anti-b3) was obtained from b3 KO mice immunized with syngeneic CD61+ platelets as previously described.9 PBS, normal mouse sera (NMS) and a monoclonal IgG isotype control antibody were used as sham controls. To passively induce ITP, all sera and antibodies were administered by an intraperitoneal (ip) injection and there optimal doses were determined by previous titration experiments. For intravenous immunoglobulin (IVIg) treatment of ITP, IVIg was administered ip at 2 g/kg, 5 minutes prior to the antibody injection.
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T A B L E 1 Details of polyclonal (serum) and monoclonal 6
antibodies used for ITP induction Name
Specificity
Host
Isotype
Dose
Anti-b3
b3 integrin
Mouse
IgG
200-250 lL
RAMS
Mouse platelet
Rabbit
IgG
5 lL
MWReg30
aIIb integrin
Rat
IgG1
2-4 lg
2C9.G2
b3 integrin
Armenian Hamster
IgG1
10
NIT-G
Glycoprotein Ib
Mouse
IgG1
1 lg
NIT-B
Glycoprotein Ib
Mouse
IgG2a
1 lg
NIT-H1
Glycoprotein Ib
Mouse
IgG1
1 lg
NIT-E
Glycoprotein Ib
Mouse
IgG2b
1 lg
Anti-b3, mouse-anti-mouse b3 serum; RAMS, rabbit-anti-mouse platelet serum.
GUO
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| Platelet counts
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Mice were bled via the saphenous vein into PBS with 10% CPDA 24 hours after antibody/sera injection, and platelet counts were measured using a Beckman Coulter Counter-LH750 haematology analyser as previously described.9
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| Statistical analysis
Statistical analyses were performed using Prism software (version 6.0; GraphPad Software, San Diego, CA). Data are presented as mean standard deviation (SD). One-way ANOVA with Tukey’s post hoc test and t test was used for data analysis, and P values of
