Targeting of platelet integrin {alpha}IIbß3 determines systemic reaction and bleeding in murine thrombocytopenia regulated by activating and inhibitory Fc{gamma}R

Bernhard Nieswandt1, Wolfgang Bergmeier1, Valerie Schulte1, Toshiyuki Takai2, Ulrich Baumann4, Reinhold E. Schmidt4, Hubert Zirngibl1, Wilhelm Bloch3 and J. Engelbert Gessner4

1 Department of Vascular Biology, Rudolf-Virchow-Zentrum, Universität Würzburg, 97078 Würzburg, Germany 2 Department of Experimental Immunology, Tohoku University, and CREST Program of JST, Institute of Development, Aging and Cancer, Tohoku University, Seiryo 4-1, Sendai 980-8575, Japan 3 Institute I of Anatomy, University of Cologne, 50931 Cologne, Germany 4 Department of Clinical Immunology, Hannover Medical School, 30625 Hannover, Germany

Correspondence to: J. E. Gessner; E-mail: gessner.johannes{at}mh-hannover.de
Transmitting editor: S. Izui


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Previous work on cellular destruction induced by several clinically relevant anti-platelet IgG antibodies suggested antigen-specific mechanisms in the development of immune thrombocytopenia in mice. mAb directed against mouse platelet GPIb{alpha} and integrin {alpha}IIbß3 were highly pathogenic, and mediated their effects via different Fc-dependent ({alpha}IIbß3) and Fc-independent (GPIb{alpha}) pathways, indicating that clearance of IgG-bound platelets is only one event in the pathogenesis of murine thrombocytopenia. Here, we demonstrate that in addition to thrombocytopenia, targeting of platelet integrin {alpha}IIbß3 results in acute systemic reaction and bleeding that is regulated by activating IgG Fc receptors (Fc{gamma}R) and the inhibitory Fc{gamma}RII. As shown by electron microscopy, anti-{alpha}IIbß3 IgG mediated initial loss of {alpha}IIbß3 integrin from platelet surfaces followed by rapid accumulation of {alpha}IIbß3 antibody-containing immune complex (IC)-like structures in spleen and liver in vivo. In FcR{gamma} chain deficiency, mice resisted bleeding, but not platelet destruction, while genetic ablation of Fc{gamma}RII resulted in uncontrolled systemic reaction and severe hemorrhage leading to enhanced mortality. Together, these results provide evidence that IC formation and engagement of Fc{gamma}R on effector cells determines the {alpha}IIbß3-specific part of the platelet pathology of the systemic reaction and bleeding in murine thrombocytopenia.

Keywords: {alpha}IIbß3 integrin, antibody, Fc{gamma} receptor, mouse, platelet


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Autoantibody-mediated cellular destruction contributes to the pathogenesis of several autoimmune diseases, and is thought to be specifically causal in the development of cytopenias such as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP). In the vast majority of patients with severe ITP, pathogenic IgG autoantibodies directed against the platelet surface receptors {alpha}IIbß3 integrin (GPIIbIIIa, fibrinogen receptor) and GPIb-V-IX (von Willebrand factor receptor) are found (13). Immune clearance of the resulting autoantibody-opsonized platelets leading to thrombocytopenia is considered as the major cause of bleeding complications in patients suffering from severe ITP (4,5). It is currently accepted that platelet destruction is mediated mainly by cells of the reticuloendothelial system and is Fc dependent (5). Initial studies in rodents suggest a prominent role of activating IgG Fc receptors (Fc{gamma}R) in this process (6,7). In humanized mouse models of Fc{gamma}R, activation of the human platelet Fc receptor, hFc{gamma}RIIA (CD32), has been reported to result in thrombosis and shock (8).

There are three classes of Fc{gamma}R on leukocytes: the high-affinity receptor, Fc{gamma}RI, and the two low-affinity receptors, Fc{gamma}RII and Fc{gamma}RIII (9,10). Both Fc{gamma}RI and Fc{gamma}RIII are multimeric receptors in association with the same FcR{gamma} chain required for assembly and signaling (11). Animal studies using FcR{gamma} or Fc{gamma}RIII mutant mice revealed that activating Fc{gamma}R, especially Fc{gamma}RIII, are involved in the initiation of immune complex (IC)-triggered inflammation and autoimmune disease (1215). Most of the Fc{gamma}RIII- and FcR{gamma}-triggered responses are balanced by the inhibitory Fc{gamma}RII when co-expressed on the same effector cell (1619). Moreover, the inhibitory Fc{gamma}RII is modulated by IVIG in the treatment of thrombocytopenia in mice (20,21).

Recent studies using newly generated panels of mAb directed against various mouse platelet antigens, including {alpha}IIbß3, GPIb{alpha}, GPIb-IX or GPV, have shown that differences in the antigenic specificity of anti-platelet IgG antibodies can determine their pathogenic activities (22,23). Most of these mAb are able to mimick the effect of cytotoxic autoantibody by triggering transient or irreversible thrombocytopenia by both Fc-dependent and -independent mechanisms in vivo. Interestingly, only the anti-{alpha}IIbß3 cluster of antibodies (JON mAb) recognizing conformational epitopes on {alpha}IIbß3 integrin is effective to induce, in addition to platelet destruction, an acute systemic reaction that potentially causes hemorrhage and bleeding. It remains to be investigated whether Fc{gamma}R can contribute to this {alpha}IIbß3-specific part of murine platelet pathology (23). In this study, we examined the role of the various Fc{gamma}R to the pathogenic effects of anti-mouse platelet {alpha}IIbß3 mAb in normal mice as compared to IgG FcR knockout mice.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The generation of FcR{gamma}–/–, Fc{gamma}RIII–/– and Fc{gamma}RII–/– mice derived from ES cells of 129 origin and backcrossed for more than eight generations with C57BL/6 mice has been described previously (11,15,16). C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). All these mice were used at 8–12 weeks of age. Experiments were conducted in accordance to the regulations of the local authorities.

Antibodies
FITC-conjugated polyclonal rabbit antibodies to human fibrinogen and rabbit anti-rat Ig were purchased from Dako (Glostrup, Denmark). Anti-mouse platelet IgG mAb of defined specificities were established by standard techniques as described previously (23,24). Anti-mouse integrin {alpha}IIbß3: JON1 (rat IgG2b) and JON3 (rat IgG1); anti-mouse GPIb{alpha}: p0p4 (rat IgG2b) and p0p5 (rat IgG1). F(ab)2 fragments were generated as described (23). Antibodies were conjugated with 5-nm colloidal gold by standard methods.

Experimental thrombocytopenia, systemic reaction and bleeding
Ether anesthetized mice received either a single dose of 3 µg/g of purified anti-mouse platelet IgG antibodies in 200 µl sterile PBS i.v. or, alternatively, 7 x 0.3 µg/g antibody i.p. were assayed at the indicated times for thrombocytopenia (as assessed by counting platelet numbers), for systemic reaction (as assessed by body temperature measurements with a rectal probe) and for bleeding [as assessed by levels of hematocrit (Ht)]. For determination of Ht, blood samples were collected into heparinized microhematocrit capillary tubes, centrifuged for 3 min at 12,000 r.p.m. in a microfuge and the percentage of packed erythrocytes was determined.

Platelet preparation and counting
Mice were bled under ether anesthesia from the retroorbital plexus. Blood was collected in a tube containing 10% (v/v) 7.5 U/ml heparin and platelet-rich plasma was obtained by centrifugation at 300 g for 10 min at room temperature. For determination of platelet counts, blood (20 µl) was obtained using siliconized microcapillaries and immediately diluted 1:100 in Unopette kits (Becton Dickinson, Heidelberg, Germany). The diluted blood sample was allowed to settle for 20 min in an improved Neubauer hemocytometer (Superior, Bad Mergentheim, Germany) and platelets were counted under a phase contrast microscope at x400 magnification.

Flow cytometry
Heparinized whole blood was diluted 1:30 with modified Tyrode’s–HEPES buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose and 1 mM MgCl2, pH 6.6) and left for 30 min at 37°C prior to stimulation. For determination of surface {alpha}IIbß3 levels, diluted whole blood was incubated with the fluorophore-conjugated mAb JON8 which binds to an epitope on the receptor different from that recognized by anti-{alpha}IIbß3 JON1, JON3 mAb used in experimental thrombocytopenia in vivo.

Electron microscopy
Tissue from spleen, liver and lung were harvested from antibody-treated male mice following cervical dislocation. Following the extraction of the organ tissue, they were immersion-fixed with 0.1 M cacodylate buffered 2% glutaraldehyde/2% paraformaldehyde for 4 h. Post-fixation was performed in 2% osmium tetroxide buffered at pH 7.3 with 0.1 M sodium cacodylate at 4°C over 2 h. Specimens were rinsed in cacodylate buffer 3 times, in 1% uranyl acetate and in 70% ethanol for 8 h. Dehydration was performed in a series of graded ethanol and specimens were then embedded in araldite. Semi-thin sections of plastic-embedded specimens were cut with a glass knife on a Reichert ultramicrotome and stained with methylene blue. Ultrathin sections (30–60 nm) were obtained with the help of a diamond knife on the microtome described above, placed on copper grids and examined with a Zeiss EM902A electron microscope (Zeiss, Oberkochen, Germany).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-{alpha}IIbß3 IgG-induced systemic reaction and bleeding, but not thrombocytopenia, is mediated by activating Fc{gamma}R
We previously generated panels of novel mAb recognizing various mouse platelet antigens and showed that antibodies against integrin {alpha}IIbß3 and GPIb{alpha} are by far the most pathogenic, resulting both in surface loss of antigen and clearance of platelets in mice (23). However, we also found that the pathogenicity of anti-{alpha}IIbß3 JON mAb is different to that induced by anti-GPIb{alpha} p0p mAb by triggering a further acute systemic reaction and bleeding. As summarized in Fig. 1, a bolus injection of 3 µg/g of purified JON1 or JON3 (anti-{alpha}IIbß3 rat mAb) induced hypothermia and significant decreases in Ht levels in C57BL/6 mice, whereas no such effects were observed with any other tested anti-platelet antibodies, including anti-GPIb{alpha} (Fig. 1B and C) (23). Moreover, we noted Fc-dependent ({alpha}IIbß3) and Fc-independent (GPIb{alpha}) events causing profound thrombocytopenia in anti-platelet IgG-treated mice (Fig. 1A) (23).



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Fig. 1. Different pathogenic effects of anti-{alpha}IIbß3 and anti-GPIb{alpha} mAb in mice. Groups of six C57BL/6 wild-type mice received a bolus injection of 3 µg/g rat IgG1 (circles), IgG2b (squares) or F(ab)2 fragments of the IgG2b (triangles) against the indicated platelet antigen in 200 µl sterile PBS i.v. (A) Platelets were counted at the indicated times, using an improved Neubauer hemocytometer. Results are shown as mean ± SD. (B) The mean values of the body temperature at the indicated time after injection are given ± SD. (C) Ht was determined at the indicated times. Results are shown as the mean Ht ± SD. The results shown are representative of three individual experiments.

 
As the pathogenic effects of anti-{alpha}IIbß3, but not anti-GPIb{alpha}, mAb are Fc dependent, we determined the role of activating Fc{gamma}R in platelet integrin {alpha}IIbß3-specific pathology using Fc{gamma}RIII–/– and FcR{gamma}–/– mice—the latter defective in activating Fc{gamma}RI and Fc{gamma}RIII (11). Anti-{alpha}IIbß3 JON antibodies induced a profound thrombocytopenia in C57BL/6 control animals, or Fc{gamma}RIII–/– and FcR{gamma}–/– mice, as documented by decreased platelet counts after i.v. injection (Fig. 2A). However, while the cytotoxic effects of the two different antibodies, JON1 (rat IgG2b) and JON3 (rat IgG1), were virtually identical in C57BL/6 wild-type mice, JON3 was less effective in both Fc{gamma}RIII–/– and FcR{gamma}–/– mice, suggesting isotype-specific differences in the pathogenicity of the two antibodies. This difference became more obvious when the systemic reaction and bleeding responses to these antibodies were analyzed in the different mouse strains. As shown in Fig. 2(B), C57BL/6 mice developed severe hypothermia within minutes upon i.v. injection of either JON1 or JON3. In contrast, in Fc{gamma}RIII–/– mice, only JON1, but not JON3, induced hypothermia, whereas FcR{gamma}–/– mice were completely protected from JON1 and JON3-induced hypothermia. A similar picture emerged when Ht were monitored in those animals. While both JON1 and JON3 induced a marked decrease in Ht in wild-type mice, only JON1, but not JON3, had an effect in Fc{gamma}RIII–/– mice (Fig. 2C). In FcR{gamma}–/– mice, neither JON1 nor JON3 had a significant effect on Ht. These findings suggest that anti-{alpha}IIbß3 antibodies elicit distinct pathogenic mechanisms for triggering thrombocytopenia or systemic reaction and bleeding, and only the latter depends on activating Fc{gamma}R. While rat IgG1 strictly requires Fc{gamma}RIII for the induction of these reactions, rat IgG2b appears to act through both, Fc{gamma}RI and Fc{gamma}RIII.



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Fig. 2. Pathogenic effects of anti-{alpha}IIbß3 mAb in Fc{gamma}RIII–/– and FcR{gamma}–/– mice. Groups of six mice received a bolus injection of 3 µg/g JON1 (IgG2b, squares) or JON3 (IgG1, circles) in 200 µl sterile PBS i.v. (A) Platelets were counted at the indicated times, using an improved Neubauer hemocytometer. Results are shown as mean ± SD. (B) The mean values of the body temperature at the indicated time after injection are given ± SD. (C) Ht was determined at the indicated times. Results are shown as the mean Ht ± SD. The results shown are representative of three individual experiments.

 
We have previously shown that repeated injections of low amounts of anti-{alpha}IIbß3 antibodies still induce a marked drop in platelet counts and bleeding, but no longer hypothermia (23). To investigate whether anti-{alpha}IIbß3 mAb-induced thrombocytopenia is the cause of bleeding complications, C57BL/6 and FcR{gamma}–/– mice were treated with 7 x 0.3 µg/g JON1 within 6 h. As shown in Fig. 3(A), such treatment resulted in severe thrombocytopenia in C57BL/6 mice and to a slightly lesser extent in FcR{gamma}–/– mice. However, while most of the control mice developed significant intestinal and s.c. bleeding, no such effects were observed in FcR{gamma}–/– mice despite their decreased platelet counts (see also Fig. 7C). The blood loss in C57BL/6 controls, but not in FcR{gamma}–/– mice, was confirmed when Ht was monitored in separate groups (Fig. 3B). Since the cytotoxic effects of anti-{alpha}IIbß3 mAb JON1 were slightly milder in the absence of the FcR{gamma} chain, it could not fully be excluded that the normal levels of Ht and the lack of bleeding in FcR{gamma}–/– mice were due to the hemostatic function of the few remaining platelets. To test this possibility, FcR{gamma}–/– mice first received anti-{alpha}IIbß3 mAb and after 6 h the remaining platelets were further depleted by injection of an anti-GPIb{alpha} mAb. As expected, this treatment resulted in a complete loss of platelets for ~4 days in FcR{gamma}–/– mice (Fig. 3C), but again the Ht levels remained virtually unchanged (Fig. 3D).



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Fig. 3. FcR{gamma}–/– mice resist anti-{alpha}IIbß3 IgG-induced bleeding complications. C57BL/6 controls (open circles) or FcR{gamma}–/– (filled circles) mice received 7 x 0.3 µg/g anti-{alpha}IIbß3 mAb within 6 h, each i.p. Platelet counts (A) and Ht (B) were determined at the indicated times. Results are shown as the mean platelet count or Ht ± SD for groups of six mice. (C and D) FcR{gamma}–/– mice received 3 µg/g anti-{alpha}IIbß3 JON1 mAb in 200 µl sterile PBS i.v. followed by 1.5 µg/g anti-GPIb{alpha} p0p4 mAb at t = 6 h. Platelet counts (C) and Ht (D) were determined at the indicated times. Results are shown as the mean platelet count or Ht ± SD for groups of six mice.

 



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Fig. 7. Increased anti-{alpha}IIbß3 IgG-induced bleeding and mortality in Fc{gamma}RII–/– mice. C57BL/6 controls (open circles) or Fc{gamma}RII–/– (filled circles) mice received 7 x 0.3 µg/g anti-{alpha}IIbß3 JON1 mAb, each i.p. Platelet counts (A) and Ht (B) were determined at the indicated times. Results are shown as the mean platelet count or Ht ± SD for groups of 10–12 mice. Death of a mouse is indicated by a cross. (C) Increased anti-{alpha}IIbß3 IgG-induced bleeding and resulting organ failure in Fc{gamma}RII–/– mice. Representative pictures from wild-type, FcR{gamma}–/– and Fc{gamma}RII–/– mice on day 3 after repeated injections of low amounts of JON1 mAb. Note the lack of bleeding in the FcR{gamma}–/– mouse, whereas the blood loss in Fc{gamma}RII–/– mice was significantly more severe than in the wild-type (WT) control, resulting in a pale appearance of the major organs, particularly liver and kidney. Such mice died from the marked blood loss and subsequent multi-organ failure.

 
Anti-{alpha}IIbß3 IgG-induced loss of platelet integrin {alpha}IIbß3 and subsequent IC formation in FcR{gamma}–/– mice
As recently suggested, the systemic reaction induced by anti-{alpha}IIbß3 antibodies may be based on the formation of IC composed of {alpha}IIbß3 and the antibody. This hypothesis was based on the observation that platelets in anti-{alpha}IIbß3-treated mice lost the integrin from the surface (23). To examine a possible involvement of Fc{gamma}R in this antibody-induced loss of {alpha}IIbß3 from the platelet surface, FcR{gamma}–/– and C57BL/6 control mice received 7 x 0.3 µg/g anti-{alpha}IIbß3 JON1 antibody and the circulating platelets were analyzed for the expression of {alpha}IIbß3 integrin at different time points after injection. In both mouse strains platelets had lost ~90% of their surface {alpha}IIbß3 (Fig. 4A) as well as surface-bound IgG (Fig. 4B) as soon as 6 h after antibody injection and the circulating platelet population remained {alpha}IIbß3-negative in all mice for at least 4 days.



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Fig. 4. Loss of surface {alpha}IIbß3–antibody complexes in FcR{gamma}–/– mice. C57BL/6 and FcR{gamma}–/– mice received 7 x 0.3 µg/g anti-{alpha}IIbß3 JON1mAb (rat IgG2b) within 6 h, each i.p. Flow cytometric detection of surface levels of {alpha}IIbß3 (A) and surface-bound JON1 (B) on platelets from control (open circles) or FcR{gamma}–/– (filled circles) at the indicated times after the first injection. Results are expressed as percent mean log fluorescence as compared to control platelets incubated with saturating amounts of JON1 in vitro for 30 min at 37°C for groups of six mice. Surface levels of {alpha}IIbß3 were determined with JON8–FITC. JON1 and JON8 recognize different epitopes on {alpha}IIbß3, and do not interfere with the binding of the other in vitro.

 
To study the process of {alpha}IIbß3 loss in vivo, wild-type and FcR{gamma}–/– mice received 3 µg/g gold (5 nm)-conjugated JON1 or control antibody i.v. and the organs were examined by electron microscopy at different time points (Fig. 5). In wild-type mice, injection of gold-conjugated JON1 induced the formation of gold particle-containing clusters of electron dense material in the close vicinity of platelets as detected 20 min after injection. These clusters were mainly found in spleen (Fig. 5A), liver and lungs, and showed a network-like formation of the electron-dense material. At 24 h after injection of the antibody, the gold particle-containing clusters were still found in these organs. At this time point, however, the density of the cluster network appeared higher when compared to the earlier time point (Fig. 5B). Interestingly, the gold particle-containing clusters were equally observed in FcR{gamma}–/– mice, confirming that the formation of these IC-like structures occurred independently of Fc{gamma}RI and Fc{gamma}RIII (not shown). In addition to the gold particle-containing clusters in the vicinity of platelets, gold particles were also found within macrophages of wild-type, but not FcR{gamma}-deficient, mice 60 min after injection, suggesting that these immune cells had phagocytosed parts of the formed IC by Fc{gamma}R-dependent mechanisms (not shown). Clustering of the gold-conjugated control antibody could not be observed in the organs of wild-type or FcR{gamma} chain-deficient mice. These results suggest that anti-{alpha}IIbß3 antibodies can induce the formation of large IC in mice and indicate that these IC are recognized by Fc{gamma}R-bearing cells.



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Fig. 5. Electron microscopy of anti-{alpha}IIbß3 IgG-induced IC formation in vivo. Representative ultrastructural images of spleen from C57BL/6 wild-type mice treated with gold-conjugated JON1 for 20 min (A) or 24 h (B). (A and B) In addition to a platelet (p), clusters (asterisks) of electron-dense material without membrane covering are shown. (Inserts) At higher magnification homogenously distributed small gold particles (5 nm) are found between the network-like distributed electron-dense material. The bar represents 250 nm (insert: 50 nm).

 
Anti-{alpha}IIbß3 IgG-induced acute systemic reaction and bleeding results in enhanced mortality in Fc{gamma}RII–/– mice
It is known that Fc{gamma}RIII-dependent activation responses induced by the FcR{gamma} chain are counter-regulated by the inhibitory receptor Fc{gamma}RII when co-expressed on the same effector cells. This balance has been established in various murine disease models, and is highlighted by the increased susceptibility of Fc{gamma}RII–/– mice to the pathogenic effects of antibodies in models of systemic anaphylaxis (25) and IC-triggered inflammation in several organs (1719). To test the hypothesis that anti-{alpha}IIbß3 antibodies trigger an IC-like response, their pathogenic effects were examined in Fc{gamma}RII–/– mice. A bolus injection of 3 µg/g of anti-{alpha}IIbß3 mAb led to similar thrombocytopenia, but significantly more severe hypothermia and increased mortality, in Fc{gamma}RII–/– mice as compared to control mice (Fig. 6). This finding demonstrates that the FcR{gamma}-triggered systemic reaction of the anti-{alpha}IIbß3 IgG-induced pathology is counter-regulated by the inhibitory Fc{gamma}RII.



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Fig. 6. Anti-{alpha}IIbß3 IgG-induced systemic reaction is enhanced in Fc{gamma}RII–/– mice. C57BL/6 controls (open circles) or Fc{gamma}RII–/– (filled circles) mice received 3 µg/g anti-{alpha}IIbß3 JON1 mAb in 200 µl sterile PBS i.v. (A) The mean values of the body temperatures of nine mice per group at the indicated times after injection are given ± SD. Death of a mouse is indicated by a cross. (B) Platelet counts of wild-type (wt) and Fc{gamma}RII–/– mice were determined at t = 1 h, using an improved Neubauer hemocytometer. Results are shown as the mean platelet count ± SD for groups of nine mice. Wild-type and Fc{gamma}RII–/– mice not receiving anti-{alpha}IIbß3 JON1 mAb all have comparable platelet counts (control).

 
To further define the biological significance of this regulation in the development of bleeding complications, C57BL/6 control and Fc{gamma}RII–/– mice received repeated injections of 7 x 0.3 µg/g JON1. This treatment produced no hypothermia (not shown) and platelet counts dropped to almost zero after 6 h in both strains of mice (Fig. 7A). Strikingly, however, Fc{gamma}RII–/– mice developed significantly more severe bleeding than the C57BL/6 controls, and 75% of the mutant mice (nine out of 12) died within 4 days after the treatment from severe bleeding (Fig. 7B) and subsequent multiorgan failure (Fig. 7C).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we have examined the specific contribution of Fc{gamma}R in experimental anti-{alpha}IIbß3 IgG-induced thrombocytopenia, systemic reaction and bleeding using knockout mice deficient in either activating or inhibitory Fc{gamma}R. Activating Fc{gamma}R are considered to play an essential role in cellular destruction integral to the pathogenesis of several immune cytopenias and this has been confirmed in a passive model of AIHA (26) where Fc{gamma}R-deficient mice resist cytotoxicity as a consequence of ineffective phagocytosis of erythrocytes due to the absence of activating Fc{gamma}R on mononuclear phagocytes (7,27,28). Initial studies in the (NZW x BXSB) F1 model of autoimmune thrombocytopenia suggested similar Fc{gamma}R-dependent mechanisms in the immune clearance of platelets (6,7). More recently, however, it has been shown that the antigenic specificity of anti-platelet antibodies when directed against different mouse platelet antigens, including GPIb{alpha} and integrin {alpha}IIbß3, is of critical importance, mediating their pathogenic effects through Fc-dependent ({alpha}IIbß3) or Fc-independent (GPIb{alpha}) mechanisms (23). Surprisingly, we found that anti-{alpha}IIbß3 IgG-induced thrombocytopenia, which is Fc dependent (Fig. 1) (23), is not abolished in mice lacking the activating Fc{gamma}RIII or the common FcR{gamma} chain (Fig. 2), indicating a contribution of additional effectors like complement in this Fc-mediated process. Preliminary experiments in C3 mutant mice show that anti-{alpha}IIbß3 IgG1 (JON3)- and IgG2b (JON1)-induced thrombocytopenia occurs independently of complement C3 activation (B. Nieswandt and J. E. Gessner, unpublished), and this may suggest redundant roles of Fc{gamma}R and complement in anti-{alpha}IIbß3 IgG Fc-induced platelet destruction. Studies in mice lacking both activating Fc{gamma}R and complement will be required to test whether complement provides an alternative pathway, as recently suggested in an antibody-dependent model of autoimmune vitiligo (29).

While anti-platelet antibodies directed against GPIb{alpha} (Fig. 1) or GPIb-IX, GPV, CD9 and linear epitopes on integrin ß3 (23) have mild to strong and irreversible effects on platelet counts, none of them induce significant bleeding in mice. This indicates that the clearance of circulating platelets alone may not be sufficient to account for bleeding complications. Importantly, only antibodies directed against conformational epitopes on the dominant platelet integrin, {alpha}IIbß3, can induce responses leading to systemic reaction and marked blood loss in mice (Fig. 1) (22,23). FcR{gamma} chain deficiency now reveals a role of activating Fc{gamma}R in the anti-{alpha}IIbß3 IgG Fc-induced processes of hypothermia and severe bleeding (Figs 2 and 3). Moreover, we observe IgG isotype dependency in this model using rat IgG1 and IgG2b subclasses in mice lacking Fc{gamma}RIII as compared to FcR{gamma}-deficient mice. Consistent with previous findings on mouse IgG isotypes (15,27), Fc{gamma}RIII mutant mice display IgG subclass-specific protection when induced by anti-{alpha}IIbß3 JON mAb of IgG1, but not IgG2b, isotypes (Fig. 2). This suggests that Fc{gamma}RIII, which is the principle activatory Fc receptor for mouse IgG1, is also specific for rat IgG1 with no role of Fc{gamma}RI (30). In contrast, rat IgG2b-induced effects are prevented in FcR{gamma}, but not Fc{gamma}RIII, mutant mice, thus showing that individual Fc{gamma}R interact differently with rat IgG isotypes in mediating platelet pathology (Fig. 2). Together, these findings suggest that anti-{alpha}IIbß3 antibodies may trigger activating Fc{gamma}R on immune effector cells in a way no other anti-platelet antibodies do by inducing the formation of antigen–antibody structures particularly efficient to be recognized by Fc{gamma}R (with relative affinities for Fc{gamma}RIII: rat IgG1 > rat IgG2b and Fc{gamma}RI: rat IgG2b > rat IgG1). However, it is important to stress that although we have previously shown similar pathogenic effects for a series of different anti-{alpha}IIbß3 antibodies (23), these results cannot be directly extrapolated to all antibodies to this receptor complex.

As part of the normal cellular function, platelets can release microparticles after activation by diverse stimuli and this phenomenon has now been recognized for most eukaryotic cells (31,32). HIV-1-related immune thrombocytopenia has been reported to be associated with circulating IC that consist of platelet membrane fragments and anti-GPIIIa IgG antibodies (33). Our finding that both FcR{gamma} chain-negative and -positive platelets have lost {alpha}IIbß3 integrin (GPIIbIIIa) from their surfaces suggests FcR{gamma}-independent IC formation (Fig. 4). Electron microscopic examinations using gold-conjugated anti-{alpha}IIbß3 antibodies demonstrate the formation of IC-like structures in the vicinity of platelets, mainly in spleen, liver and lung (Fig. 5). IC-like structures were equally found in wild-type and FcR{gamma} chain mutant mice, suggesting that activating Fc{gamma}R act downstream of this process. This hypothesis is supported by the finding that gold clusters were present in macrophages of wild-type, but not FcR{gamma} mutant mice, which strongly indicates that the complexes are recognized by Fc{gamma}R expressed on these cells.

It is well known that IC can activate immune cells, including macrophages and mast cells, by Fc{gamma}R-dependent mechanisms, thereby eliciting an acute inflammatory response (34,35). Furthermore, IC-dependent activation responses mediated by Fc{gamma}RIII and the FcR{gamma} chain are regulated by the inhibitory Fc{gamma}RII (1719). A similar coupling is also found here in anti-{alpha}IIbß3 IgG-induced hypothermia and bleeding. The genetic deletion of Fc{gamma}RII results in enhanced sensitivity leading to uncontrolled acute systemic reaction and bleeding associated with profound mortality (Figs 6 and 7). These findings demonstrate the balance between Fc{gamma}RII inhibition and FcR{gamma}-triggered activation on effector cells as the major immunoregulatory event in the {alpha}IIbß3-specific pathology in mice.

Taken together, the results presented here suggest the existence of distinct pathogenic mechanisms that may determine the severity of hemorrhage in thrombocytopenia in mice. At least two different events are required for bleeding, one of which involves {alpha}IIbß3 integrin and activating Fc{gamma}R. IgG opsonization of platelets and their subsequent destruction represents the initial step in thrombocytopenia (20). However, platelet clearance appears not strictly dependent on activating Fc{gamma}R and does not necessarily lead to bleeding complications in mice. Platelet destruction results in severe bleeding when integrin {alpha}IIbß3 is the target of the pathogenic antibody. Targeting of {alpha}IIbß3 by anti-{alpha}IIbß3 IgG antibodies induces the formation of IC efficient to trigger systemic reaction through engagement of activating Fc{gamma}R on effector cells. The combination of thrombocytopenia and FcR-dependent downstream events is critical for fatal hemorrhage in mice. In accordance, a more severe systemic reaction, hemorrhage and profound mortality occurs as a consequence of changes in the balance of activating Fc{gamma}R and the inhibitory Fc{gamma}RII.


    Acknowledgements
 
We are grateful to K. Rackebrandt for excellent technical assistance, J. Bouten for expert assistance in the preparation of the photographs, M. Mörgelin for help with the gold conjugation of antibodies, members of our labs for helpful comments and discussions on the manuscript, and U. Barnfred for constant support throughout the study. This work was supported in part by grants of the Deutsche Forschungsgemeinschaft (DFG) to B. N. (NI556/2-1) and J. E. G. (Ge892/7-2). B. N. is a Heisenberg Fellow of the DFG.


    Abbreviations
 
AIHA—autoimmune hemolytic anemia

Fc{gamma}R—Fc receptor for IgG

FcR{gamma}{gamma} chain of activating Fc{gamma}RI and Fc{gamma}RIII receptors

Ht—hematocrit

IC—immune complex

ITP—immune thrombocytopenic purpura


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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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