Correspondence to: Simon Karpatkin, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel:212-263-5609 Fax:212-263-0695 E-mail:simon.karpatkin{at}med.nyu.edu.
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Abstract |
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Patients with human immunodeficiency virus 1associated immunological thrombocytopenia (HIV-1ITP) have markedly elevated platelet-bound immunoglobulin (Ig)G, IgM, and C3C4, as well as serum circulating immune complexes (CICs) composed of the same. Affinity purification of IgGs from their CICs with fixed platelets reveals high-affinity antibody (Ab) against platelet glycoprotein (GP)IIIa 4966, which correlates inversely with their platelet count. However, sera from these patients have little to no anti-GPIIIa activity. To investigate this, we assayed serum, purified serum IgG, and CIC-Ig from these patients. This revealed ~150-fold greater Ab activity in purified serum IgG, and ~4,000-fold greater reactivity in CIC-IgG. This was shown to be associated with the presence of antiidiotype Ab2 (both IgG and IgM) sequestered in the CIC-IgG. The IgM antiidiotype was predominantly blocking Ab, as demonstrated by specificity for F(ab')2 fragments of antiGPIIIa 4966 of HIV-1ITP patients and inhibition of reactivity with peptide GPIIIa 4966, not with a control peptide. The IgM antiidiotype was not polyreactive. Similar measurements were made in nonthrombocytopenic HIV-1infected patients. Their serum reactivity was not measurable, but serum Ig and CIC-IgG against platelet GPIIIa 4966 was present, although considerably lower than that found in HIV-1ITP patients (26- and 35-fold lower, respectively). In addition, their IgM antiidiotype reactivity was 12-fold greater than that found in HIV-1ITP patients. The IgM antiidiotype Ab titer of both cohorts correlated with in vivo platelet count (r = 0.7, P = 0.0001, n = 32). To test the in vivo effectiveness of the IgM antiidiotype, thrombocytopenia was induced in mice with 25 µg of affinity-purified antiGPIIIa 4966 (mouse GPIIIa has 83% homology with human GPIIIa and Fc receptors for human IgG1). Maximum effect was obtained at 46 h after intraperitoneal injection into Balb/c mice with a platelet count of ~30% baseline value. Preincubation of the anti-GPIIIa Ab with control IgM at molar ratios of IgM/IgG of 1:7 before intraperitoneal injection had no effect on the in vivo platelet count, whereas preincubation with patient IgM antiidiotype improved the platelet count to 5080% of normal. Thrombocytopenia could be reversed after addition of IgM antiidiotype 4 h after induction of thrombocytopenia. Thus, CICs of HIV-1infected patients contain IgM antiidiotype Ab against anti-GPIIIa, which appears to regulate their serum reactivity in vitro and their level of thrombocytopenia in vivo.
Key Words: platelet, HIV, autoimmunity, antiidiotype antibody, AIDS
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Introduction |
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Immunological thrombocytopenia is a common complication of HIV-1 infection. The incidence in HIV-1infected patients of HIV-1associated immunological thrombocytopenia (HIV-1-ITP)1 is 021% at onset and increases to 30% or more with the development of AIDS (1) (2). Kinetic data on platelet survival strongly suggest that early-onset HIV-1ITP is secondary to increased peripheral destruction of platelets, whereas patients with AIDS are more likely to have decreased platelet production (3). Patients with early-onset HIV-1ITP have a thrombocytopenic disorder that is clinically indistinguishable from classic autoimmune thrombocytopenia (ATP), seen predominantly in females (4) (5) (6) (7). However, HIV-1ITP is different from classic ATP with respect to the predominant male incidence and the markedly elevated platelet-associated IgG, IgM, and C3C4, as well as presence of circulating serum immune complexes (CICs) composed of the same (5) (6). These complexes contain anti-F(ab')2 Abs (8) as well as HIV-1related Abs (9) (10). Affinity purification of IgGs from their CICs with platelets has revealed high-affinity IgG1 Ab against the platelet integrin glycoprotein (GP)IIIa peptide 4966 (11) (12). This serum anti-GPIIIa Ab correlates inversely with platelet count (r = 0.71; reference (12)) and induces severe thrombocytopenia in mice (12), which can be prevented and/or reversed with GPIIIa 4966 peptide (reference (12); mouse GPIIIa is 83% homologous with human GPIIIa, and macrophages have Fc receptors for human IgG1).
However, we have recently observed that sera from HIV-1ITP patients have considerably less antiGPIIIa 4966 reactivity compared with ~150-fold greater reactivity in purified IgG from serum and ~4,000-fold greater reactivity sequestered in their serum CICs. This suggested the possibility of blocking or antiidiotype Ab against anti-GPIIIa in these patients.
This report documents the presence of blocking IgM antiidiotype antibody (Ab2ß and/or Ab2) versus antiGPIIIa 4966 in these patients, which correlates with their platelet count (r = 0.7, P = 0.001, n = 32) and reverses in vivo induced thrombocytopenia in mice.
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Materials and Methods |
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Population.
The population consists of 37 early-onset HIV-1infected patients without AIDS (19 homosexuals and 18 drug abusers). 22 were thrombocytopenic, and 15 had normal platelet counts. Five control sera were obtained from healthy laboratory personnel. Seven sera were obtained from classic ATP patients.
Purified IgG.
IgG was prepared from serum by ion-exchange chromatography (13).
F(ab')2.
F(ab')2 fragments were prepared from purified IgG by pepsin digestion as described (13), and were shown to be free of Fc fragments by SDS-PAGE as well as ELISA (13).
Immune Complexes.
Immune complexes (ICs) were prepared from serum by polyethylene glycol precipitation (5). Precipitates were dissolved in one fifth of their serum volume in 0.01 M PBS, pH 7.4.
Isolation of IgG and IgM from ICs.
IgG and IgM were isolated and purified as described (11). In brief, polyethylene glycol (PEG)-ICs were applied to a staphylococcal protein A affinity column (Sigma-Aldrich). The bound complex was washed with PBS and eluted with 0.1 M glycine buffer, pH 2.5. The eluted material was applied to an acidified sephadex G-200 gel filtration column (Amersham Pharmacia Biotech) preequilibrated with the same elution buffer. Effluents of the IgG peak were isolated, neutralized, dialyzed against PBS, and applied to a rabbit anti-IgM affinity column (ICN Pharmaceuticals, Inc.) prepared from Affi-Gel 10 (Bio-Rad). The flow-through material was free of contaminating IgM by immunoblot and ELISA. Effluents of the IgM peak were isolated, neutralized, dialyzed against PBS, and applied to an antiFc receptor affinity column to remove rheumatoid factor. (Fc fragments were prepared by papain digestion (11) and affinity purified on a staphylococcal protein A column; the acid eluate was verified by SDS-PAGE and was coupled to Affi-Gel 10). The flow-through IgM was devoid of rheumatoid factor, as determined by inability to bind to a second Fc column.
Affinity Purification of Antiplatelet IgG.
Antiplatelet IgG was affinity purified with 108 platelets fixed with 2% paraformaldehyde for 2 h at room temperature, followed by overnight gentle rocking at 4°C, then acid elution and neutralization, as described (11). The IgG subclass, determined by radial immunodiffusion (The Binding Site), was IgG1 with both k and l light chains.
Affinity Purification of Antiplatelet GPIIIa 4966.
Peptide GPIIIa 4966, CAPESIEFPVSEARVLED (synthesized by Quality Controlled Biochemicals), was coupled to an affinity column with the heterobifunctional cross-linker sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate as recommended by the manufacturer (Pierce Chemical Co.; cross-links the resin with NH2-terminal cysteine of the peptide), and was incubated with 0.4 ml of affinity-purified IgG overnight at 4°C. The column was then washed, eluted at pH 2.5, and neutralized as described (12).
ELISA.
Antibody reactivity was measured by solid-phase ELISA (12) (13) using serial doubling dilutions of IgG or IgM on U-shaped polyvinyl microtitre plates (Curtin-Matheson Scientific) preincubated overnight at 4°C with 200 ng of peptide GPIIIa 4966 or F(ab')2 fragment of antiGPIIIa 4966 in PBS, and was blocked with 2.5% BSA in PBS. A minimum of two different F(ab')2 fragments were used for each experiment. The first Ab, used to detect IgG binding, was a 1:500 dilution of goat F(ab')2 antihuman IgG ( chain specific) coupled to alkaline phosphatase (Sigma-Aldrich). The second Ab, used to detect IgM binding, was a 1:1,000 dilution of goat F(ab')2 antihuman IgM (µ chain specific) coupled to alkaline phosphatase (ICN Pharmaceuticals, Inc.). Appropriate enzyme substrate was added, and color was read in an automated micotitre plate reader at 405 nm. In some experiments, bound antiGPIIIa 4966 was preincubated with GPIIIa 4966 peptide for 2 h at room temperature before testing for antiidiotype Ab binding.
IgM Ab Titer.
IgM Ab titer refers to the reciprocal of the lowest concentration of IgM Ab (µg/well) capable of binding to its antigen, determined by extrapolation of the linear portion of the binding curve to zero binding.
Induction of Thrombocytopenia in Mice with AntiGPIIIa 4966.
Human affinity-purified antiGPIIIa 4966 (25 µg) was injected intraperitoneally into BALB/c mice (Taconic Farms), and blood was drawn from the retroorbital sinus at various times. In some experiments, antiGPIIIa 4966 Ab was preincubated with either control IgM or antiidiotype IgM before intraperitoneal injection; in other experiments, control or antiidiotype IgM was given after 4 h of thrombocytopenia. Platelet counts were determined from 20 µl of blood drawn into Unopettes (no. 5855; Becton Dickinson) containing optimal anticoagulant concentration and diluent for quantitating platelet count by phase microscopy.
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Results |
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Fig 1 demonstrates a comparison of the relative binding reactivity of serum, serum IgG, and purified IC-IgG for peptide GPIIIa 4966 in a representative experiment of five different patients (Table 1, HIV-1ITP). 50% detection sensitivity for the respective Ab cohorts were ~125, 0.8, and 0.03 µg/well. Thus, serum IgG has ~150-fold greater reactivity than serum, and IC-IgG has approximately sevenfold greater reactivity than serum IgG (~4,000-fold greater than serum). Similar studies performed on sera of nonthrombocytopenic HIV-1infected patients also detected antiplatelet GPIIIa 4966 in their serum IgG and CIC-IgG, but at considerably lower levels (25- and 35-fold less, respectively; Table 1; HIV-1 controls). This suggested the possibility of blocking or antiidiotype Ab in serum.
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In contradistinction to HIV-1ITP patients, minimal to absent serum antiplatelet reactivity was noted in seven classic ATP patients, with no enhancement of reactivity noted with serum IgG or IC-IgG (data not shown).
Presence of IgG Antiidiotype Ab versus AntiGPIIIa 4966.
Fig 2 A demonstrates binding of purified IC-IgG Ab from five different HIV-1ITP patients to F(ab')2 fragments of affinity-purified antiGPIIIa 4966. 50% binding was observed at ~2 µg/ml. Similar results were obtained with a second F(ab')2 fragment (data not shown). No binding was obtained with the same five IC-IgG preparations against two different control F(ab')2 fragments (one of which is shown in Fig 2). No binding was obtained with five different control IC-IgG preparations (data not shown).
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Fig 3 A demonstrates poor to partial blocking of binding of IgG antiidiotype Ab to antiGPIIIa 4966 with peptide GPIIIa 4966. Thus, only 20% of Ab binding could be inhibited at a peptide/F(ab')2 molar ratio of 1,024:1, and therefore designated blocking (P < 0.05 for last three concentrations of peptide; one-tail Student's t test).
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Presence of IgM Antiidiotype Ab versus AntiGPIIIa 4966.
Fig 2 B demonstrates binding of IgM Ab to antiGPIIIa 4966. 50% binding was observed at ~1.25 µg/ml. Similar results were obtained with a second F(ab')2 fragment (data not shown). IgM antiidiotype specificity was next examined against five different control F(ab')2 fragments and four different antigens to determine whether this IgM could represent polyclonal germline IgM secreted by CD5+ B1 cells (14). No binding was obtained with 16 positively reacting IC-IgM preparations against five different control F(ab')2 fragments. One of these experiments is shown in Fig 2 B. No binding to antiGPIIIa 4966 was obtained with five different control IgM preparations made from control subject IC-IgM (Fig 2 B). No binding was obtained with four different proteins: ovalbumin, soybean trypsin inhibitor, thyroglobulin, or carbonic anhydrase (Fig 4).
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Fig 3 B demonstrates considerable blocking of binding of IgM antiidiotype Ab to antiGPIIIa 4966 with peptide GPIIIa 4966. Thus, 50% of Ab binding could be inhibited at a peptide/F(ab')2 molar ratio of ~1:6.4, and therefore be designated blocking antibody (Ab2ß and/or Ab2). No blocking was noted with irrelevant peptide CGYGPKKKRKVGG at a peptide/F(ab')2 ratio of 1,024:1. IgM antiidiotype Ab did not bind to peptide GPIIIa 4966 (five experiments, data not shown).
Fig 3 C demonstrates blocking of binding of antiGPIIIa 4966 to platelets by IgM antiidiotype, not by control IgM or IgG antiidiotype. Thus, 50% of Ab binding to platelets could be inhibited at an IgM/IgG molar ratio of ~1:10.
Correlation between IgM Antiidiotype Ab versus AntiGPIIIa 4966 and Patient's Platelet Count.
The above data suggested that the reason for the relatively weak serum antiGPIIIa 4966 reactivity with its Ag was because of the presence of IgM blocking antiidiotype Ab in its serum. We therefore reasoned that if this were true and pathophysiologically relevant, then a positive correlation should be obtained between the antiidiotype titer and the patient's platelet count. This proved to be the case. Fig 5 demonstrates such a correlation of r = 0.71 (P = 0.001, n = 32). Specific measurements and clinical data are described in Table 1, lanes 19. In these two cohorts, IgM antiidiotype Ab was 12-fold greater in nonthrombocytopenic patients. A similar log mean IgM antiidiotype Ab titer of 1:3,643 was found in 10 additional nonthrombocytopenic HIV-1 patients.
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IgM Antiidiotype versus AntiGPIIIa 4966 Reverses In Vivo Thrombocytopenia Induced in Mice by AntiGPIIIa 4966.
Our previous study demonstrated that human antiGPIIIa 4966 Ab could induce significant thrombocytopenia in mice, with nadir at 4 h when injected intraperitoneally (control IgG had no effect; reference (12)). We therefore tested the ability of the IgM antiidiotype Ab versus antiGPIIIa 4966 to reverse this effect in vivo. Again, this proved to be the case. Thus, Fig 6 A demonstrates a 70% drop in platelet count induced by 25 µg/ml of antiGPIIIa 4966, with reversal to 5080% of normal by preincubation with IgM antiidiotype/antiGPIIIa 4966 ratios of 1:7, respectively. Neither control IgM nor IgG-antiidiotype, at similar ratios, had any effect. To rule out the possibility that the IgM antiidiotype Ab was not operating through increased clearance of the antiplatelet Ab, experiments were also performed after induction of the thrombocytopenia at 4 h. Fig 6 B demonstrates reversal of thrombocytopenia with the IgM antiidiotype Ab, not with the IgG antiidiotype, thus confirming that the IgM antiidiotype was interfering with the binding of anti-GPIIIa to platelets.
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Discussion |
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These data clearly indicate the presence of IgG and IgM antiidiotype (Ab2) against antiGPIIIa 4966 in HIV-1ITP patients. Their absence in classic ATP patients suggests that the mechanism of thrombocytopenia is different in both autoimmune disorders. The antiidiotype in HIV-1ITP patients was both Ab2 as well as blocking (Ab2ß and/or Ab2
) for both isotypes, with blocking IgM Ab2 predominating over IgG Ab2. Several lines of evidence support the conclusion that blocking IgM Ab2 is responsible for the impaired serum reactivity of antiGPIIIa 4966: (a) purification of serum IgG and IC-IgG increased anti-GPIIIa reactivity ~150- and 4,000-fold, respectively; (b) IgM purified from PEG-IC bound to antiGPIIIa 4966 in a specific manner (no binding with control IgM); (c) IgM Ab could be blocked from binding to antiGPIIIa 4966 with the Ag for antiGPIIIa 49-66 (not with an irrelevant Ag); (d) antiGPIIIa 4966 could be blocked from binding to platelets with IgM antiidiotype, not with control IgM or IgG antiidiotype; and (e) in vivo thrombocytopenia induced in mice with antiGPIIIa 4966 could be reversed with purified IC-IgM, not with purified serum IgM or IC-IgG.
These data strongly suggest that the IgM antiidiotype as well as level of antiGPIIIa 4966 IgG Ab play a role in regulating early-onset autoimmune HIV-1ITP in vivo. A correlation of platelet count with IgM antiidiotype of r = 0.7 supports this suggestion, as do our previous observations on the presence of an inverse correlation between antiGPIIIa 4966 antibody and platelet count in early-onset HIV-1 infection (thrombocytopenic versus nonthrombocytopenic; reference (12)). It should be recognized that multiple factors are likely to regulate the platelet count in HIV-1ITP patients. These include platelet production, platelet survival, and relative phagocytic function of the reticuloendothelial system. It is therefore not surprising that the correlation coefficient is <1.
The presence of antiGPIIIa 4966 Ab in nonthrombocytopenic patients, albeit at 2635-fold lower reactivity than in HIV-1ITP patients, is of interest and suggests that low levels of reactivity may be present in most early-onset HIV-1infected patients. It is possible that this may represent molecular mimicry between antiHIV-1 Abs and platelet GPIIIa. Indeed, this has been reported for antiHIV-1gp120 Ab (15).
Our findings on the presence of blocking IgM antiidiotype in HIV-1ITP patients is reminiscent of previous observations on IgM Abs blocking natural polyreactive low-affinity Abs in mice as well as humans (16) (17) (18) (19). However, our antiGPIIIa 4966 autoantibody preparation is different, in that it is highly specific (11) and contains high-affinity Ab (Kd = 12 nM; reference (11)). In addition, the purified serum IgG of HIV-1ITP patients is 150-fold more reactive than serum, compared with the 35-fold greater reactivity reported for purified IgG blocking Ab in normal subjects (18). It is possible that antiGPIIIa 4966 was originally a polyreactive natural Ab that underwent somatic mutation and selective pressure by antigens (HIV-1), as has been suggested for the development of autoimmune disease (17). This is supported by the presence of lower affinity (Kd = 712 nM) Ab as well in our antiGPIIIa 4966 preparation (11). The same selective pressure could also apply for natural, polyreactive, low-affinity IgM "antiidiotype" Ab.
Alternatively, the pathogenic potential of low-affinity IgM Ab has recently been demonstrated in a study comparing monoclonal mouse IgM anti-RBC Ab with its IgG class-switch variant (20). In these studies, the RBC binding activity of the IgM Ab was 1,000 times that of its IgG class-switch variant and was related to its pentameric structure, which promoted binding, agglutination of RBCs, and hemolytic anemia. These data indicate that affinity maturation of autoantibodies may not be required for generation of autoantibodies capable of inducing clinical pathology. The same applies for the reactivity of the IgM antiidiotype of our study.
Although the role of antiidiotype Ab in the regulation of the immune response is controversial, a case can be made for its dysregulation in the pathophysiology of some autoimmune diseases: (a) patients with severe, uncontrolled SLE have high levels of anti-DNA Abs and low levels of anti-F(ab')2 Abs, whereas patients with quiescent disease have the reverse (21); (b) patients with systemic vasculitis have antiidiotype Abs against antimyeloperoxidase and antineutrophil cytoplasmic antigen, with rise in antiidiotype titer as disease activity subsides (22) (23); and (c) a hemophiliac patient with a serious antifactor VIII (antihemophilic factor) inhibitor developed antiidiotype Ab against antifactor VIII, which coincided with recovery and the disappearance of the inhibitor (24). A case for antiidiotype dysregulation is indirectly supported by other observations: (a) patients with thyroid autoimmunity have naturally occurring antiidiotype IgM Ab against antimicrosomal Abs (25); (b) patients with myasthenia gravis have naturally occurring antiidiotype Abs against acetylcholine receptor Ab (26); and (c) intravenous -globulin infusions containing "antiidiotype" Ab are often effective in the treatment of patients with autoimmune thyroid disease (25), systemic vasculitis (22) (23), myasthenia gravis (27), and kawasaki syndrome (28).
Our results contribute to and extend these observations in a more definitive manner in HIV-1ITP. Our observations demonstrate the presence of high-affinity antiGPIIIa 4966 Ab (11) (12), specific blocking IgM antiidiotype Ab, a positive correlation between IgM antiidiotype and platelet count, and most importantly, a reversal of in vivo antibody-induced thrombocytopenia with its antiidiotype. These data support the concept that dysregulation of antiidiotype Ab can play a role in the development of autoimmune disease in HIV-1ITP.
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Footnotes |
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1 Abbreviations used in this paper: ATP, autoimmune thrombocytopenia; CIC, circulating immune complex; GP, platelet glycoprotein; HIV-1ITP, immunological thrombocytopenia associated with HIV-1infection; IC, immune complex.
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Acknowledgements |
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This work was supported by National Institutes of Health grants HL-13336-26 and DA-04315-1A, and by the Dorothy and Seymour Weinstein Platelet Research Fund.
Submitted: 20 September 1999
Revised: 7 April 2000
Accepted: 14 April 2000
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References |
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