In-vitro inhibition of antiplatelet autoantibodies by intravenous immunoglobulins and Rh immunoglobulins.YS Mehta, SS Badakere
Institute of Immunohematology (ICMR), KEM Hospital, Parel, Mumbai.
Autoimmune thrombocytopenia (AITP) is caused by autoantibodies to platelet glycoprotein antigens. Intravenous immunoglobulin (i.v.IgG) and Rh immunoglobulin infusions have found great significance in the treatment of AITP patients not responding to corticosteroids and other modes of therapy. In our study, it was observed that immunoglobulins (i.v.IgG & Rh), and their Fab fragments inhibited the binding of antiplatelet autoantibodies to normal platelets, from 15.8 to 90.7% and 25.6 to 90.08% respectively; whereas, their Fc portion did not show any inhibition. The presence of specific anti-idiotypic antibodies to antiplatelet autoantibodies was established by using monoclonal antibodies to Glycoprotein IIb/IIa and Glycoprotein Ib/IX, as the specific idiotype source. The i.v.IgG and Rh immunoglobulin products reacted with the monoclonal antibodies, only through their Fab and not through the Fc portions, thereby confirming its specific anti-idiotype activity.
Keywords: Antibodies, Anti-Idiotypic, immunology,Antibodies, Monoclonal, immunology,Autoantibodies, drug effects,Blood Platelets, immunology,Chronic Disease, Drug Evaluation, Preclinical, Human, Immunoglobulins, Fab, therapeutic use,Immunoglobulins, Intravenous, therapeutic use,Platelet Glycoprotein GPIIb-IIIa Complex, immunology,Platelet Glycoprotein GPIb-IX Complex, immunology,Purpura, Thrombocytopenic, Idiopathic, blood,immunology,therapy,Rho(D) Immune Globulin, therapeutic use,
Intravenous immunoglobulin, and Rh immunoglobulins, have been used as therapeutic agents for the treatment of immune thrombocytopenias. One of the mechanism of action of these preparations, that allows the opsonized platelets to evade phagocytosis by the macrophages, is blocking of the Fc receptors on the macrophages. The other mechanisms of action proposed for these products is the inhibition of the platelet autoantibodies binding to the platelet antigens by anti-idiotypic action of the IVIgG and Rh immunoglobulins. Thus, administration of these products has shown long lasting remissions, especially in some refractory cases.
This study was undertaken
1. to show that MgG and Rh immunoglobulins inhibit the binding of antiplatelet autoantibodies with normal washed 'O' group platelets,
2. to show that this inhibition was due to the Fab portion and not Fc mediated, and
3. to show that the inhibition of antiplatelet autoantibodies was due to a specific anti-idiotype effect, by the use of monocional antibodies to specific platelet glycoproteins Gp IIb/IIa and Gp Ib/IX.
In this study, we selected 5 patients of chronic AITP. AITP was diagnosed on the basis of a consistently low platelet count raised platelet associated immunoglobulins and normal or increased megakaryocytes in the bone marrow. The patients' sera when they first presented and had not been treated were taken for the inhibition assay.
Eight inhibitors (MgG and Rh immunoglobulin products) from 3 different sources, with various batches of the same product were used. PAIgG was estimated by the ELISA method described by Hegde et al.
Inhibition of Patients' and Control Sera Using IVIgG and Rh immunoglobulin Inhibitors:
All the inhibitors were so diluted as to have a protein concentration of 1 mg/ml in 0.01 M phosphate buffered saline (PBS). The patients' sera were then allowed to react with these diluted inhibitors at room temperature (RT) for 1 hour so as to allow the anti-idiotypic antibodies to react with the idiotypes (i.e. the antiplatelet autoantibodies). The reaction mixtures were then centrifuged at 3000 rpm for 10 mins at 4 deg. C to remove any precipitate formed. In one aliquot, normal saline was added so to serve as a negative control e.g. no inhibition. The reaction mixtures were then allowed to interact with washed normal donor 'O' group platelets suspended in PBS at RT for 1 hour. The platelets were then washed using PBS and the platelet associated IgG estimated. Normal donor sera were used as controls [Figure:1].
The inhibition was seen as a decrease in the PAIgG value of the inhibited sample as compared with the uninhibited control (normal saline). The percentage inhibition was calculated by taking each individuals uninhibited sera PAIgG value as 100%. [Figure:2]
Thus, % inhibition = 100 - 100 x PAIgG value (inhibited)
PAIgG value (uninhibited)
Detection of anti-idiotype antibodies to antiplatelet antibodies
Monoclonal antibodies to platelet glycoproteins GP IIb/IIa and GP Ib/IX were used as the specific idiotype source. 50 ?g of these monoclonal antibodies (5?g/ml) were coated onto the ELISA plates using 0.5 M carbonate bicarbonate buffer overnight at 4 deg. C. The excess was washed using normal saline tween 20 (NS. T20) and the wells blocked with 250 ?l 5% non fat dried milk powder (2 hours/37 deg. C). The MgG and Rh Immunoglobulin preparations and their papain digested products diluted to l mg/ml (50 ?l) were then reacted with the monoclonal antibodies (idiotypes) coated on the ELISA plate wells at RT for 2 hours. The excess was washed off with NS. T20 and the bound anti-idiotype antibodies were detected using anti-human globulin conjugated to alkaline phosphatase (FIT for 1 hour). After thoroughly washing out the excess, the substrate p-nitro phenyl phosphate l mg/ml in 0.05 M carbonate bicarbonate buffer pH 9.8 was added and the colour allowed to develop in the dark. The reaction was stopped with 3N Sodium Hydroxide 50 ?l and the optical density read at 405 nm on ELISA reader. While normal saline served as blank normal donors sera served as the negative control.
Preparation and Purification of Fab and Fc Fragments
The MgG and Rh immunoglobulins were digested using papain enzyme to prepare Fab and Fc fragments using the standard technique described by Porter et al. The purified fragments [Figure:3] were then used for inhibiting the antiplatelet autoantibodies and to analyse which fragment carried the anti-idiotype activity.
In this study we have established the presence of anti-idiotype antibody activity in the intravenous immunoglobulin and Rh immunoglobulin preparations by two different methods.
Firstly, when sera of patients of AITP, containing antiplatelet autoantibodies is reacted with a fixed number of washed 'O' group platelets it would interact to produce measurable antigen-antibody complexes depending on the amount of PAIgG. Addition of IVIgG or anti-D preparations, which supposedly contained anti-idiotype antibodies, to the above mixture would then show a variable decrease in the amount of auto-antibody than can still bind the platelets. This decrease caused in the levels of PAIgG [Figure:1] by the various inhibitors studied was found to vary from product to product and the percentage of inhibition was different for each AITP patients sera ranging from 15.8 to 90.7% [Figure:2]. There was no decrease in the PAIgG values for normal donor sera reacted with the same platelets. Also, it can be seen in [Figure:1] and [Figure:2] that gamma globulin precipitate from a single healthy donor serum used as control do not show any inhibition.
Also, in [Figure:3], it can be seen that inhibition of antiplatelet autoantibodies from binding to the platelets is mediated through the Fab fragments and not the Fc portion of these Ig preparations. The Fab fractions of the different immunoglobulin products inhibited the binding of antiplatelet antibodies to platelets, the inhibition ranging from 25.6 to 90.08% whereas no significant inhibition was seen by the Fc fraction of these products. Again the levels of inhibition was seen to vary from product and even amongst the different AITP patient's sera
Secondly, when we used monoclonal antiplatelet antibodies to GP IIb/IIa and GP lb/IX as specific source, to confirm the presence of anti-idiotype antibodies to antiplatelet autoantibodies, it was observed that all the preparations reacted with the monoclonal antibodies coated onto ELISA plates. This conclusively showed the presence of anti-idiotype antibodies. The papain digested fragments of these immunoglobulins showed that the Fab fragment of these immunoglobulins reacted with the monoclonal antibodies, whereas' no reaction occurred with the Fc fragment of the same products.
Thus, results of both the inhibition and the reaction with the specific monoclonal idiotype source confirm the presence of anti-iclicitype antibody activity to antiplatelet GP IIb/IIa and GP Ib/IX antibodies in most cases of AITP.
Recently glycoproteins GP IIb/IIa and lb/IX have been shown to be the antigenic determinants against which the autoantibodies have been shown to be directed in a majority of the cases of AITP. All antibody molecules have been shown to possess in their variable region idiotypic determinant which themselves act as antigens and lead to production of anti-iclicitype antibodies,. Anti-idiotype antibodies are considered to play an immunoregulatory role in controlling the immune response.
Several mechanisms of action have been suggested for the therapeutic action of IVIgG and Rh immunoglobulins,,, the commonest being the blocking of the Fc receptors on the macrophages modulation of the T-lymphocytes leading to depressed immunoglobulin production including production of platelet specific antibodies. It has also been shown that these preparations could have anti-idiotype antibodies to antiplatelet autoantibodies and thus might be probably inhibiting the opsonization of the platelets. Earlier studies, have shown the inhibition of monoclonal antiplatelet antibodies by the IVIgG and anti-D preparations and speculated the presence of anti-idiotype antibodies.
In our study, the inhibition of antiplatelet autoantibodies in the patients' sera from binding to ‘O’ group normal donor platelets was seen to vary from 15.8 to 90.7% [Figure:2] Furthermore, when the inhibition studies were carried out using the Fab and Fc fragments of the same preparations, it was seen that the Fab fraction of these immunoglobulins inhibited the binding from 25.6 to 90.08% [Figure:3] while the Fc fragments showed no significant inhibition. The percentage of inhibition as observed by Lisa Cardo et al, for IVIgIS (using monoclonal antibodies) was 55 to 60% and for Anti-D, it ranged from 50 to 80%, which is quite comparable to the inhibition recorded in our study. Gamma globulins precipitated from single normal donors and normal saline served as negative controls. Also, normal donor sera were reacted with normal donor 'O' group platelets with and without the inhibition and these did not show any significant changes in their binding to the platelets.
Monoclonal antibodies to GP IIb/IIa and GP Ib/IX were used as the specific source of the idiotype to test for anti-idiotype antibody activity in these preparations in our study. It was observed that the IVIgG and Rh immunoglobulin preparation and their Fab fragments reacted with the monoclonal antibodies coated onto ELISA plates while no reaction was seen with the Fc fragments. Normal controls did not show any such reaction. Thus, it can be interred from our study, that IVIgG and Rh immunoglobulins, probably, also act by inhibiting the antiplatelet autoantibodies from binding to their respective platelet antigens, and this could be one of the reasons for some of the complete remissions seen with the use of such products. Also, based on our findings we can say that anti-idiotype antibodies probably play a significant role in immunomodulation of AITP.