New model of transient strain-dependent autoimmune thrombocytopenia in mice immunized with rat platelets (1)
Mouse immunization with allogeneic platelets triggers the production of alloantibodies that recognize mostly donor platelet MHC antigens. Monoclonal anti-mouse platelet antibodies induced after immunization of rats with mouse platelet or endothelial antigens and monoclonal autoantibodies derived from (NZW × BXSB)F1 animals allow analysis of the mechanisms leading to thrombocytopenia, but not to the development or regulation of the autoimmune response.
CBA/Ht mice were immunized by weekly intraperitoneal injection, without adjuvant, of platelets isolated from Wistar rats. Platelet count showed a moderate decrease that reached a maximum 3 weeks after the first rat platelet administration. The thrombocytopenia developed progressively and was transient, with platelet counts returning to normal levels, usually after 5 weeks, despite continuous immunization.
immunized mice treated with clodronate-containing liposomes, but not with control PBS-containing liposomes, recovered normal platelet counts. This clearly showed that platelet phagocytosis was involved in the transient thrombocytopenia that followed immunization with rat platelets.
the presence of antibodies associated with the platelets of mice immunized with rat platelets when compared to platelets obtained from control animals (O.D. 1.07 +/- 0.04 for immunized mice vs 0.383 +/- 0.01 for control animals, p = 0.0159). the presence of antibodies at the surface of platelets obtained from immunized, but not from control, mice.
The pathogenicity of antiplatelet autoantibodies elicited by immunization of CBA/Ht mice with rat platelets was assessed by injecting them into normal recipients.
The platelet count also decreased in SJL/J mice at 3 weeks after the rat platelet immunization, but the difference was not statistically significant. In contrast, no thrombocytopenia occurred in BALB/c, DBA/2, and C57BL/6 mice. Binding of antibodies to normal mouse platelets differed drastically. No binding of antibodies eluted from BALB/C platelets could be detected either with BALB/ C or with CBA/Ht normal platelets. In contrast, antibodies eluted from CBAHt platelets bound similarly BALB/C and CBA/Ht platelets.
The ability to destroy antibody-coated platelets was first compared in these two strains by administration of an anti-CD41 mAb. This treatment resulted in a similar thrombocytopenia in CBA/Ht and BALB/c mice.
After separation on 10% acrylamide gel in nonreducing conditions, several rat platelet proteins, with apparent molecular weight of +/-22, 41, 104, and >230 kDa were bound by antibodies eluted from platelets of both CBA/Ht and BALB/C animals. A mouse platelet protein of approximately 140 to 150 kDa was recognized by antibodies eluted from CBA/ Ht, but not BALB/C, platelets. CBA/Ht mice recognized similarly a band of approximately 150 to 160 kDa on blots loaded with CBA/Ht and BALB/ C platelet proteins. It was possible to determine that the larger band of approximately 150 to 160 kDa that was recognized comigrates with CD42b (platelet glycoprotein Ib), whereas the +/-95- to 100-kDa band comigrates with CD61 (platelet glycoprotein IIIa).
The thrombocytopenia observed in immunized mice was caused by the antiplatelet autoantibodies. Although a substantial proportion of these autoantibodies were of the IgG2 subclasses, it may be postulated that their pathogenicity depends mainly on their antigenic specificity. Platelet glycoprotein IIIa (CD61), but not platelet glycoprotein IIb (CD41), also was recognized by autoantibodies elicited by immunization with rat platelets. A transient thrombocytopenia was observed after immu- nization with rat platelets of CBA/Ht (H-2k), 129/Sv (H-2b), and to a lesser extent SJL/J mice (H-2s), but not of BALB/ C (H-2d), C57BL/6 (H-2b), and DBA/2 (H-2d) animals.