John Freedman

IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity.

Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody‐dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region‐reactive (anti‐idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet‐specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre‐established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti‐idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody‐sensitized red blood cells. F(ab′)2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti–idiotype interactions are not required.

p38 MAPK is activated but not necessary in porcine von Willebrand factor-dependent platelet activation

We have investigated the role of p38 mitogen‐activated protein kinase (MAPK) in von Willebrand factor (VWF)‐dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF‐dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1u2003min post‐addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S‐transferase‐MAPK activated protein kinase‐2. To determine if p38 MAPK was necessary for porcine VWF‐induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF‐induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580‐inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.

Monoclonal antibody‐mediated inhibition of the human HLA alloimmune response to platelet transfusion is antigen specific and independent of Fcγ receptor‐mediated immune suppression

Presensitization of donor platelets with allo‐specific immunoglobulin (Ig)G results in a diminished immune response against subsequent transfusions of platelets. To understand better the mechanism of how alloantibody presensitization results in a decreased alloimmune response, we have used murine monoclonal antibodies directed to polymorphic and non‐polymorphic regions of human leucocyte antigen (HLA) as well as platelet‐specific molecules. Here, we demonstrated that presensitization with anti‐human HLA class I antibodies, as well as β2‐microglobulin‐specific antibody, protected against alloantibody production to five subsequent untreated platelet challenges. Use of complement fixing, non‐fixing or F(ab′)2 fragments of HLA‐specific antibody also resulted in complete inhibition of alloantibody production. This protection was not seen when the platelets were presensitized with monoclonal antibodies to CD42a (GPIX), CD32 (low‐affinity IgG/Fcγ receptor) or murine IgG and was thus independent of B‐cell FcγRII‐mediated immune suppression. The inhibition observed was independent of HLA alloantigenic specificity as antibodies directed at the β2‐microglobulin portion of HLA class I were as effective as antibodies against any of the HLA‐α regions (either polymorphic or non‐polymorphic) of class I. This work demonstrates that monoclonal antibody‐mediated suppression of the human HLA alloimmune response to platelet transfusion is antigen specific and is independent of FcγRII‐mediated immune regulation, complement fixing or HLA alloantigenic specificity.

Antibody‐mediated inhibition of the human alloimmune response to platelet transfusion in Hu‐PBL‐SCID mice

Severe combined immune deficient (SCID) mice were engrafted with human (Hu) peripheral blood lymphocytes (PBL) from a previously alloimmunized donor and transfused with HLA‐mismatched platelets. We have previously shown this to be a useful model for platelet transfusion. These engrafted mice (Hu‐PBL‐SCID mice) produced high levels of alloantibody in response to standard platelet preparations. However, when the first platelet challenge was presensitized with anti‐HLA antibody and then transfused there was a marked reduction in the amount of alloantibody produced to five subsequent untreated platelet transfusions. Platelets pretreated with platelet‐specific anti‐HPA‐1a (PLA1) sera did not induce a decrease in the anti‐HLA alloantibody response. This demonstrated that platelet‐induced HLA alloimmunization can be blocked by anti‐HLA antibody‐sensitized cells.