Recipient humoral immunity against leukoreduced allogeneic platelets is suppressed by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase

Abstract

Leukoreduced allogeneic platelet transfusions have been previously shown to initially stimulate an in vitro cellular cytotoxicity and subsequently Induce the formation of immunoglobulin G (IgG) antidonor alloantibodies. To further characterize these responses and determine if they are related, recipient BALB/c H-2d mice were treated with aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and transfused weekly with 2 x 10(8) C57BL/6 H2b platelets. In control, non-AMG-treated mice, transfusion significantly (P < .01) increased serum levels of interferon-gamma (IFN-gamma) by day 1 posttransfusion (PT). IFN-gamma returned to pretransfusion levels by day 3 PT, and its production was not affected by AMG treatment. Serum interleukin-4 (IL-4), on the other hand, was undetectable before and during the transfusion protocol. By day 3 PT, recipient spleen cells could mediate in vitro anti-P815 (auto), anti-EL4 (allo), and anti-R1.1 (third-party MHC) cytotoxicity, and these responses were maximal by day 7 PT. Concurrently, a significant reduction in the vitro ability of recipient splenocytes to respond to Concanavalin A (ConA) was observed; this was not seen with lipopolysaccharide (LPS) stimulation. Elevated levels of NO2- were found in the ConA culture supernatants from transfused mice at day 3 PT. Serum antidonor alloantibodies were detected by the fifth platelet transfusion. AMG treatment of recipient mice significantly inhibited the transfusion. Induced cytotoxicity and ConA-stimulated NO2- production, and restored ConA-induced proliferation to normal levels. AMG appeared to selectively inhibit platelet-induced alloantibody production in that it did not affect antibody production induced by transfusions with 10(5) allogeneic leukocytes or by immunization with a foreign protein antigen, human gamma globulin, in adjuvant therapy. These results indicate that an in vivo AMG-sensitive mechanism is essential for recipients to initiate a humoral IgG immune response against allogeneic platelets.

CTLA4‐Ig prevents alloantibody production and BMT rejection in response to platelet transfusions in mice

“…CD4 + T-cell activation correlates with alloantibody responses to transfused PLTs. 23,25,27,28 In addition, depletion of CD4 + T cells abrogates alloantibody induction by LR-PLT transfusion. 29 Finally, CD4 + T cells are required for the induction of T cells involved in BMT rejection across mHA barriers.…”

“…LR-PLT products were harvested as previously described, 11 and transfusion dose and schedule were based on existing protocols in mice. [23][24][25] Briefly, donor PLT-rich plasma was isolated by centrifugation and was passed over a neonatal high-efficiency leukoreduction filter (Purecell PL, Pall Corp., Port Washington, NY). LR-PLT transfusions contained 1 ¥ 10 8 total PLTs and were transfused through the tail vein.…”

A novel immunosuppressive pathway involving peroxynitrate‐mediated nitration of platelet antigens within antigen‐presenting cells

“…Previous reports have suggested that the production of NO by iNOS within recipient APCs plays a significant role. 14,15 The role of iNOS/NO in the immune system comprises both regulatory and effector functions. 29 The former includes immunosuppressive effects (e.g., inhibition of lymphocyte proliferation) and the latter includes immunopathologic effects (e.g., tissue destruction) and immunoprotective activities (e.g., killing of microbial pathogens).…”

“…On the other hand, although it cannot be ruled out that the additional lack of hydrogen peroxide production in the GP91 PHOX KO mice may also play a role in the above processes, PLT transfusions are associated with high interferon responses. 14,15 When interferon is present, iNOS is significantly activated and generates large quantities of NO, and because of the high affinity of NO with superoxide, the ONOOpathway may be preferentially activated. [36][37][38] When NO reacts spontaneously with superoxide to form ONOO -, the latter is very efficient at nitrating and nitrosylating tyrosine (tyr-NO2) and cysteine (cys-SNO) residues, respectively.…”

“…12,13 This indirect IgG response could be completely suppressed by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), suggesting that the recipient's innate immune mechanisms were critical for stimulating PLT antibodies. 14,15 It was further shown that NO production within recipient APCs was stimulated by the donor PLT's ability to interact with recipient NK cells early after transfusion and induce them to secrete interferon gamma which subsequently activated iNOS. 16 Taken together, it appears that NO may act as a central mediator within APCs that determines the recipient's ability to respond to PLT transfusions; however, the mechanisms of how this reactive nitrogenous species regulates PLT alloantibody development remains unclear.…”