A Preliminary Study On Induction Of Immune Tolerance By Oral Administration Of Platelet Antigen In An Experimental Model Of Immune Thrombocytopenia

Idiopathic thrombocytopenic purpura(ITP),also referred to as autoimmune thrombocytopenic purpura(AITP),is a relatively common cause of abnormal bleeding.It is an organ-specific autoimmune disorder associated with failure of immune tolerance and disturbance of humoral and cellular immunity resulting in production of anti-platelet autoantibodies or stimulation of cytotoxic T lymphocytes to enhance platelet clearance and/or impair platelet production.Therapy remains opportunistic and empirical.Approximately 25%-30%of patients are characterized as refractory when they fail to respond to standard-dose corticosteroids and splenectomy or require unacceptably high doses of corticosteroids to maintain a safe platelet count. Alternative therapies,such as immunosuppressant and bone marrow transplantation, often have potential toxicities and imperfect effects.Therefore,a more efficient and less harmful approach for this difficult-to-treat disorder would be a considerable advance.In principle,autoimmune disorders arise because of the failure to eliminate or deactivate autoreactive lymphocytes,which is reflected in a deficiency of central and/or peripheral tolerance induction mechanisms.Thus,purging autoreactive lymphocytes from the immune repertoire or restoring the working of immune tolerance is an option for controlling the onset and development of autoimmune disorders.Oral tolerance(OT)is the immunologic mechanism by which the mucosal immune system maintains unresponsiveness to the myriad of antigens in the mucosal environment which might otherwise induce untoward immune responses.OT is a potent way of inducing immune tolerance to specific antigen involved in autoimmune diseases.In the last 20 years,OT has been already successfully tested in a number of experimental models of autoimmune diseases including experimental autoimmune encephalomyelitis(EAE),collagen and adjuvant-induced arthritis(CIA,AA), diabetes in the non-obese diabetic(NOD)mouse,experimental autoimmune uveitis (EAU),and experimental autoimmune myasthenia gravis(EAMG).The mechanisms of OT are not completely defined but are thought to involve two primary ways:low doses of antigen favor the induction of active suppression mediated by regulatory T (Treg)cells,including CD4⁺CD25^highTr cells,CD8⁺CD28^- Ts cells,NKT cells andγδT cells,whereas high doses of antigen favor either clonal deletion or anergy of specific T cells.Based on the long history of OT and the safety of the approach,which generally void of the side effects associated with conventional immunosuppression due to Ag-specific mechanism of action,human trials have been initiated in multiple sclerosis(MS),rheumatoid arthritis(RA),autoimmune uveitis and typeâ… diabetes. Positive results have been observed in phaseâ…¡trials and new trials are underway. Although clinical efficacy resulting in an approved drug hasn't yet been completely demonstrated,as well as many areas of this strategy remain to be investigated such as the formulation of the antigen,what can be concluded from these initial trials is that there has been no systemic toxicity or exacerbation of disease and results in humans have paralleled in many respects what has been observed in animals.On the basis of these findings,ITP,classed as an organ-specific autoimmune disease,appears to be an appropriate candidate for an oral tolerance study.A preliminary study of oral tolerance in ITP using autologous platelets has been carried out.Despite suffering from these drawbacks including difficulties of collection of autologous platelets and limitations on the storage of platelets,potential advantages were more attractive such as the readily accessible autoantigen and objectively assessment(platelet count).Furthermore,recommendations have been brought forward on the studies of oral immune tolerance induction using allogeneic platelets, cultured autologous platelets or purified GPâ…¡b-â…¢a.An ideal experimental model bearing much resemblance to the course and pathophysiology of human ITP could serve as a potential tool for the research of ITR In the past decades,several experimental models of immune thrombocytopenia have been developed,such as autoimmune thrombocytopenic purpura-Prone(NZW×BXSB) F1 mice.Our study is to develop an ideal animal model that simulate human ITP based on the foreign experience and to evluate the effect of oral administration of platelet antigens on this experimental model.Partâ… .Development of a murine model of immune thromboeytopeniaObjective:To develop an ideal animal model that simulate human ITP.Methods:CBA/CaJ mice were immunized by weekly intraperitoneal injection, without adjuvant,of platelets isolated from Wistar rats.The platelet count and mean platelet volume(MPV)were weekly measured by automated cytometer.We also detected the plasma levels of IFN-γ,IL-2,IL-4,IL-10 by enzyme-linked immunosorbent assay(ELISA).Presence of platelet-associated immunoglobulin G (PAIgG)was analyzed by flow cytometry(FCM).Anti-platelet reactivity was determined by in vitro lymphocyte proliferation assay.In addition,bone marrow examination was performed,in order to observe the morphocytology of megakaryocytes.Results:After the first rat platelet administration,platelet count of the mice showed a significant decrease that reached a maximum 8 weeks(P＜0.01).At this time, significant differences in both MPV and PAIgG were observed between immunized mice and control animals inoculated with saline(P＜0.05).The plasma levels of IL-2, IFN-γ,IL-4 and IL-10 were found to be significantly increased in immunized mice, compared to control animals inoculated with saline(P＜0.05).Additionally,bone marrow examination revealed normal or only slightly increased numbers of megakaryocytes with no dysplastic features on light microscopy.Conclusions:A murine model bearing much resemblance to the course and pathophysiology of human ITP could serve as a potential tool for the research of ITP.Partâ…¡.Expression of murine GPIbα(CD42b)GST fusion proteins and its activity to induce oral tolerance in the murine model of immune thrombocytopeniaObjective:To assess the potential of oral administration of recombinant platelet antigens to be a safe and efficacious means to prevent or treat thrombocytopenia in the experimental model of immune thrombocytopenia.Methods:Four different portions of murine GPIbαwere expressed as recombinant glutathione S-transferase(GST)fusion proteins in the bacterial expression plasmid vector,pGEX-6P-1.Specificity of the GST-fusion proteins was confirmed on immunoblots probed with goat anti-GST polyclonal antibodies and rat anti-mouse GPIbαmonoclonal antibody.Purified preparations were obtained through affinity chromatography.For induction of oral tolerance,mice were divided into different groups including blank control,vehicle control,and tolerogen group.The mice in the tolerogen group were given consecutive oral doses of recombinant GPIbα-GST fusion proteins,at a low(rGP[L])or middle(rGP[M])or high(rGP[H])dosage,10 days before the first immunization.The platelet count,MPV,PAIgG were detected, respectively.Besides the measurement of plasma levels of IFN-y,IL-2,IL-4,IL-10, we determined the plasma level of TGF-]3.Additionally,the proportion between CD4⁺CD25^highTreg cells and CD4⁺T cells in periphery blood,spleen and mesenteric lymph node were analyzed by FCM.Results:All recombinant fusion proteins were successfully purified and used as oral tolerogen.After the oral administration of recombinant GPIbα-GST fusion proteins, the development of thrombocytopenia was postponed and the abnormalities of MPV and PAIgG were corrected,respectively.The results of measurement of CD4⁺CD25^highregulatory T cells demonstrated that in comparison with blank control, %CD4⁺CD25^high/CD4⁺ was significantly decreased in vehicle control(P＜0.05), while those in tolerogen group—rGP[H]was increased in comparison with vehicle control,however,which was still lower than blank control.Further study indicated that imbalance of Thl and Th2 in the experimental model was corrected and the production of TGF-B was enhanced in the tolerogen group.Conclusions:The oral administration of specific autoantigens,namely GPIbα,could ameliorate the course of ITP in this experimental model.Mechanisms of this action presumedly attribute to the regulatory cell-driven tolerance,resulting in the correction of immune deviation.These findings provide new insights into therapeutic strategies for ITP.