The Role And Mechanism Of The Inhibition Effect Of Platelet Destruction By Anti-CD44 Antibody In ITP

Immune thrombocytopenia(ITP),a common bleeding disorder,used to be named as idiopathic thrombocytopenia purpura,is an autoimmune disorder in which both increased platelet destruction and insufficient platelet production are involved.Low platelet counts increase the risk for bleeding,which can lead to severe intracranial haemorrhage in 1-5%of patients.ITP patients many undergo long-term therapeutic regimens to manage platelet counts,and suffer a marked decrease in quality of life.ITP pathogenesis is very complicated.Studies of ITP pathogenesis show that the direct dissolution of antigen-specific antibodies mediated platelet destruction plays an important role in ITP patients,among whom the failure of immune tolerance leads to the production of autoantibodies specific to the glycoprotein ?b/???b/?a(75%)on platelet membrane.Besides there are also a small portion of autoantibodies binding to GP?a/?a?GP??GP? and GP?.Platelet destruction following autoantibody binding has generally been considered to occur in the spleen,through binding of the Fc portion of immunoglobulins on the platelet surface to Fc?R?a and Fc?R?a on tissue macrophages of the reticuloendothelial system,which leads to the decrease of platelet counts and survival period,and the increase of bleeding risk in ITP patients.In addition,cytotoxic T cells(CTL)mediated platelet destruction and an immune-mediated megakaryocyte maturation abnormality and abnormal apoptosis also result in decreased platelet production in ITP patients.Phagocytosis is a critical component of both innate and adaptive immunity and can play key roles in the first line of defense against invading microorganisms as well as in tissue homeostasis and remodeling.Dysregulation of phagocytosis can lead not only to impaired host immune responses to pathogens,but may also lead to alterations in immune tolerance and to the development of chronic inflammation and autoimmunity.The phagocytic process can be initiated by a wide range of surface receptors which often involve two main groups:non-opsonic receptors that are capable of direct recognition and binding of target particles,and opsonic receptors that recognize opsonins,such as Abs and complement.Of these opsonic receptors,Fc?receptors(Fc?Rs),which bind the Fc portion of IgG,and complement receptor 3(CR3),which binds the C3bi component of complement,are the best described.At present,recommended first-line treatments for ITP include immunosuppressive and immunomodulatory drugs(e.g.corticosteroid therapy,intravenous immunoglobulin therapy(IVIg)and anti-Rh immunoglobulin(D)treatment).All three agents mentioned above can competitively bind to Fc receptors on macrophages,block the binding of platelets and monocyte/macrophages,and prevent the anti-platelet antibody coated platelets retention in the spleen,reducing the platelet destruction by macrophages,inhibiting macrophage chemotaxis and the delayed hypersensitivity,improving blood capillary permeability,ameliorating symptoms of hemorrhage.However,these treatments may lead to serious side effects,such as glucocorticoids can lead to cortical hyper-function syndrome and osteoporosis.Intravenous immunoglobulin treatment has advantages like high IgG content and good stability in blood.Furthermore,IVIg can rapidly increase IgG level in vivo,platelet can begin to rise after 24h of administration,and up to peak in 1 week.However,the patients' response to treatment is temporary,after 3 to 4 weeks of treatment,the platelet count reduces to the pretreatment level.Only a few patients show persistence reaction.For those patients with poor therapeutic effect,the first choice of second line treatment is splenectomy.ITP has long course,easy to relapse.Some patients who lack response to the existing first-line or second-line therapy or relapse in a short term,may develop into persistent refractory ITP.CD44 is a widely expressed cell surface polymorphic glycoprotein in a number of tissues,including leucocytes,keratinocytes,chondrocytes,many epithelial cell types,and some endothelial and neural cells.CD44 is the cell surface receptors for extracellular matrix molecules like hyaluronic acid,fibronectin,collagen,and fibrin.CD44 plays an important role in many physiological and pathological processes that include cell-cell and cell-matrix interactions,cell migration,lymphocyte activation and extravasation,and presentation of growth factors,cytokines.Activation of CD44 has previously been shown to regulate the inflammatory response.For example,ligation of CD44 with short fragment HA can induce a number of pro-inflammatory cytokines.CD44 also directly regulates neutrophil migration in that CD44-deficient cells demonstrate impaired migration in vitro.CD44 interaction with HA can also regulate in vitro lymphocyte adhesion.There is increasing evidence to suggest that CD44 plays a pivotal role in autoimmune diseases and its expression is increased in synovial cells in patients with rheumatoid arthritis,which correlates with synovial inflammation.The administration of antibodies against CD44 can significantly reduce inflammatory processes in murine models of collagen-or proteoglycan-induced arthritis and experimental autoimmune encephalomyelitis.Abs to CD44 have been successfully used in the treatment of several animal models of autoimmune and inflammatory diseases,including experimental autoimmune encephalomyelitis,collagen-or Ab-induced arthritis,autoimmune diabetes,experimental autoimmune uveitis,experimental colitis,experimental pulmonary eosinophilia,and cutaneous delayed-type hypersensitivity.The mAb rat anti-human/mouse CD44,IM7,which recognizes an extracellular epitope on the CD44s,has been extensively used in many of these studies,and robust anti-inflammatory effects have been reported with the administration of the Ab in these disease models.In comparison,the mAbs KM81,KM201,and KM114 have only been shown to have limited anti-inflammatory activity in arthritis.In previous studies,we provided evidence that CD44 antibody KM114 ameliorates thrombocytopenia in SCID mice.Mice injected with anti-platelet antibody demonstrated significant thrombocytopenia compared to unmanipulated mice.Pretreatment of mice with either IVIg or KM114 protected mice against thrombocytopenia.This suggests that CD44 antibody can function similarly as IVIg and the rapid effects of this antibody are not primarily functioning via interaction with CD44 molecular on B or T cells in murine ITP models.These results provide a new direction for the treatment of ITP.In addition,IgG is purified from plasma,which has high costs and limited production.CD44 monoclonal antibody,in contrast,can save the cost of treatment for patients,have significant research value and great prospect.In this study,we detected the effect of CD44 monoclonal antibody pretreatment on phagocytosis of macrophage by both flow cytometry and confocal microscopy.Evidence suggest that anti-CD44 monoclonal antibody IM7 and KM114 can inhibit Fc?R mediated platelet phagocytosis in macrophages.Compared with KM114,same concentration of IM7 has better inhibitive influence on macrophage phagocytosis.In addition,data shows that this inhibition effect doesn't work on the binding step,we assume that anti-CD44 antibody may affect the internalization process during phagocytosis.However,only IM7 can inhibit the phagocytosis of red blood cells by macrophages.Objective:The broad anti-inflammatory properties of the CD44 mAb and our previous researches prompted us to investigate whether this antibody can inhibit macrophage phagocytosis.To assess the effect of anti-CD44 antibodies on FcyR-mediated phagocytosis,we selected two types of anti-CD44 antibodies that either proved to worked in ITP murine models,KM114 and IM7.First,we co-culture the antibody-coated/uncoated RBC/PLT with macrophages,to simulate the pathological process of ITP in vitro.Then,we pretreat the macrophages with IM7 or KM114,to evaluate their effects on phagocytosis under different isotypes and concentration of anti-CD44 mAbs.Furthermore,we check the binding abilities of different isotypes and concentration of anti-CD44 mAbs,to explore the underlying mechanism.Methods:1 Culture murine macrophage cell line RAW264.7 and human leukocyte monocyte cell line THP-1 with RPMI 1640 completed media.2 Stimulate the THP-1 cell with PMA overnight,to differentiate to macrophages.3 Collect peritoneal macrophage from wild type CD1 female mice.4 Collect blood from wild type CD1 female mice,isolate the RBCs and PLTs.Use Coulter Counter to count PLTs and flow cytometer to count RBCs.5 Opsonized RBCs and PLTs separately with TER-119 and Mwreg30,and co-culture RBCs/PLTs with macrophages for at 37°C for 30mins.6 Evaluated phagocytosis of TER-119 opsonized mouse RBC by RAW macrophages using microscopy.Quantification of phagocytosis was done by counting the number of internalized RBCs in 200-300 macrophages from each sample and the phagocytic index(PI)was calculated according to the following formula:PI =(total number of internalized RBCs/total number of counted macrophages)*100.7 Phagocytosis of Mwreg30 opsonized CMFDA labeled platelets was evaluated by flow cytometry using the MACSQuant analyzer,and data were analyzed using FlowJo software.Using main fluoresce of CMFDA to represent the quantity of internalized PLTs.8 Pretreat the macrophages with different concentration of IM7/KM114,then co-cultured with anti-CD41 antibody(Mwreg30)opsonized PLTs.9 Evaluated phagocytosis of antibody opsonized SRBC by murine peritoneal macrophages using confocal macroscopy.Fluorescent images were collected using the Zeiss LSM 700 confocal microscope system.Zen Black software was used for image acquisition,and ImageJ2 software was used for digital image processing.PI was calculated as blow,PI =(total number of internalized RBCs/total number of counted macrophages)*100.10 Evaluated phagocytosis of anti-CD41 antibody opsonized mouse PLTs by RAW cells using confocal macroscopy.Fluorescent images were collected using the Zeiss LSM 700 confocal microscope system.Zen Black software was used for image acquisition,and ImageJ2 software was used for digital image processing.PI was calculated as blow,PI =(total number of internalized PLTs/total number of counted macrophages)*100.Also,binding index(BI)was calculated as total number of bond PLTs/total number of counted macrophages)*100.11 Anti-CD44 antibody binding assay.Pretreat the macrophages with different concentration of IM7/KM114.Labeled anti-CD44 mAbs that bound to macrophages with PE-conjugated goat anti-rat antibody.The binding abilities of anti-CD44 mAbs were detected by flow cytometry.Results:1,Antibody-opsonized RBCs/PLTs can be recognized and internalized by macrophages.Data shows that phagocytosis index(PI)of TER-119 opsonized mouse RBCs are 157.2 for RAW cells and 89.63 for THP-1 cells,significantly higher than non-opsonized ones(RAW cells PI=0;THP-1 cells PI=0.49,P<0.0001).Also,same results are gained from flow cytometry detecting the Phagocytosis of PLTs,which the mean fluoresce of macrophage co-cultured with opsonized PLTs is much higher than the control(1849 vs 458,P<0.001).2,Evaluate the effect of IM7/KM114 pretreatment on macrophage phagocytosis of RBCs.Data shows in control groups PI was 157.2 for RAW cells and 89.63 for THP-1 cells;at IM7 0.1 u g/ml PI was 157.2,30.49;at 1?g/ml and 10?g/ml,PI were 0,which are significantly different to the control(P<0.0002).And this inhibitive effect shows in a dose-dependent manner.KM114,on the contrary,has no effect on the RBCs phagocytosis for both cell types(at lOug/ml PI was 139.72 or 64.2,P>0.55).3,Both IM7 and KM 114 can inhibit the phagocytosis of opsonized PLTs in RAW264.7 cells in a dose-dependent manner.Besides,IM7 works better than same concentration of KM114(P<0.02).At the concentration of 0.1 ?g/ml,IM7 and KM114 both show partial inhibition effect(P<0.001).IM7 can reach almost totally inhibition of phagocytosis at the concentration of 1 ?g/ml(P<0.0001).KM114 can produce some extent of inhibition effect at 1 ?g/ml and 10 ?g/ml(P<0.0001).And KM114 at 10 ?g/ml gives similar inhibitive effect as IM7 at 1 u g/ml(P=0.71).4,Anti-CD44 antibodies have no effect on the binding of opsonized PLTs and macrophages.Both the pretreatment of isotypes and lower concentration groups of anti-CD44 antibodies have no significant difference to controls(P>0.5).Only in the IM7 higher concentration groups,there were more PLTs binding to the surface of the macrophages.5,The binding abilities of IM7 and KM114 are different.Within the same isotype,the binding ratio goes up with the concentration of anti-CD44 mAb.For IM7,binding ratios can reach up to 90%at 5 ?g/ml.At the same concentration,IM7 definitely binds better to macrophages than KM114(P<0.001).For KM114 there are no significant differences to control at the concentration from 0.1 ?g/ml to 5?g/ml(P=0.87).Conclusion:1,Antibody opsonized RBCs/PLTs can be recognized and internalized by macrophages,which mimic the pathogeneses of ITP in vivo.2,IM7 pretreatment can significantly inhibit the Fc?R-mediated phagocytosis of RBCs by both RAW264.7 and THP-1 derived macrophages in a dose-dependent manner,while KM114 doesn't work.3,Both IM7 and KM 114 pretreatment can significantly inhibit the FcyR-mediated phagocytosis of PLTs by RAW264.7cells in a dose-dependent manner.And,IM7 works better than the same concentration of KM 114.It provides a new method to treat ITP in the future.4,Anti-CD44 antibody has no effect on the binding of PLTs and macrophages,we presume the anti-CD44 antibodies work on the internalization step during macrophages phagocytosis.5,At the same concentration,IM7 binds better to macrophages than KM114,which may explain why it works better in the phagocytosis assay.