Study On The Pathogenetic Mechanism Of Destruction And/or Suppression Of Hematopoiesis By Auto-IgG On Bone Marrow Cells In Pancytopenia Patients With Positive BMMNC-Coombs Test

OBJECTIONSTo explore the pathogenetic mechanism of destruction and/or supp-ression of hematopoiesis by auto-antibodies(IgG)on bone marrow cellsin pancytopenia patients with positive BMMNC-Coombs Test(immunorelatedpancytopenia,IRP).METHODSIRP cases,severe aplastic anemia (SAA)and normal controls wereenrolled in this study.The categories of auto-antibodies(IgG,IgM)onBMMNC (CD34+/CD15+/GlycoA+ hematocytes)were assayed by fluorescenceactivated cell sorting (FACS).First Section The auto antibodies(IgG)in the"EI"on the BM smears of forty-eight cases with IRP,ten SAA andeleven normal controls were detected by immuno-histofluorescence (IF)staining.Clinical and laboratory data of all cases and controls werealso analyzed respectively.Second Section Sixty-one cases with IRP,tenSAA and thirteen normal controls were enrolled in this study.The quantity(the percent of CD68+/CD45+)and the expression of activated antigens (thepercent of CD69+CD68+/CD68+)of macrophages in the bone marrow of allcases and controls were measured by FACS and the function of macrophageswere evaluated according to the phagocytosis ratio and index of red bloodcells(CRBC)phagocytosed by macrophages which was cultured in vitro.Third Section EPO receptors(EPOR)on GlycoA(+)BMMNCs of IRP patientswith IgG(+)on GlycoA(+)BMMNCs were measured by FACS.The hemopoieticprecursors of BM were cultured in different levels of erythropoietin (EPO)in vitro and colony-forming units-erythoid (CFU-E)were observed at7-day-culture. RESULTSFirst Section (1)Mouse anti-human IgG could be detected on the BMsmear of 14 cases(29.17%),which deposited on the junction betweenmacrophagesanderythroblasts in the"EI"s,IgGon theGlycoA(+)cellsthese 14 cases were all founded.The initial clinical feature of the 14cases with auto antibodies(IgG)in the EI was anemia〔severe anemia (64.29%),middle degree anemia (35.71%)〕,concentration of hemoglobin〔(59.6±16.2)g/l〕in peripheral blood of cases with auto antibodies(IgG)inthe EI were lower than those〔(83.4±25.0)g/l〕of 34 cases without autoantibodies(IgG)in the EI (P＜0.05),but the percent ofreticulocyte(Ret)count〔(2.0±0.8)%〕,the ration of erythroid in the bone marrow fromsternum〔(44.1±13.9)%〕and the level of IBIL of the formers〔(9.4±4.7)mmol/l)higher (P＜0.05)(P＜0.05);Cases with auto antibodies (IgG)in the EI had good reponse to high doses of IVIG (HDIVIG)or /andGlucocorticoids therapies.Total curative rate at 3 and 6 month(indivi-dually 57.14%,85.71%)of cases with auto antibodies(IgG)in the EIwere better than those of the cases without auto antibodies(IgG)in theEI (P＜0.05)respectively.Second Section the quantity,expression ratioof activatedantigen,phagocytosisratioandindex〔(0.57±0.30)%,(40.30±18.49)%,(37.56±15.20)%和(0.75±0.34)〕of BM macrophages in IRPpatients were respectively significantly higher than those in all thecontrol cases〔(0.46±0.08)%,(32.44±19.37)%,(28.26±10.46)%和(0.59±0.39)〕(P＜0.05).And the quantity present high-positive correlationwith the expression ratio of activated antigen phagocytosis ratio andindex of BM macrophages (r=0.89,p＜0.01;r=0.43,p＜0.01;r=0.40,p＜0.01).Patients with IRP were classified into two subgroups according to the quantity of macrophages:Group A(M_Φ≥0.5%)(34casese)and Group B(M_Φ＜0.5%)(27cases),32 cases (94.12%)with auto antibodies(IgG)in Group A,only 2 cases(7.41%)with auto antibodies(IgG)in Group B.There weresignificant differences in expression ratio of activated antigen,phagocytosis ratio and index of macrophages between Group A〔(49.19±16.63)%,(46.62±13.38)%,(0.91±0.36)〕andGroupB〔(29.11±14.30)%,(28.67±12.59)%,(0.61±0.30)〕(P＜0.05).Thirty-four cases of IRP wasdivided into two subgroups according to the quantity of M_Φ:high levelgroup(≥0.75%)(25cases)and Low level group (＜0.75%)(9cases),24 casesin M_Φhigh level group with auto antibodies(IgG)on one hemotopoietic celllineage,l on two lineages,while 8 cases in M_ΦLow level group with autoantibodies(IgG)on two cell lineages,and l on three cell lineages.Theexpression ratio of activated antigen,phagocytosis ration and index ofmacrophages were much higher in high level group〔(56.12±15.11)%,(60.22±12.51)%,(1.23±0.23)〕than those〔(44.58±18.16)%,(43.32±9.24)%,(0.84±0.24)〕in low level group(P＜0.05).The count of RBC,concentration of HbandBPCin peripheral blood of high level group wererespectively lower than those in low level group(P＜0.05).While thepercentage of Ret,the level of TBIL and the ratio of erythroid of sternalbone marrow in high level group were higher than those in low levelgroup(P＜0.05).Third Section The level of EPO receptors(EPOR)onGlycoA(+)BMMNCs of IRP patients with IgG(+)on GlycoA(+)BMMNCs〔(4.16±0.15)%〕was lower than that of normal controls〔(6.04±0.48)%〕(P＜0.05).There were two different relationships between CFU-E and EPO level.Therewere twenty patients in Group 1 and four cases in Group2.The equationtest of goodness of fit in Grouplwasy=l.OO28x-2.0056,(P＜0.05,g＜0.05). There was no linearity correlation between CFU-E and EPO level (F=0.25,P=0.6264,g=0.16)in Group2.The equation test of goodness of fit in normalcontrol was y=0.68915x-1.9783 (P＜0.05,g＜0.05).CONCLUSION(1)Macrophage connected with erythroblasts by auto antibodies(IgG)in the"EI"s of some of IRP patients.These"EI"s were not niches oferythroblastic developmentment and differenttiation but where macroph-ages devoured and destroyed erythroblasts in vivo.Macrophages didn't nurse but devour erythroblasts.The pathogentic mechanism of IRP withauto antibodies(IgG)in the EI may be that macrophages devoured morehematopoietic cells.(2)The quantity,expression ratio of activatedangtigen and phagocytosis ratio and index of macrophages were enhancedin IRP patients with auto antibodies(IgG).When the numbers of hematopoi-eric cells with auto antibodies(IgG)of bone marrow phogocytosed bymacrophages which were activated were more than those of the hematopo-ietic cells which are produced by bone morrow,bone marrow failure canoccur.Macrophages might not involved in destruction of bone marrow inIRP with auto antibodies(IgM)or cold autoantibodies.(3)Maybe autoantibodies (IgG)which combinated with EPOR on hematopoietic cells blockEPO-EPOR signation and hematopoietic cells could not proliferate anddifferentiate,then the absence of EPO causes bone marrow failure.