Nearly one-third of IgM antilymphocyte autoantibody-positive sera from patients with systemic lupus erythematosus (SLE) contain IgM antibodies to one or more 180-220-kD molecules (p180, p190, p205, and p220) in blots of glycoproteins purified from T cells by wheat germ agglutinin affinity chromatography. Identity of these IgM targets with multiple isoforms of CD45 was established by their specific depletion from T cell glycoproteins by immunoprecipitation with T191, a monoclonal antibody (mAb) that reacts with an epitope common to all CD45 isoforms. Although the anti-CD45 autoantibodies recognize higher molecular weight isoforms primarily, antigenic specificity in this system is quite heterogeneous and includes multiple distinct CD45 isoforms on different types of T cells that are, at least in part, different from those reactive with mAbs 2H4 and UCHL-1. Because CD45 is a major membrane protein tyrosine phosphatase that plays a critical role in antigen- induced T cell activation, the present data may be relevant to some of the antilymphocyte antibody-mediated immunologic abnormalities that characterize SLE and related autoimmune diseases

Fernsten, Philip

Jarjour, Wael

Mimura, Toshihide

Winfieldc, John B.

T Mimura

P Fernsten

W Jarjour

J B Winfield

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Autoantibodies specific for different isoforms of CD45 in systemic lupus erythematosus.

Carolina Digital Repository

BriefDefinitive ReportAutoantibodies Specific for Different Isoformsof CD45 in Systemic Lupus ErythematosusBy Toshihide Mimura, Philip Fernsten, Wael Jarjour,and John B . WinfieldFrom the Division of Rheumatology and Immunology and the Thurston 'Arthritis ResearchCenter, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514SummaryNearly one-third of IgM antilymphocyte autoantibody-positive sera from patients with systemiclupus erythematosus (SLE) contain IgM antibodies to one or more 180-220-kD molecules (p180,p190, p205, and p220) in blots of glycoproteins purified from T cells by wheat germ agglutininaffinity chromatography. Identity of these IgM targets with multiple isoforms of CD45 wasestablished by their specific depletion from T cell glycoproteins by immunoprecipitation withT191, a monoclonal antibody (mAb) that reacts with an epitope common to all CD45 isoforms .Although the anti-CD45 autoantibodies recognize higher molecular weight isoforms primarily,antigenic specificity in this system is quite heterogeneous and includes multiple distinct CD45isoforms on different types of T cells that are, at least in part, different from those reactive withmAbs 2144 and UCHL1. Because CD45 is a major membrane protein tyrosine phosphatase thatplays a critical role in antigen-induced T cell activation, the present data may be relevant to someof the antilymphocyte antibody-mediated immunologic abnormalities that characterize SLE andrelated autoimmune diseases.S tudy of the cell-type specificity of IgM antilymphocyteautoantibodies (cold lymphocytotoxins) in SLE has es-tablished an overall preferential reactivity with peripheralCD4+ T cells expressing high levels of CD45RA isoformsdefined by monoclonal anti-2144 (suppressor-inducer pheno-type) (1, 2) and with activated T cells (3) . Observations inthis system further suggest that such autoantibodies depletecirculating CD4+/CD45RA+ T cells in patients with ac-tive disease and inhibit antigen-induced T cell proliferationand suppressor-inducer function in polyclonal B cell activa-tion in vitro (2-4) . Collectively, the available data argue thatautoantibodies to lymphocytes contribute substantially to the .complex immunoregulatory abnormalities that characterizepatients with SLE and related systemic autoimmune disorders(5, 6) . Progress in this area has been limited, however, bythe paucity of unambiguous information concerning the mo-lecular nature of the target antigens involved . In the presentinvestigation, several -200-kD glycoproteins shown previ-ously to be IgM antilymphocyte autoantibody targets in thisdisorder (7) were identified as isoforms of the CD45 molec-ular complex (leukocyte common antigen) . Because differentisoforms of CD45 define functionally important lymphocytesubpopulations and are pivotal in T cell regulation of cellsignaling during the immune response via their protein tyro-sine phosphatase activity (8-10), these data may be relevantto basic mechanisms underlying immunologic dysfunctionin SLE and other autoimmune diseases .653Materials and MethodsPatients and Serum.	Venous blood was obtained from 43 patientsattending the University ofNorth Carolina Lupus Clinic who metthe revised criteria of the American College ofRheumatology forclassification as SLE (11), and from 10 normal subjects. After sepa-ration from blood, serum was aliquoted and stored at -70°C. Eachserum aliquot was heated at 56°C for 1 h immediately before useto inactivate complement .Cells. E6-1, a CD4+/CD45RA+/TCR-tx/a . Jurkat T cellline phenotypically similar to resting peripheral lymphocytes, andPEER, a CD4 -/CD8- /CD45R0+/TCR-a/f3+ cell line, werecultured in RPMI 1640 medium containing 10% FCS, glutamine,and antibiotics .Special Immunological Reagents. T191, an IgG2a mAb to con-ventional CD45 (12) was provided byDr. R. Mittler, Bristol-MeyersCo., Wallingford, CT Anti-2144, a mAb to CD45RA (13), wasa gift from Dr. C. Morimoto, Harvard Medical School, Boston,MA. UCHL1, a mAb to CD45RO (14), was purchased from DakoCorp., Santa Barbara, CA.Indirect Immunofluorescence and Flow Cytometry. Binding ofr Absand SLE IgM to the T cell lines was detected by indirect im-munofluorescence at 4°C using FITC-conjugated F(ab')2 goatanti-mouse IgG or anti-human IgM (Chapel Laboratories, Coch-ranville, PA) (1) .Lectin Affinity Chromatography. Glycoproteins from E6-1 andPEER were purified from 0.5% Renex 30 detergent cell lysatesby elution from agarose-conjugated wheat germ agglutinin (WGA)(Vector Laboratories, Burlingame, CA) with 10% N-acetyl-glucosamine (7) .J . Exp. Med . © The Rockefeller University Press " 0022-1007/90/08/0653/04 $2 .00Volume 172 August 1990 653-656Preclearance Experiments.	Allisoforms of CD45 were pre-clearedfrom E6-1 and PEER WGA eluates by immunoprecipitation withT191 and protein A-Sepharose (Zymed Laboratories, San Francisco,CA), as described in detail previously (15). Control immunoprecipi-tations were performed with UPC10, a monoclonal IgG2a. SLEpatient sera were depleted ofantilymphocyte antibodies by incuba-tion with viable E6-1 cells, as described previously (7).Electrophoresis andImmunoblotting. Detergent cell lysates, lectineluates, or -32-fold enriched plasma membranes (16) in Laemmlisample buffer were subjected to electrophoresis on 6 or 8% SDS-polyacrylamide slab gels under reducing conditions, and electro-eluted to nitrocellulose . The blots were probed with appropriatedilutions ofSLE patient serum or normal human serum and stainedwith biotinylated anti-human IgM or IgG antibodies (Tago Inc.,Burlingame, CA) and avidin-biotinylated horseradish peroxidase(Vectastain ABC kit ; Vector Laboratories, Inc., Burlingame, CA),as described in detail previously (7, 15) .Results and DiscussionNearly one-third (10/29) of SLE patient sera positive forIgM antilymphocyte antibodies against E6-1 in indirect im-munofluorescence assays contained IgM that stained one ormore "400-kD proteins (gp -200) on blots ofE6-1 plasmamembranes or WGA-purified glycoproteins (Fig . 1 a) . IgMantibodies to gp -200 were not demonstrable in 10 controlsera from normal subjects or in 14 antilymphocyte autoanti-body-negative SLE sera . When blots of glycoproteins fromE6-1 and a second T cell line, PEER, were separated on 6%SDS-polyacrylamide gels using extended electrophoresis times,gp -200 was resolved into four proteins ofMr 180-220,000 .As illustrated for representative SLE patients in Fig. 1 b, anti-gpFigure 1 . IgM antibodies in SLE patient sera recognize different-200-kD T cell glycoproteins. (a) Equal concentrations of protein inenriched plasma membranes (lane 1), whole cell lysate (lanes 2 and4), or glycoproteins purified from E6-1 cells by WGA affinity chro-matography (lane 3) were separated on 8% SDS-polyacrylamide gels,blotted to nitrocellulose, and probed with IgM from SLE patients St(lanes 1 and 2) and Wa (lanes 3 and 4), as described in Materials andMethods. (b) Blots were prepared with E6-1 or PEER WGA eluatesseparated on 6% SDS-polyacrylamide gels using prolonged elec-trophoresis times, and probed with antilymphocyte antibody-positiveSLE sera (St, lanes 1, 6, and 7; Wa, lane 2 ; Le, lane 3 ; and Tu, lanes8 and 9), antilymphocyte antibody-negative SLE serum (lane 4), ornormal human serum (lane 5) .654 Autoantibodies to CD45 in Systemic Lupus Erythematosus-200-positive sera stained proteins of 190, 205, and/or 220kD with E6-1 glycoproteins (lanes 1-3, 6), but, with oneexception, did not react with PEER glycoproteins (e.g., com-pare staining of E6-1 and PEER by St serum in lanes 6 and7, respectively). The exceptional serum (patient Tu) containedIgM that stained several E6-1 proteins (lane 8) and a 180-kDprotein with PEER (lane 9) . The general absence of anti-gp-200 reactivity with PEER glycoproteins in the panel ofSLE sera studied probably explains the dramatically lowerIgM indirect immunofluorescent staining of PEER (32 ±19% IgM-stained cells) relative to E6-1 (62 ± 28% IgM-stained cells) .The similarity between the molecular mass of the four SLEIgM antibody targets and that of the p220/p205/p190/pl80isoforms of CD45 known to be expressed on different popu-lations of lymphocytes (17) suggested that the SLE IgM an-tibodies were directed against individual isoforms of CD45expressed differently on E6-1 and PEER . In indirect im-munofluorescence experiments to test this possibility, E6-1was stained by T191, a mAb that recognizes all CD45 iso-forms, and by anti-2H4 (CD45RA specific), but not byUCHL1 (CD45RO specific) ; conversely, PEER reacted withT191 and UCHL1, but not with anti-2H4 . Taken together,these data strongly suggested that the IgM anti-gp -200antibodies in the SLE sera recognize specific isoforms of CD45 .The identity of gp -200 with CD45 was established bydepleting CD45 from WGA eluates of E6-1 andPEER withT191. Immunoblots of CD45-depleted glycoproteins no longerwere stained by SLE IgM (Fig . 2 a) . These data, togetherwith the results of the immunofluorescence experiments andthe informative staining patterns of Tu IgM for E6-1 andPEERglycoproteins, indicate unequivocally that SLE IgM recog-nized isoform-specific determinants of CD45 expressed onthe cell surface, rather than the intracytoplasmic domain ofFigure 2 . Extra-cytoplasmic domain specificity of IgM autoanti-bodies in SLE sera for different isoforms of CD45 . (a) WGA eluatesof E6-1 or PEER were pre-cleared of CD45 with mAb T191 (evenlanes) or control IgG2a (odd lanes) and examined for residual reac-tivity with SLE IgM by immunoblotting (patient McD, lanes 1 and2 ; patient Tu, lanes 3 and 4) . (b) Serum from SLE patient Wa wasexhaustively absorbed with packed human erythrocytes, as a control(lane 1), or viable E6-1 cells (lane 2) and then examined for residualreactivity with CD45, as described in Materials and Methods.this molecule, which is identical among all isoforms. Con-sistent with this interpretation was the observation that ex-haustive absorption of SLE sera with viable E6-1 cells removed,in parallel, both IgM antibody that stained CD45 on blotsand IgM staining of T cells by indirect immunofluorescence(Fig. 2 b) .It is likely that anti-CD45 autoantibodies recognize aminoacid sequence determinants, at least in part, because glycosi-dase F treatment of E6-1 glycoproteins, while reducing theestimated molecular mass of the CD45 isoforms, did not ap-preciably alter their capacity to be stained with SLE IgM (datanot shown) . Epitope mapping will be required to define ex-actly the fine specificity of anti-CD45 autoantibodies, butit can be deduced from the present data that at least someof the reactive epitopes are distinct from those recognizedby anti-2H4 on p220/p205 and by UCHIA on p180 . Thus,of eight SLE sera studied in detail, four reacted only withp220 and p205 CD45RA isoforms (13), but the other foursera reacted with p205 alone (one serum), p220 alone (twosera), p205, p120, and p180 (one serum), and p220, p205,and p190 (one serum) . Of interest, the dominant specificityReferences2 .3 .4 .5 .of SLE autoantibodies for CD45RA isoforms appears to besimilar to that of mouse mAbs, which also recognize CD45RAepitopes primarily (13) .The anti-CD45 autoantibodies described in the present in-vestigation may contribute to some of the immunologic ab-normalities in SLE (5) and other disorders (18), e.g ., deple-tion of functionally important T cell subpopulations (4, 18,19) and defective T cell activation in response to antigenicstimuli (20, 21) . Thus, CD45 is absolutely required for Tcells to enter the cell cycle in response to stimulation withantigen (22) . Its cytoplasmic domain has protein tyrosine phos-phatase activity, which plays an essential role in signal trans-duction (8, 23) . mAbs to different isoforms of CD45 inhibitor enhance lymphocyte activation in vitro (12, 23-25), prob-ably by interfering with the capacity of CD45 to interactwith other cell surface proteins in regulating the phosphory-lation of molecules important in cellular activation after trig-gering of the TCR/CD3 complex (9, 10) . Experiments todelineate the effects ofanti-CD45 autoantibodies on lympho-cyte function and to define their relevance to the pathogen-esis of SLE will be particularly challenging.We thank Melody Shaw for expert technical assistance, Dr. Robert Mittler (Bristol-Myers Co., Walling-ford, CT) for providing anti-CD45 mAb T191, and Dr. Chikao Morimoto (Harvard Medical School,Boston, MA) for providing anti-2H4 .This work was supported by National Institutes of Health grants R01 AM-30863, T32 AR-7416, andP60 AR-30701, and by a Biomedical Research Center grant from the Arthritis Foundation .Address correspondence to John B. Winfield, Division of Rheumatology and Immunology, School ofMedicine, CB 7280, 932 Faculty Laboratory Office Building, The University of North Carolina at ChapelHill, Chapel Hill, NC 27599 .Received for publication S February 1990 and in revised form 24 May 1990.Yamada, A., P.L. Cohen, and J.B. Winfield . 1985 . Subsetspecificity of anti-lymphocyte antibodies in systemic lupuserythematosus. Preferential reactivity with cells bearing theT4 and autologous erythrocyte receptor phenotypes . ArthritisRheum. 28:262 .Tanaka, S., T. Matsuyama, A.D . Steinberg, S.F. Schlossman,and C . Morimoto . 1989 . Antilymphoryte antibodies againstCD4`2H4* cell populations in patients with systemic lupuserythematosus. Arthritis Rheum . 32:398 .Yamada, A., and J.B. Winfield. 1984 . Inhibition of solubleantigen-induced T cell proliferation by warm-reactive antibodiesto activated T cells in systemic lupus erythematosus . J. Clin .Invest. 74:1948 .Winfield, J.B., M. Shaw, A . Yamada, and S . Minota . 1987 .Subset specificity of anti-lymphocyte antibodies in systemiclupus erythematosus. II . Preferential reactivity with T4' cellsis associated with relative depletion of autologous T4' cells .Arthritis Rheum . 30:162 .Winfield, J.B. 1987 . Anti-lymphocyte antibodies : specificityand relationship to abnormal cellular function . In Systemic655 Mimura et al .	Brief Definitive ReportLupus Erythematosus. R.G . Lahita, editor, John Wiley & Sons,Inc ., New York . 305-331 .6 . Morimoto, C., and S.F. Schlossman. 1987 . Antilymphocyteantibodies and systemic lupus erythematosus . ArthritisRheum .30:225 .7 . Mimura, T., P. Fernsten, M. Shaw, W Jarjour, and J.B.Winfield . 1990. Glycoprotein specificity of cold-reactive anti-lymphocyte antibodies in systemic lupus erythematosus. Ar-thritis Rheum . In press.8 . Tonks, N.K ., H . Charbonneau, C.D. Diltz, E.H . Fischer, andK.A . Walsh . 1988 . Demonstration that the leukocyte commonantigen CD45 is a protein tyrosine phosphatase. Biochemistry.27:8695 .9 . Clark, E.A ., and J.A . Ledbetter. 1989 . Leukocyt e cell surfaceenzymology : CD45 (LCA, T200) is a protein tyrosine phos-phatase. Immunol. Today. 10:225 .10 . Mustelin, T, K.M. Coggeshall, and A . Altman . 1989 . Rapidactivation of the Tcell tyrosine protein kinase pp56kk by theCD45 phosphotyrosine phosphatase. Proc Natl. Acad. Sci. USA.86:6302 .11 . Tan, E.M., A.S. Cohen, J.F. Fries, AT Masi, D.J. McShane,N.F. Rothfield, J.G. Schaller, N. Talal, and R.J . Winchester.1982 . The 1982 revised criteria for the classification of systemiclupus erythematosus. Arthritis Rheum. 25:1271.12 . Mittler, R.S ., R.S . Greenfield, B.Z . Schacter, N.F. Richard,and M.K. Hoffmann . 1987 . Antibodies to the common leu-kocyte antigen (T200) inhibit an early phase in the activationof resting human B cells. j Immunol. 138:3159.13 . Streuli, M., C. Morimoto,M. Schrieber, S.F. Schlossman, andH. Saito. 1988 . Characterization of CD45 and CD45Rmono-clonal antibodies using transfected mouse cell lines that express individual human leukocyte common antigens.J. Immunol.141:3910.14 . Terry, L.A ., M.H . Brown, and P.C.L . Beverley. 1988 . Themonoclonal antibody, UCHL1, recognizes a 180,000MW com-ponent of human leukocyte-common antigen, CD45. Immu-nology. 64 :331 .15 . Minota, S., S. Koyasu, I . Yahara, andJ.B. Winfield . 1988 . Au-toantibodie s to the heat-shock protein hsp90 in systemic lupuserythematosus. J. Clin. Invest. 81 :106.16 . Crumpton, M.J ., andD. Snary. 1974 . Preparation and proper-ties oflymphocyteplasma membranes. In Contemporary Topicsin Molecular Immunology, Vol. 3. G.L . Auda, editor. PlenumPublishing Corporation, New York . 27-56.17 . Thomas, M.L . 1989 . The leukocyte common antigen family.Annu . Rev. Immunol. 7:339 .18 . Morimoto, C., D.A . Hafler, H.L . Weiner, N.L . Letvin, M.Hagan, J. Daley, and S.F. Schlossman . 1987 . Selective loss ofthe suppressor-inducer T-cell subset in progressive multiple scle-65 6 Autoantibodies to CD45 in Systemic Lupus Erythematosusrosis. Analysis with anti-2H4 monoclonal antibody. N. Engl.J. Med. 316:67 .19 . Morimoto, C., A.D. Steinberg, N.L . Letvin, M. Hagan, TTakeuchi, J. Daley, H. Levine, and S.F. Schlossman. 1987. Adefect of immunoregulatory T cell subsets in systemic lupuserythematosus patients demonstrated with anti-2H4 antibody.J. Clin . Invest. 79:762.20 . Rosenthal, C.J., and E.C . Franklin . 1975 . Depression of cellular-mediated immunity in systemic lupus erythematosus . ArthritisRheum. 18 :207 .21 . Gottlieb, A.B., R.G. Lahita, N. Chiorazzi, andH.G. Kunkel .1979 . Immune function in systemic lupus erythematosus. Im-pairment of in vitro T cell proliferation and in vivo antibodyresponse to exogenous antigen. J. Clin. Invest. 63:885 .22 . Pingel, JT, and M.L . Thomas . 1989 . Evidence that theleukocyte-common antigen is required for antigen-induced Tlymphocyte proliferation . Cell. 58:1055.23 . Ledbetter, J.A., N.K . Tonks, E.H . Fischer, and E.A . Clark.1988 . CD45 regulates signal transduction and lymphocyte ac-tivation by specific association with receptor molecules on Tor B cells . Proc. Natl. Acad. Sci. USA. 85:8628.24 . Ledbetter, J.A ., L.M . Rose, C.E . Spooner, P.G . Beatty, P.J.Martin, andE.A . Clark. 1985 . Antibodies to common leuko-cyte antigen p220 influence human T cell proliferation bymodifying IL 2 receptor expression . J. Immunol. 135:1819 .25 . Schraven, B., M. Roux, B. Hutmacher, and S.C. Meuer. 1989 .Triggering ofthe alternative pathway of human Tcell activa-tion involves members of the T 200 family of glycoproteins .Eur. J. Immunol. 19:397 .

Autoantibodies specific for different isoforms of CD45 in systemic lupus erythematosus

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Autoantibodies specific for different isoforms of CD45 in systemic lupus erythematosus

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