Anti-F(ab')2 antibodies in thrombocytopenic
patients at risk for acquired immunodeficiency
syndrome.
J R Yu, … , E T Lennette, S Karpatkin
J Clin Invest.
1986;
77(6)
:1756-1761.
https://doi.org/10.1172/JCI112498
.
22 homosexual or narcotic addict patients at risk for acquired immunodeficiency syndrome
(AIDS) or with AIDS, were studied for the presence of antiimmunoglobulin antibodies and
circulating immune complexes (20 were thrombocytopenic, 6 had AIDS). Circulating
immune complex levels were 10-fold higher than levels in normal subjects. IgG anti-F(ab')2
antibodies were noted in homosexual as well as narcotic addict patients. Of 16 homosexual
patients, 7 had IgG anti-F(ab')2 antibody of moderate to marked titer with broad reactivity
against autologous, homologous, and control F(ab')2 fragments. Three others demonstrated
limited reactivity against one or two F(ab')2 fragments. The remaining six patients were
negative. Six of six narcotic addict patients had IgG anti-F(ab')2 antibody, five with limited
reactivity, one with broad reactivity. In contrast, neither elevated circulating immune
complexes nor anti-F(ab')2 antibodies were detectable in six autoimmune thrombocytopenic
patients. Anti-F(ab')2 antibody could be affinity purified from serum or circulating immune
complexes. Anti-F(ab')2 reactivity correlated with circulating immune complex levels, r =
0.83, P less than 0.01.
Research Article
Find the latest version:
Anti-F(ab')2 Antibodies
in
Thrombocytopenic Patients
at
Risk
for Acquired Immunodeficiency Syndrome
Ji-Ren Yu,* Evelyne T.
Lennette,*
and Simon Karpatkin**Department ofMedicine,NewYorkUniversity Medical School,NewYork,NewYork10016;
and Virolab, Inc., Berkeley, California94710
Abstract
22homosexual ornarcotic addict patients at risk for acquired immunodeficiency syndrome (AIDS) or with AIDS, were studied
for
the presence ofantiimmunoglobulin antibodies and circulating immune complexes (20 were thrombocytopenic, 6 had AIDS). Circulating immune complex levels were10-fold
higher thanlevels
in normal subjects. IgGanti-F(ab')2
antibodies were noted in homosexual as well as narcotic addict patients. Of 16 ho-mosexualpatients, 7 had IgGanti-F(ab')2 antibody of moderate to marked titer with broad reactivity against autologous, ho-mologous, and controlF(ab')2
fragments. Three others demon-strated limited reactivity against one or two F(ab')2 fragments. The remaining six patients were negative. Six of six narcotic addict patients had IgGanti-F(ab')2 antibody, five with limited reactivity, onewith
broadreactivity.Incontrast, neither elevatedcirculating
immunecomplexes noranti-F(ab')2 antibodies
were detectable insix autoimmune thrombocytopenic
patients.Anti-F(ab')2
antibody could beaffinity purified
from serum or circulating immune complexes.Anti-F(ab')2
reactivity cor-related with circulating immune complex levels, r=0.83,
P<0.01.
Introduction
Individuals at risk for the acquired immunodeficiency syndrome
(AIDS):'
sexuallyactive homosexuals(1-3), narcotic addicts (4), andmultiply transfused hemophiliacs
(5,6) developanew syn-dromeof
idiopathic thrombocytopenic
purpura (ITP),which
isclinically indistinguishable from classic
autoimmunethrom-bocytopenic
purpura(ATP) (7).
However,immunologic studies
Thispaper was presented at the98th Annual Meeting of the American Association of Physicians, 5 May 1985, and published in abstract form (1985. Clin. Res. 33:61 IA).
Dr.Yuis aVisiting Scientist from the First Teaching Hospital, Beijing Medical College, Beijing, China. Address reprint requeststoDr. Kar-patkin.
Received for publication 20 November 1985 and in revised form 7 February 1986.
1.Abbreviations usedinthispaper:ADE,adenovirus; AIDS,acquired immunodeficiency syndrome; ATP, autoimmune thrombocytopenic purpura;CMV,cytomegalovirus; EBV,Epstein-Barr
virus; HSITP,
ho-mosexual ITP; HSV, herpessimplex virus;ITP,idiopathic thrombocy-topenicpurpura;KLH,keyholelimpethemocyanin;
NITP,narcotic ad-dicts with ITP;PEG,polyethylene glycol;PMSF,phenylmethylsulfonyl
fluoride.
(1-4, 6) reveal significant differences between the new syn-drome(s) and classic ATP. Thrombocytopenic patientsatrisk for AIDS havemarkedly elevated platelet-bound IgG and com-plement (C3C4) compared withATPpatients, markedly elevated incidence and level ofcirculating immune complexes (deter-mined by the polyethylene glycol [PEG] precipitation method) compared with ATP patients, and serum antibody against the AIDS-related retrovirus. Recent studies (2) evaluating the im-munologic mechanisms involved have suggested that this dis-order may be mediated by circulating immune complexes in homosexual ITP (HSITP); and by circulating immune com-plexes as well asspecific antiplatelet 7S IgG in narcotic addicts
(NITP) (4).
Our present report describes studies on immune complexes inpatients at risk for AIDS and reveals the presence of Ig anti-F(ab')2 antibodiesintheserum andcirculatingimmune com-plexesof these patients.
Methods
Population. Thepopulationstudied foranti-F(ab')2 antibody included 16 sexually active homosexuals(10 withthrombocytopeniaalone,4with thrombocytopenia and AIDS [3 with Kaposi's sarcoma, 1 with Pneu-mocystis cariniipneumonia],2with AIDS [Kaposi's sarcoma] and normal platelet counts);6narcotic addicts with thrombocytopenia;7classic fe-maleATPpatients; and 5 controlsubjects.The clinical manifestations presented by thesepatients are essentially the same as those described inprevious publications (1-4, 7).
Procedures. Viral studies for detection ofantigenaswellasantibody wereperformed for Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpessimplex virus (HSV), rubeola virus, and adenoviruses (ADE)using standard indirect immunofluorescenttechniques.
Antigen-containing platelets orPEG-precipitatedimmunecomplexes were washedtwice withcentrifugation at 3,000 g in0.01 M phosphate-buffered saline (PBS), pH 7.4,containingImg/ml bovineserumalbumin andspottedonglassslides, whichweredivided intotwogroups. One groupwastreated with 0.1 Mglycine buffer,pH 3.0 for 15 min, and rinsed twice with PBStoremove dissociated antibodies. Both treated anduntreated fractionswerefixedinacetoneand tested for viralantigens using fluoresceinisothiocyanate-conjugatedrabbit antiseraspecific for eachofthemoietiestobetested.
Antibody specificities in sera or in immune complexprecipitates were tested asfollows:Eachsamplewasdiluted in 0.1 Mglycine buffer, pH 3.0at 1:2or1:8 and heatedat56°C for30mintodissociate endog-enouscomplexes.ThepH of each samplewasadjustedtopH7.0 and tested forantibody activity. Two standard serologic procedureswere
used, indirectimmunofluorescence antibody stainingand immune ad-herencehemagglutination (8).
Platelet eluates wereprepared from 1 X 108 washed platelets (9)
sus-pended in PBS. 2 ml ofetherwasadded and thesample vigorously shaken for2 minbeforebeingincubatedina37°Cwaterbath for 30 minwithshaking.Thesamplewasthencentrifugedfor15 minat2,500 g,and the eluate(aqueoussolution)removed withaPasteur
pipette
and stored at-20°Cpriorto use.Circulating immune complexeswereestimatedbyamodified PEG precipitation procedure (10). 50 ul oftest serum wasaddedto150
ild
of J.Clin.Invest.© TheAmericanSociety for ClinicalInvestigation,Inc. 0021-9738/86/06/1756/06 $1.00
0.2 MNa2EDTA, pH 7.4. 1 ml of 6% PEG in 0.1 Msodium borate buffer, pH 8.4,wasadded andincubatedat4°Covernight.Theprecipitate wascentrifugedat3,000 gat4°Cfor 10 min and thesupernatant dis-carded. The precipitatewasthen washedtwice in 5% PEG in borate buffer,anddissolved in 200 ul PBScontaining0.02% sodium azide. The solutionwasassayed forprotein usingthe Lowryprocedure.When sub-jectedtoelectrophoresisandstained forproteinwith amidoblack, semi-quantitativeestimation ofprotein peaksforIgG, IgM,andcomplement components(determined byimmunoelectrophoresis) represented - 85-90% ofstainable material. The presence ofIgG, IgM,and C3C4 was
independentlyconfirmedwithhighly specificsolid-phaseimmunoassays (4). Themeanvalueofcirculatingimmunecomplexesfor ninehealthy controlsubjectswas0.21±0.20(2 SD) mg/ml.
Purified IgGwaspreparedfrom crudeserumglobulinfractions ob-tained from 50% saturatedammonium sulfate
precipitation
followed by dialysisagainst0.1 Mphosphatebuffer,pH 7.6. The samplewasthen appliedto adiethylaminoethyl-52 column,equilibratedwith thesamebuffer. Theflow-throughmaterial containedpurified IgG.
Serumfractions enriched withIgMwereobtained aftergel-filtration ofserum on aG-200Sephadexcolumnequilibratedwith PBSby
utilizing
thevoid volume fraction.
F(ab')2 fragmentswereprepared bypepsin digestionofIgGatpH 4.0 (I1),followedbydialysis againstPBS plus 0.01% sodiumazide, fol-lowedbyaffinity chromatographyon aninsolublestaphylococcalprotein Acolumnequilibratedin thesamebuffer. The nonadherentmaterial is purified F(ab')2asdeterminedbysodiumdodecylsulfate-polyacrylamide gelelectrophoresis (10),aswellas asolid-phaseimmunoassay performed witharabbit anti-humanIgG-Fcantibody.
Anti-F(ab')2IgGactivitywasmeasuredby solid-phaseimmunoassay. 40lA ofa
5-Mg/ml
solution ofF(ab')2f
fagments
isappliedto12-microtiter wells(induplicate), incubatedatroomtemperature for 1 h, and then washed three times with PBS-azidecontaining1% bovineserumalbumin (1%BSA-PBS). 12 serial twofold dilutions of 40,ulofpurified IgG in1% BSA-PBS (startingwith 5Mg/ml)
were thenapplied toeach well and allowedtoincubate for 1 h. Nonadherentantibodywaswashed three times with 1%BSA-PBS,and 40Mlof radioactive staphylococcalprotein A(30 mCi/mg, Amersham Corp., Arlington Heights,IL), 150,000 cpm,applied
toeach well for 1 h.The wellswerethen washed three timesasabove and assayed forradioactivity.In someexperiments, non-radioactiveproteinAwasaddedtotheF(ab')2fragments to block anypossible
residual Fc domainsbyaddition of 40MlofproteinA(2,ug/ ml).Ineachexperiment:(a)patientIgGwas runagainst patient F(ab')2fragment
aswellascontrolF(ab')2 fragment;(b)ControlIgGwas run against autologousorhomologous controlF(ab')2, aswell aspatient F(ab')2.Thecountsper minute obtained for each serial dilution ofpatient IgGagainst patientF(ab')2
weredivided by thecountsperminuteobtained forcontrolIgGagainst autologouscontrolF(ab')2,andaratio obtained. Values> 1.3wereconsideredpositive, since this reflectsa2-SDchange ofreplicatemeasurementsmadeonthesameIgGvs.F(ab')2.Apositive resultwasdefinedasthehighest ratio obtained bytwoconsecutive di-lutions,bothof whicharepositive(> 1.3).Anti-F(ab')2
IgMactivitywasmeasured byusing40-Ml
aliquotsof theIgM-enrichedfraction of serum, nonradioactiveproteinA(2MLg/ml)
toblock anycontaminating IgG (<4%of total IgM+IgGasdetermined byimmunoassay
[4]),rabbit anti-humanIgM(heavy
chainspecific, di-luted1:800, CappelLaboratories,
Cochranville,PA), and125I-protein
A. Thesensitivityof the IgM assaywas0.5 ng per well.Affinity purificationofanti-F(ab')2fromserumIgGwasaccomplished bycouplingpurified F(ab')2to asolid-matrixAffi-gelcolumn(Bio-Rad Laboratories, Richmond, CA). Affi-gel 10waswashedon a scintered glassfilter with ice-coldisopropanol, followedby ice-cold distilled water,
asrecommendedbythemanufacturer. F(ab')2wasdialyzed against0.1 M2[(N-morpholino)ethanesulfonicacid]buffer, pH 6.5 containing0.1 mMphenylmethylsulfonylfluoride (PMSF)
overnight.
1mlofF(ab')2 (5 mg/ml)wasthenincubated with 1mlof packed Affi-gel 10overnight. Unattachedreactive siteswereneutralized with 1MTris-HCIbuffer, pH 6.5 for1h and thenwashed.Al-ml
column was prepared fromaplasticsyringe,
andIgG applied
tothe columnwiththeeffluent reapplied
twotimes. The columnwasthen washedextensivelywith0.1 MNaHCO3, pH 8.0,until theoptical density readingof the effluentat280 smwas
zero. Boundantibodywasthen elutedwith 0.1 M glycine buffer,pH 2.5, and the effluent fractions neutralized immediately withNaHCO3. The peak fractionsweredialyzedagainst water, and lyophilizeduntil furtheruse.
Affinity purification of F(ab')2 from immune complexeswasattained with thefollowing procedure: Affi-gel coupledtoF(ab')2,asabove,was
washed with glycine buffer, pH 2.5, containing 0.1 M PMSF until the washsolution had anoptical density at 280umof zero and pH of 2.5. Theisolated immune complex precipitatewasdissolved in0.1 Mglycine buffer, pH 2.5, containing protease inhibitors: 0.1 mM PMSF, 5 mM EGTA, and 10 mM benzamidine. The acidified immune complex so-lution and Affi-gel coupled to
F(ab')2
weremixed 1:1 by volume and dialyzed overnight against 0.1 M NaHCO3, pH 8.0, containing protease inhibitors. The pH of the suspension in the dialysis sac should be 8.0. By this maneuver, the solid-phaseF(abV)2
competes for the solubilized anti-F(ab')2 IgG of the immune complex as the pH is raised. Thecontents were thenpouredinto aplastic syringe column and washed with 0.1 M NaHCO3 plus protease inhibitorsuntil the effluent had an optical density reading of zero. The boundanti-F(ab`)2 antibodywasthen eluted as abovewitb0.1 Mglycinebuffer, pH 2.5, and neutralized with NaHCO3. Kappa and lambdalight chains of affinity-purified F(ab')2antibody weredetected bysolid-phase immunoassayusingrabbitanti-kappachain antibodyorrabbit anti-lambda chain antibody at a dilution of 1:300 (Calbiochem-Behring Corp., American Hoechst Corp., San Diego,CA), and1251I-protein
A,asin the assay cited above foranti-F(ab')2reactivity.
Results
The presence of "anti-antibodies"wassuggested from prelimi-narystudies designed to analyze platelets and immune complexes for viral antigens, towhich theyhad been exposed, and their respective antibodies. These are cited below.
Viral
studies ofplatelets. Preliminary viral
detectionstudies offixed washedplatelet preparationsof sixHSITP patients re-vealed absence of CMV, HSV,orADE antigensontheirplatelets
(under conditionswhereinpositive controlsreacted with the test antisera), despitethe presence of antiviral antibodies forthese antigensin their sera: CMVantibodytiter(range, 1:32-1:256); HSV (1:32-1:256);ADE(< 1:8-1:64);EBV(1:320-1:640). Sim-ilarly, platelet eluates of eight HSITP patientswerenegative for antibodies against EBV, CMV, HSV, or ADE.Viral studies
of immune complexes. As with the
platelet preparations, viral antigens for EBV, CMV, HSV, andrubeola virus were absent on four fixed immune complex preparations ofHSITP patients. However, anti-viral antibody analysisofthe same immune complexes did reveal antibodies against EBV, CMV, HSV,andrubeola virus,thatcorrelated with thepresence ofthese antibodiesintheirrespective sera(Table I). No such correlationwasnoted withpreparationsfromthreehealthy con-trolsubjects whose immune complex preparationswerenegative in the presenceof positive
serum titers.These
observations
suggested that someimmune complexes may be composed of antibodies directed against other antibodies.Anti-F(ab')2 IgG antibodies in homosexual patients.
Whenpurified
IgGfrom
10 HSITP patients (A-V) and two Kaposi's sarcomapatients
with normal platelet counts (E, F) was tested againstapanelof F(ab')2 fragments from four HSITP patients (A-D) and two patients with Kaposi's sarcoma alone (E, F) moderateto markedanti-F(ab')2 reactivity was noted with the IgG fractions of five patients (A-I) (Table II). This was associated withfairly
broadreactivity,
whereinantibody
wasdirected
against
TableI. AntiviralAntibodyTiters in SeraandImmuneComplexes ofHomosexual Thrombocytopenic Patients and Control Subjects *
Patients Controls
Virus Pi P2 P3 P4 Cl C2 C3
HSV
serum 128 <8 512 128 512 32 128
complex 128 <8 <8 128 <8 <8 <8
CMV
serum 2,048 256 256 128 32 <8 <8
complex 2,048 <8 128 128 <8 <8 <8
EBV(VCA)
serum 1,280 5,120 10,240 640 320 320 80
complex 2,560 20,480 20,480 1,280 <20 <20 <20
EBV(EBNA)
serum 160 160 320 80 80 80 80
complex 320 1,280 640 320 <5 <5 <5
Rubeola
serum 64 256 64 32 ND ND ND
complex 256 512 128 8
VCA, viral capsid antibody;EBNA,EpsteinBarrnuclear antigen;ND, notdone. *Reciprocal viral titersaregiven forpatients(Pl-P4)and controls
(Cl-C3)
inserumsamplesaswellasimmunecomplexesobtained from thesameserumsamples. Immune complex precipitateswere dilutedtothesamevolumeofserumfrom whichtheywereobtained.well as patient F(ab')2. Two IgG fractions (R, S) demonstrated
limited
reactivity against
one or two of the panel of four to sixF(ab')2 fragments.
Three IgG fractions (T-V) demonstrated noreactivity. Similar
results wereobtained
with the two patients with Kaposi's sarcoma (E, F). Negative results were obtained when six IgM-enriched fractions were tested against the samepanel of F(ab')2 fragments,
using a rabbit anti-IgM antibody ca-pableof detecting
0.5 ng of IgM anti-F(ab')2 reactivity per well (data not shown). Negative results were also obtained forrheu-matoid factor when the sera of 10 patients weretested by the routine clinical laboratory at our institution.
Fourpatients with AIDS and
thrombocytopenia
were alsostudied (Table III).
Patient II demonstrated broadreactivity
against
theentire
panelof F(ab')2 fragments,
which included threepatients with Kaposi's
sarcomaand ITP(FF, GG, HH),
onenarcotic addict with
ITP(X) andtwocontrols (G,
H). Patient
II'simmune
complex wasdissociated
at low pH andaffinity
purified
onsolid phaseF(ab')2
ofpatient
GG (seeMethods).
TheTable II. BindingRatiosofHomosexualIgG to
F(ab')2
Fragments ofHomosexual Patients and Control Individuals ComparedtoBinding of ControlIgG to ControlF(ab')2
Fragments *PurifiedIgG immunoglobulin of homosexuals
F(ab)2 Control
fragments A B C D I R S T U v E F G
A 6.6 2.6 1.8 4.0 1.4 0.8 1.1 0.8 0.9 0.9 1.5 1.5 1.1
B 6.6 2.5 1.8 2.8 1.2 1.3 1.3 0.9
C 4.4 1.9 1.8 9.4 1.6 7.5 1.5 1.1 1.1 1.1 1.3 1.6 0.9
D 3.2 1.7 1.3 2.2 1.0 1.4 1.7 1.0
E 3.1 1.7 1.5 2.0 2.5 - 1.3 2.0 0.8
F 7.0 1.7 1.4 6.4 1.3 0.7 0.6 0.6 0.8 1.2 1.4 1.3
G control 3.4 2.5 2.0 1.2 2.5 1.2 1.0 - 1.0 1.1 1.0
Hcontrol 6.2 2.9 1.6 6.0 2.7 1.6 1.3 1.6 1.0
Q control - - 0.9 0.9 0.9 1.0
*F(ab')2 fragments(0.2 ,ug)ofsexually active homosexual patients and control subjects (G, H, Q) were adsorbed tomicrotiter plates thatwere
then reactedwithpurifiedIgG of patientsorcontrolsubjectsat12serial dilutions ofIgG, startingat0.2gg.
1251-protein
A wasthen added and assayed for radioactivity. Thecpmobtained for each serial dilutionwasdividedby thecpmobtained fromasimultaneouslyruncontrolexperi-mentinwhichcontrolF(ab')2 fragmentswerereactedwithautologouscontrolIgG atserial dilution.Thenumbersgiven refertothemeanof the highest ratios obtained foraminimum oftwoconsecutive dilutions.Aratio>1.3is considered positive.It wasdetermined from the2SD mea-surementobtained from sixreplicate determinations with six differentcontrolsubjects. Each ratio is themeanof2-5experiments. F(ab')2 frag-ments werepassed throughaproteinAcolumn and shown to befreeofIgG asdeterminedbybothSDS-PAGEandradioimmunoassay. -,not
TableIII.BindingRatiosofAIDS HSITP Patient'sIgGto
F(ab')2
Fragmentsof
Patients and ControlsComparedtoBinding
of
ControlIgGtoControlF(ab')2
Fragments*Purified IgG immunoglobulin of AIDS patients with ITP
F(ab')2
fragments II FF GG HH
FF 1.7(2.5) 0.8 1.0 0.8
GG 2.8(6.4) 1.0 1.1 1.0
HH 2.7 (4.5) 0.9 1.2 1.1
X 2.5(5.4) Gcontrol 4.2 H control 4.2
*Methodology same asinTable2.PatientIIisahomosexualwith Pneumocystis carinii pneumonia; patients FF, GG,and HH are ho-mosexualswith Kaposi'ssarcomaandITP;patientXhasNITP; sub-jects G andH arecontrols. Numbersin parentheses refertoratios ob-tained withaffinity-purified
anti-F(ab')2
obtained from the dissociated immunecomplexesof patient IIabsorbed tosolid-phaseF(ab')2 of GG.eluate had greater
binding
than thepatient's
serumfor
F(ab')2fragments
FF,GG, HH, and X.Anti-F(ab')2
IgGantibodies
inNITPpatients.When purified IgGfractions of five NITP patients were tested against a panel of F(ab')2 fragmentsfrom
various patients and controls, all five reacted, butwith limited reactivity, particularly against F(ab')2fragments
ofpatients
AandF(Table IV).Patient DD's purified serumIgG
wasaffinity-purified
onsolid-phase F(ab')2 from
pa-tient
A. Thereactivity of
thehighly purified anti-F(ab')2
IgGincreased
its ratio to 9.0. Thisaffinity-purified
antibody was shown to have both kappa and lambdalight chains.
Asixth
patient,
JJ, hadbroadreactivity against
the panelof
6 F(ab')2fragments
of Table III, with ratios ranging from 2.2 to 2.9 (data not shown).Absence of anti-F(ab')2 IgG antibodies
in ATPpatients. In contrast, when theIgGfractions of six thrombocytopenic
ATPTableIV.Binding Ratios ofNarcotic Addict IgG to
F(ab')2
FragmentsofPatients and Control Individuals Compared to Bindingof
ControlIgG toControlF(ab)2
Fragments*PurifiedIgGimmunoglobulinof narcotic addicts
F(ab)2
fragments AA BB CC DD EE
A 4.9 4.8 4.9 5.4
(9.0)4
6.0Z 0.8 0.8 0.8 0.7 0.8
V 1.5 1.0 1.0 1.1 1.1
E 1.0 1.0 1.0 1.2 2.1
F 2.4 2.0 1.8 1.6 1.8
W 1.0 1.0 1.2 1.5 1.2
X 0.9 0.8 0.8 0.8 0.8
HControl 1.4 1.1 0.9 1.0 1.0
QControl 0.9 1.0 - 0.9 0.9
*Methodologysame asinTable
II.
Patients A, Z, V have Kaposi'ssarcomaand ITP; patients E and F have Kaposi's sarcoma, patients W and Xhave narcotic addictITP, subjects H and Q are controls.
± Whenadsorbed to solid-phase F(ab')2 of patientAand eluted, the ra-tiowas9.0.
patients
weretestedagainst
thesamepanel
ofF(ab')2 fragments
asin Table I,noreactivity
wasnoted. Reactivitywasnotnoted when theIgG fractions of four ATP patientsweretestedagainst
apanel
ofF(ab')2 fragments
fromsevenATPpatients (Table
V).
Correlation
of anti-F(ab')2 IgG
antibodies withpresenceof
immunecomplexes inserum. 18HSITP and NITP patients hadcirculating
immunecomplex
measurementsperformed
onserathat
averaged
2.16mg/ml±0.48 (SEM),
or 10-fold the value noted in healthy control subjects (0.21±0.20) (4). These were tested for anti-F(ab')2IgG
reactivity. The binding ratios for spe-cificIgGs
withbroadspecificity against F(ab')2 fragments
wereaveragedtoobtainameanreactivity index. Mean reactivity index correlated withserumimmune complex
level,
r =0.83, P<0.01,
n = 16, using Spearman's rank correlation coefficienttest.
Specificity
of anti-F(ab')2 IgG antibodies. The broad specif-icity of anti-F(ab')2 Ig antibodies, noted particularly in homo-sexual patients, suggested that thesewere notantiidiotype an-tibodies. To confirm this suspicion, experimentswereperformed
withtwodifferentpurified multiple
myeloma monoclonalF(ab')2
fragments. Anti-F(ab')2IgG
from patients A and II reacted equally well with both monoclonal F(ab')2 fragments. Anti-F(ab')2 Ig from patient A reacted equally well with both mono-clonal F(ab')2 fragmentsaswellasF(ab')2 fragments from patients A andBaswell as control H (Table VI). These dataindicate lack of idiotype specificityofanti-F(ab')2antibodies forF(ab')2fragments.
Discussion
These data indicatethe presence ofhigh titerIgG anti-F(ab')2 antibodies in the seraand immune complexes of thrombocy-topenic patientsatriskforAIDS,orwithAIDS,thatcorrelates
TableV.Binding RatiosofATPIgGtoF(ab')2Fragments ofHSITP Patients, Controls, and A TPPatientsCompared toBinding of Control IgGtoControlF(ab')2 Fragments*
F(ab)2
fragments PurifiedIgG immunoglobulin of ATP patients
Homosexuals J K L M N 0 P
A 0.8 0.9 0.9 0.9 1.0 0.9
B 0.9 1.1 0.7 0.7 1.1 0.8
C 1.1 1.3 0.7 1.3 1.1 0.8
-D 0.9 0.9 0.9 1.0 0.9 0.8
E 1.2 1.1 1.2 1.0 1.0 1.2
-F 1.1 1.0 0.9 1.0 0.9 1.0
Gcontrol 0.8 0.9 0.7 0.9 1.0 0.7
ATPPatients
J 0.8 0.9 - 1.1 0.9
K 1.2 1.1 - 1.2 1.6
L 1.0 1.1 - 0.8 0.9
M 0.9 0.9 - 0.8 0.9
N 1.1 1.0 - 1.0 0.9
O 1.0 1.1 - 0.8 1.0
P 1.0 1.5 - 1.1 1.0
TableVI.Binding RatiosofHomosexualPatients' IgG to F(ab')2 Fragments ofMonoclonalIgGofMultiple MyelomaPatients, ComparedtoBindingofControl IgG to Control F(ab')2 Fragments *
PurifiedIgG immunoglobulin of homosexual patients
F(ab')2fragments A II
MIgG
1 (kappa) 2.4 2.9MIgG 3(kappa) 2.2 2.1
A 2.2
B 2.5
Hcontrol 2.8
* MIgG IandMIgG 3referto
F(ab')2
fragmentsofpurified mono-clonal IgGobtained fromtwopatientswithmultiple myeloma.A and B arepatients with HSITP;IIisahomosexualpatientwithKaposi's sarcomaandITP;Hisacontrolsubject.Theratios refertothemeansof 2-5experiments.
with the concentration of circulating immune complexes (data
determined
by the PEG method). Thisobservation
was suggestedfrom
preliminary studies
onimmune complexes in these patientsthat
revealedspecific antibodies
against a host of viral antigens, with absence of the specific antigens. In contrast, patients withclassic autoimmune thrombocytopenia
haveneither
circulating
immune
complexes detected by this method, nor anti-F(ab')2antibodies.
However, similaranti-F(ab')2
antibodies have recently beenreported in the seraof patients with
rheumatoid arthritis (12-15) and systemic lupus erythematosus (12, 15-17), disordersof immune regulation
that areassociated
with the presence ofcirculating
immune complexes as well as autoantibodies.The markedly elevated platelet-bound IgG, IgM, and
C3C4
in these HSITP or NITP patients (2, 4) has suggested a role for immune complexes in theinduction of thrombocytopenia.
Itislikely
that someof
the platelet-bound IgG and C3C4 represent immune complexes and that someof
the immune complexes may be composed of IgG anti-F(ab')2 complexes. Indeed, anti-Fab'antibodies
have recently been demonstrated in thecircu-lating immune
complexes of patients withrheumatoid arthritis
(18),
andanti-F(ab')2 antibodies
inindividuals
immunizedagainst
keyholelimpet hemocyanin
(KLH) (19).Theanti-IgG antibody found in these patients was not
rheu-matoid factor
(IgM against IgG-Fc), nor IgM anti-IgG, but rather IgGanti-F(ab')2.
Thereactivityfor
F(ab')2 was broad withreac-tivity against control F(ab')2
aswell asautologousorhomologousF(ab')2
fragments for most of the homosexual patients studied. However,3of 12 HSITP and 5 of 6 NITP patients appeared to have limited reactivity against specific F(ab')2fragments.
Thissuggested
thepossibility
that someof
theseanti-F(ab')2 antibodies
might
beantiidiotypes.
Attempts to demonstratespecificity
wereunsuccessful,
since F(ab')2fragments from
two differentpurified
monoclonalIgG preparations of multiple
myelomapatients
re-acted equally well with differentpositive reacting anti-F(ab')2
preparations.
Nevertheless, sinceaffinity-purified anti-F(ab')2
antibodieswerepolyclonal,
(kappa and lambdalight chains),
thepossibility of specific antiidiotype
antibody has not been ruled out. For example, if anti-AIDS-associated retrovirusan-tibody
canbeaffinity purified, it
would be ofinteresttodetermine
whetheranti-F(ab')2 antibodies
could blockbinding of
anti-AIDS-associated retrovirus antibody to its epitopes on the virus. Indeed,IgG anti-F(ab')2 antibodies with blocking activity against anti-DNA antibody (16, 17), as well as antitetanus toxoid an-tibody (17) have recently been reported in patients with SLE; whereas anti-F(ab')2 antibodies of patients with rheumatoid
ar-thritis did
notblock anti-DNA antibody of SLE patients (16). Inaddition, individuals immunized with KLH have anti-F(ab')2antibodies
against anti-KLH antibody in their circulating im-munecomplexes (19). The etiology of the circulating immune complexes as well as the autoimmune and alloimmune anti-F(ab')2 antibodies in these patients at risk for AIDS, or with AIDS,is not readily apparent. Experimental anti-antibodies have been produced after immunization of rabbits with antigen as well asantigen-antibody complexes, however (20-22). It has been postulated thatimmune
complexformation
results inunfolding
of sites
onthe autologous IgG molecule that could expose buriedantigenic determinants
notrecognized
by the host (20). Indeed, human IgGanti-F(ab')2 or anti-IgG antibodies have been noted in normal subjects (23, 24) that could be adsorbed out or dis-sociated with immune complexes (23, 24), or F(ab')2 fragments (23), but not with intact IgG (23). Repeated antigenic stimulation in patients at risk for AIDS could lead to the veryhigh
levelof
circulating immunecomplexes
noted, which inturnmaycon-tribute
to theformation of
anti-F(ab')2 antibodies,
thus perpet-uating immune complex formation. It ispossible
that thecir-culating
immunecomplexes
andanti-F(ab')2result from expo-sure tothe AIDS-associated retrovirus aswellas other viruses. Inaddition, since
AIDSis
adisorder of
immuneregulation,
it islikely
thattheseanti-F(ab')2 antibodies,
whicharepresentat lowlevels
insome normalcontrol
subjects (12, 16, 23),
have losttheir
immunoregulatory
controlmechanisms.
Adisorder of downregulation of anti-F(ab')2 antibody production
thusmight
reflect perturbation of
theTcell-B cell networkby
theAIDS-associated
retrovirus.
Itispossible
that some, ifnotall,
of thepostulated
mechanismsareresponsible
for the elevated circu-lating immune complexes andanti-F(ab')2 antibodies
inpatients
atriskfor
AIDS,
orwith AIDS.Acknowledgments
The authorsareindebtedtoDr.BlasFrangioneof New YorkUniversity Medical School for the gift oftwo monoclonal myelomaantibodies, IgG1 and IgG3.
Supported by grant HL-13336-16 of the National Heart, Lung and BloodInstitute and grant
C-00534
from the New York State Department of HealthAcquired Immune Deficiency SyndromeInstitute.Dr.Yuwassupported in part byagrant from thePeople'sRepublicof China.
References
1.
Morris,
L.,A. Distenfeld, E.Amorosi, and S.Karpatkin. 1982. Autoimmunethrombocytopenic purpura in homosexualmen.Ann. Int. Med.96:714-717.2. Walsh, C. M.,M.A.Nardi, and S.Karpatkin. 1984. On the mech-anism ofthrombocytopenicpurpurainsexually-activehomosexualmen.
N.Engl.J.Med. 311:635-639.
3.Walsh, C. M.,R.Krigel,E. T.Lennette, and S.Karpatkin.1985. Thrombocytopenia in homosexualpatients: prognosis,responseto ther-apyandprevalence ofantibodytotheacquired immunodeficiency syn-drome-associated retrovirus.Ann.Intern.Med. 103:542-545.
4.Savona,S.,M. A.Nardi,E. T.Lennette, and S.Karpatkin.1985. Thrombocytopenicpurpura in narcotics addicts.Ann.Intern.Med. 102: 737-741.
5.Ratnoff, 0. D., J. E. Menitove, R. H. Aster, and M. M. Lederman. 1983.Coincident classic hemophilia and 'idiopathic' thrombocytopenic purpura inpatients undertreatmentwithconcentratesofanti-hemophilic factor(Factor VIII). N.Engl. J. Med. 308:439-442.
6. Karpatkin, S., and M. A. Nardi. 1985. On the mechanism of thrombocytopenia in hemophiliacs multiply-transfused withAHF con-centrates.Blood.66:A.
7. Karpatkin, S. 1985. Autoimmune thrombocytopenic purpura. Semin.Hematol. 22:260-288.
8. Lennette, E.T. 1979.Applications of immune adherence hem-agglutination assaytodiagnostic serology. InDiagnosisof Viral Infections: The Roleof the Clinical Laboratory.D. A.Lennette, S.Specter, andK. Thompson, editors.University Park Press, Baltimore. 73-83.
9. Van Leeuwen, E. F., J. ThM. van der Ven, C. P. Engelfriet, and A. E.von demBorne. 1982. Specificity of autoantibodies in autoimmune thrombocytopenia. Blood. 59:23-26.
10.McDougall, J.S., P. B. Redecha, R. D. Inman, and C. L. Christian. 1979.Binding of immunoglobulin G aggregates and immune complexes in human sera to staphylococci containing protein A. J. Clin. Invest. 63: 627-636.
11.Nisonoff, A., F. C. Wissler, L. N. Lipman, and D. L. Woernley. 1960.Separation of univalentfragmentsfrom the bivalent rabbit antibody molecule by reduction of the disulfide bonds. Arch. Biochem. Biophys. 89:230-244.
12.Heimer, R.,L.D.Wolfe, and J. L. Abruzzo. 1982. IgM and IgG anti-F(ab')2antibodies in rheumatoid arthritis and systemic lupus ery-thematosus.Arthritis Rheum. 25:1298-1306.
13. Birdsall, H. H., M. D. Lidsky, and R. D. Rossen. 1983. Anti-F(ab')2antibodies in rheumatoid arthritis. Arthritis Rheum. 26:1481-1492.
14.Persselin, J. E., and R. H. Stevens. 1985. Anti-Fab antibodies in humans. Predominance of minorimmunoglobulinGsubclasses in rheu-matoid arthritis. J. Clin. Invest. 76:723-730.
15.Nasu, H., D. S. Chia, D. W. Knutson, and E. V.Barnett. 1980.
Naturally occurringhuman antibodies tothe F(ab')2 portion of
IgG.
Clin. Exp. Immunol. 42:378-386.
16.Nasu, H., D. S.Chia, 0.Taniguchi,and E. V. Barnett. 1982. Characterization of anti-F(ab')2 antibodies in SLE patients. Evidence for cross-reactingauto-anti-idiotypic antibodies. Clin. Immunol. Immuno-pathol. 25:80-90.
17.Silvestris,F.,A.D.Bankhurst,R. P.Searles,andR. C.
Williams,
Jr. 1984. Studies of anti-F(ab')2 antibodies and possibleimmunologic controlmechanismsin systemic lupus erythematosus. Arthritis Rheum. 27:1387-1396.18.Birdsall,H.H.,M.D.Lidsky,and R. D. Rossen. 1983. Anti Fab' antibodies in rheumatoid arthritis. Measurement of the relativequantities incorporated in soluble immune complexes inseraand supernatants from culturedperipheralblood lymphocytes. Arthritis Rheum. 26:1481-1492.
19.Birdsall, H. H., and R. D. Rossen. 1983. Characterization ofanti-F(ab')2antibodies in human sera; identification of soluble immune com-plexes that contain hidden anti-KLH and blockinganti-immunoglobulins following immunization with keyhole limpet haemocyanin. Clin.Exp. Immunol. 53:497-504.
20. Williams, R.C., Jr., and H. G. Kunkel. 1963. Antibodies to rabbit gamma globulin after immunizing with various preparations of autologous gamma-globulin. Proc. Soc. Exp. Biol. Med. 112:554-561.
21. Abruzzo, J. L., and C.L.Christian. 1961. The induction of rheu-matoid factor-like substance in rabbits. J. Exp. Med. 114:791-806.
22. Aho, K., and 0. Wagner. 1961. Production of 'anti-antibodies' in rabbits. 1961.Ann.Med.Exp. Biol. Fenn. 39:79-87.