Polymorphism of lymphocyte
function-associated antigen-1 demonstrated by a lupus
patient's alloantiserum.
K D Pischel, … , V L Woods Jr, H G Bluestein
J Clin Invest.
1987;
79(6)
:1607-1614.
https://doi.org/10.1172/JCI112996
.
We have found a human serum, E27, obtained from a multiply transfused patient with
systemic lupus erythematosus, which immunoprecipitates the lymphocyte function
associated antigen-1 (LFA-1). The immunoprecipitated molecules were identified as the
LFA-1 alpha and beta chains by their comigration on PAGE, two-dimensional
SDS-PAGE, and by sequential clearance experiments. Serum E27 did not immunoprecipitate
LFA-1 from autologous cells, though LFA-1 molecules were present. In contrast, serum E27
immunoprecipitated LFA-1 from most but not all normal donor lymphocytes. Thus, serum
E27 defines two serological phenotypes of LFA-1. 95% of normal individuals tested
exhibited the LFA-1 phenotype precipitated by serum E27. Serum E27 appears to be
directed at determinants of the LFA-1 alpha-chain and not the beta-chain since it
immunoprecipitated LFA-1 molecules but not the Mac-1 molecules. Additional evidence for
the alpha chain specificity was provided by immunoprecipitation of mouse-human
heterohybridoma cells. LFA-1 was immunoprecipitated by serum E27 from mouse-human
heterohybridoma cells expressing the human alpha-chain, not from a hybrid cell line
expressing the human beta-chain. Together these findings demonstrate an antigenic
polymorphism of the human LFA-1 alpha-chain molecule.
Research Article
Find the latest version:
Polymorphism of Lymphocyte Function-associated Antigen-1 Demonstrated
by
a
Lupus Patient's Alloantiserum
Ken D.Pischel, Steve D. Marlin, TimothyA.Springer, Virgil L. Woods, Jr., and Harry G. Bluestein
Division ofRheumatology, DepartmentofMedicine, Universityof California MedicalCenter, San Diego, California92103; Dana FarberCancer Institute, Harvard Medical School,Boston,Massachusetts02115
Abstract
We havefound a human serum, E27, obtained from a multiply transfused patient with systemic lupus erythematosus, which
immunoprecipitatesthelymphocytefunction associated
antigen-1 (LFA-1). Theimmunoprecipitated molecules were identified
as the LFA-1 alpha and beta chains by theircomigration on
SDS-PAGE, two-dimensional SDS-PAGE, and by sequential clearance experiments. Serum E27 did notimmunoprecipitate
LFA-1 from autologous cells, though LFA-1 molecules were present. Incontrast, serumE27 immunoprecipitated LFA-1 from most but notall normal donor lymphocytes. Thus, serum E27
defines twoserological phenotypes of LFA-1. 95% of normal individuals tested exhibited the LFA-1 phenotype precipitated by serum E27.Serum E27 appears to be directed at determinants of the LFA-1 a-chain andnottheE-chainsince it
immunopre-cipitated LFA-1 molecules but not the Mac-i molecules. Ad-ditionalevidence for thealpha chainspecificitywasprovidedby immunoprecipitation of mouse-human heterohybridoma cells. LFA-1 was immunoprecipitated by serum E27 from mouse-hu-man heterohybridoma cellsexpressingthe humana-chain,not
from a hybrid celllineexpressingthe human
f-chain.
Together these findings demonstrate an antigenic polymorphism of the humanLFA-1 a-chain molecule.Introduction
The lymphocyte function associated
antigen-i (LFA-1)'
mole-culesarecellsurfaceglycoproteins importantinleukocyte adhe-sion processes. Theyareheterodimers comprisedofan a-poly-peptide chain of 177,000molwtandafl-polypeptide
chainof95,000mol wt,noncovalently linked together (1).LFA- I isone
ofa
family
ofstructurally similarmolecules that also includesp150,95,and Mac-I (the complement receptorCR3). Mac-I is
recognizedby monoclonalantibodies (MAbs)Mac- 1, Mo 1, and OKM1, andiscomposed ofadistinct a-chain of165,000 mol
wtassociatedwitha95,000-mol wt(3-subunit. TheLFA-I and Mac-I
,-subunits
areapparently identicalasindicatedby
iden-tical isoelectric
focusing
patterns(2)
andidenticalpeptide
mapsAddressreprintrequests to Dr.Pischel.
Receivedfor publication27October 1986 andinrevisedform17February
1987.
1.Abbreviations usedinthis paper:LFA-1,leukocyte function associated antigen-1;MAb, monoclonalantibody;NW, nylon wool;SLE,systemic
lupus erythematosus; 2D-PAGE, two-dimensional gelelectrophoresis;
2-ME, 2-mercaptoethanol.
J. Clin. Invest.
X TheAmericanSocietyfor ClinicalInvestigation,Inc.
0021-9738/87/06/1607/08 $1.00 Volume79,June 1987, 1607-1614
(3, 4). The LFA-1, Mac-I family also contains a third
hetero-dimer,p150,95 composed ofadistinct 150,000mol wta-chain linked with the common 95,000-mol-wt (-chain. Themembers
ofthis family are all heterodimers sharing a common(-chain
complexed with a unique a-subunits distinguished by
size,
iso-electric focusing point, and peptide patterns(2).
The LFA-1 molecules are expressed on the surface of lym-phocytes, thymocytes, monocytes, granulocytes, and bone
mar-row cells (5). The Mac-i molecules are present on human monocytes,granulocytes, and natural killer cells, but areabsent
(or minimally present) on lymphocytes. The p150,95 hetero-dimerhas been less wellcharacterized but it appears to be
rec-ognized by the MAb Leu M5 and is present upon monocytes and, in low density, on granulocytes and large granular
lym-phocytes.
Little structural variability of any of these molecules has
beendescribed in man. Recently, a polymorphism of the CR3 complement receptor that bindsC3bihas been reported (6). A murine MAb was found to immunoprecipitate CR3 from 14% ofindividuals. That MAb did not react with LFA-1 and was thoughttoreactwith anepitope on the Mac-I a-chain.
No structural variationof the human LFA-1 molecules has beenpreviously reported. We haveidentified a human serum, obtainedfrom a patient with systemic lupus erythematosus who hadpreviously received multiple blood transfusions, that im-munoprecipitates LFA-I from lymphocytes of most but not all
individuals. This serum appears to identify a phenotypic vari-ationof the human LFA-1 molecules.
Methods
Cells andlymphocytepreparations.Peripheralblood mononuclearcells (PBMs)wereobtained byFicoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) density gradientcentrifugation(7),pelleted,andwashed thrice with RPMI. These cellswereeither useddirectlyorfurther pro-cessedbypassagethroughanylon wool(NW) column(8)toyieldNW T-enriched cells (T cells) before iodination.
Mouse X human heterohybridoma cells expressing LFA-l hybrid
molecules have beenpreviouslydescribed (9). HumanPHAblasts, ob-tainedfrom theparentofaLFA-l Mac-l deficientpatientand themurine cellline BW5147werefused andhybridsselected for theexpressionof humana-orhuman(3-chainsby flowcytometry. The MBXBW5+cells retain thehumanchromosome 16 andexpresshybridLFA- I molecules composed ofhumana-subunits associated withmouse(3-subunits.The MBXBW5-hybridswerederived fromtheMBXBW5'but have lost the human chromosome 16 andno longerexpress the human LFA- 1
a-chain.The
MB#33
hybridsretainhumanchromosome21 andexpress hybrid LFA-l moleculescomposed ofhuman(-chain withmousea-chainsand theMB(3-cellswerederived fromtheMB(33+cells buthave lost the human chromosome 21 and nolongerexpressthe human
(3-chain (9).
Radiolabeling. Lymphocytesweresurface iodinated with
lactoper-oxidase byamodificationofthe method of Bauretal.(10)aspreviously
(New England Nuclear,NorthBillerica,MA) and 10 Mllactoperoxidase
(Calbiochem-Behring Corp., SanDiego,CA, grade, 100 IU/ml) and 25
Alof 0.03%H202.After 5 min 5
Al
oflactoperoxidaseand 10 !d of0.03% H202 were addedand at10 min 10Ad
of 0.03% H202wasadded.After 15min thereactionwasstoppedby the addition of cold RPMI, cells centrifuged and washed six times with media.Viabilitywas>95%before andafter iodinationasjudgedby trypan blue exclusion.Antibodies. Serum E27 was obtained from apatientwith systemic lupuserythematosus(SLE). Theserum washeatinactivatedat560Cfor 30min, aliquoted, and frozenat -80'C untiluse. MAbTSI/22 and MAb TSI/18areLFA-l chainspecificmonoclonal antibodiespreviously described(2, 12) thataredirectedata-chain and(3-chain determinants,
respectively. The MAb OKM 1, whichrecognizesanepitopeonthe Mac-1 molecule, was purchased from OrthoDiagnostics (Raritan,NJ).
Extraction and immunoprecipitation. Surface iodinated cellswere usually incubated withserum orMAbpriortothesolubilization of the cell membranewith nonionic detergent. 50Mlofserum or1 Mlof MAb asciteswereincubated with intact cells for 30 minat40Candthe cells washed once withmedia. Cells were then lysedwithTris-bufferedsaline containing 0.5% NP40 andimmunoprecipitatedaspreviouslydescribed
(11).Briefly, lymphocyteswerelysedbysuspendingcells in 0.15MNaCl,
0.01 MTrispH 7.4,0.5%NP-40 (TBS-NP-40) containing 0.05mM N-a-p-tosyl-L-lysine choromethyl ketone (TLCK, Sigma Chemical Co., St. Louis, MO), 0.06mML-I-tosylamide-2-ketone (TPCK, Sigma Chemical Co.) and 0.2mMphenylmethylsulfonylfluoride(PMSF,SigmaChemical Co.). After incubation for 30 minat40C,cellularlysateswere
ultracen-trifugedat100,000gfor 30 min at 4VC andaliquotswereprecleared by incubationwith100 Mlof 10% Sansorbin(Staphylococcusaureuslacking protein A,Calbiochem-BehringCorp.)or50Mlof Sepharose4Bfor15 min followed bycentrifugation (microfugeB, Beckman Instruments, Inc., Palo Alto,CA) for4min. The immune complexeswerecollected with 50,l of 10% S. aureus Cowan's strainI(SAC)(Calbiochem-Behring
Corp.),orwith 10 MlSepharose-proteinA,orwith 10Mlofaffinity purified
rabbit anti-mouse kappa chainconjugatedtoSepharose4B(I mg/ml) accordingtothemanufacturer's instructions(PharmaciaFineChemicals),
pelleted and washed six times with TBS-NP-40.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Washedimmunoprecipitateswereeluted and denatured with 50 Ml 1%SDS and 5%2-mercaptoethanol(2-ME) byheatingat100°C for 1-2minandanalyzed by 8or10%Laemmli SDS-PAGE(13)along with14Cmolecular weight markersmyosin (200,000 mol wt), phosphor-ylaseB(92,000 mol wt), bovine serum albumin(68,000 mol wt), oval-bumin (45,000 mol wt),chymotrypsin (25,000molwt),beta-lactoglobulin (18,400molwt), andcytochrome c(12,400molwt) (Bethesda Research Laboratories, Gaithersburg, MD). Gelswerefixed in 10% acetic acid,
109%2-isopropyl alcohol then dried andautoradiographedwithprefogged XAR-5 film (Eastman Kodak, Rochester, NY) usingan intensifying screen (Cronex Lighting-Plus, Dupont, Wilmington, DE) for 1-7 d at-800C.
O'Farrell two-dimensionalgelelectrophoresis(2D-PAGE). 2D-PAGE
wascarried out after the method ofO'Farrell (14) as previously described
(11).Sampleswereelutedfrom the SAC or Sepharose immunoadsorbents by incubation with 8Murea, 5% 2-ME for 60minat220Candapplied toprefocused6%acrylamidetube gels with 10mMphosphoric acid (pH 3.9) and 50 mM Tris base (pH 9.9) electrode solutions, and focused for
10h at1,000V.The gels were thenequilibrated with SDS-PAGE diluent buffer and run on 8% SDS-PAGE in the second dimension and fixed and processedasdescribed above forone-dimensional SDS-PAGE.
Results
Serum E27immunoprecipitates two polypeptides that
co-elec-trophoresed
with the LFA-1 a-andj#-chains.
In order to detectlymphocyte surface molecules reactive with the lupus serum E27,
immunoprecipitationexperimentswere performed. NW purified
Tcells wereisolated, and their cell surface proteins were iodinated
with
lactoperoxidase
and reactedwithserumfromlupus patient
E27.After detergent-solubilization, the iodinated surface mol-ecules boundto antibodywere immunoprecipitated and
ana-lyzed bySDS-PAGE (Fig. 1). SerumE27immunoprecipitated two iodinated cellsurface molecules whose molecular weights wereestimated to be 175,000 and 95,000 (lane 5). In addition, nonspecificbands of 80,000 and 50,000 molwt are seeninthe serumE27precipitateand also innegative control samples (lanes Iand2). InsomeexperimentsserumE27alsoprecipitated chains of43,000 and 12,000 molwtwhichcomigrated with HLA chains (data notshown). Because the twolarger E27-immunoprecipi-tated molecules had similar molecular weightstothe subunits ofLFA-1, their electrophoretic migrationwascompared with that ofimmunoprecipitates produced by LFA-l specific
mono-clonal antibodies TS1/22 and TSI/18 (Fig. 1, lanes 3 and 4). The 175,000-mol-wt band immunoprecipitated with E27 co-electrophoresed with the LFA-l a-chain and the 95,000-mol-wt bandcoelectrophoresed with the LFA-Ip-chain. This suggested that serum E27 contained antibody reactive with the LFA-l
molecules.
To morecritically compareLFA- 1 and the molecules pre-cipitated withE27, theywereanalyzed by O'Farrell 2D-PAGE. Surfaceiodinated cellswerereacted withserumE27or LFA- 1-specific MAb and immunoprecipitates prepared. The samples weredenaturedwith urea, reduced with 2-ME and subjected to isoelectricfocusing for 10 h followed by SDS-PAGE in the second
1
23
4
5
MW(KD)
200
92
68
PPT
AB:
LFA
RX:
--o-
LFA-I
a
--
LFA-1,/
fb
g
3
a
E27
_--++
++Figure1.Comparisonoflymphocytesurfaceantigens precipitated
with humanserumE27and LFA-Iby SDS-PAGE. Radioiodinated lymphocyteswerereactedwith (lane1)noAb; (lane2)controlMAb;
(lane 3) anti-LFA-l a-MAbTS1/22; (lane 4)anti-LFA-ftMAbTSI/
18;and(lane 5) humanserumE27 andimmunoprecipitates prepared. Immunoprecipitateswerereduced andrun on8% SDS-PAGE. Molec-ularweightmarkersareshownatleft.
1608 Pischel,Marlin,Springer, Woods, and Bluestein
-dimension and autoradiography. The molecules immunopre-cipitated by the patient'sserumcomigratedexactlywiththe LFA-1 a-and 13-chains precipitated with the LFA-1 specific MAb (Fig. 2). Each of the 1 75,000-mol-wt chains focused between a pHrange of 4.3 to 4.6 and each of the 94,000-mol-wt chains focused at positions from pH 4.6 to 5.0. This resultstrengthened
thesuggestion that serumE27containedantibodies to the LFA-1 molecules.
Preclearance ofLFA-J moleculesremovesE27 reactive mol-ecules. The identity of the molecules precipitated by E27 as
LFA-1 wasconfirmed by preclearance experiments. Aliquots of
ra-dioiodinated lymphocyteswerefirst incubated with either MAb specific for the LFA-l a-chain, LFA-lI3-chain, or a control MAb, andthen theAb-complexed molecules were removed. Next, the precleared extracts were immunoprecipitated with serum E27
or LFA- 1 specific MAb andanalyzed by SDS-PAGE. Clearance with MAb to LFA-1 a-or1-subunitsresultedin the substantial removal of the LFA-1 molecules(Fig. 3, lanes 2, 3, 5, 6) and the nearly completedepletionof the 175,000-mol-wt molecule normallyimmunoprecipitatedby serumE27(Fig. 3, lanes 8 and 9).Together the electrophoretic comigrationofE27-precipitated moleculeswith LFA-1 subunits and clearance of E27 chains by LFA-l MAb established the identity of the two E27 reactive molecules as thea-and13-chainsof LFA- 1.
SerumE27isalloreactive and not autoreactive. Lupus patient E27, whose serum contained the anti-LFA-1 antibodies, had received numeroustransfusions10-20yragofor severe recurrent hemolytic anemia. This raised the issue ofwhether the anti-LFA-1 antibodies detected in her serum were a form of
auto-- IEF- +
MW(KD) A. NAB
TSl/22
200
_muibug.
-.-LFA-I
a
92-,00
200-40
.g5k.
1 2 3
MW(KD)
200-a
-
/3-
68-CLEAR AB:
PPT AB:
,N
a_8
a
4
5 6
45
:/3
:7 8
9
N
a
,X3,
E27
Figure3.Sequentialimmunoprecipitationof LFA-1 and E27-reactive molecules. Radiolabeledlymphocytelysateswerealiquotedand sam-plespreclearedeither with control MAb boundtoSepharose(lanes1, 4, 7),anti-LFA-I a-MAb TS1/22 (lanes 2, 5, 8),oranti-LFA-1
13-MAbTSI/18 (lanes3, 6, 9).Immunoprecipitateswerethen pre-paredfrom theprecleared lysates with either the anti-LFA-1 aMAb TS1/22 (lanes1-3), anti-LFA- 1 3-MAbTS1/18(lanes 4-6),orserum E27(lanes7-9) andrun onreduced 7% SDS-PAGE.
reactive antilymphocyte antibodies,such as arepresentin some
lupussera, or werealloreactive antilymphocyteantibodies. To
testthis, PBMs from the patient andanormal individual were
isolated, radioiodinated, and reacted with serum E27.
Immu-noprecipitates from the patient and control individual were
comparedbySDS-PAGE(Fig. 4).SerumE27 was found to
pre-1 2
34
5 6
LFA-1p
a-4.
a--L [27
-LFA-1 a
--LFA-13 92
--Figure2.Comparison of E27-immunoprecipitatedmolecules and LFA-I chainsonO'Farrelltwo-dimensionalgels.Radiolabeled
lym-phocyteswereimmunoprecipitated with anti-LFA-Ia-specificMAb TS1/22(A) andserum E27 (B); run on twodimensionalO'Farrell gels andautoradiographed. Isoelectric focusingwasfirstconductedfrom
lefttoright followed by reduction with 2-MEandSDS-PAGE from toptobottom.
CELLS:
NEl
AB:
OKM1
NE,
E27
NE,
LFA*1
Figure4.Testof autologous cells forLFA-1 reactivitywithserum
1 2
3
4 5 6
7
8
9101112131415161718
...LA.1F
.. : . , ... : . :.:;::i;Isk M
*
l,.ai,..-.*f---.,*
tm * r ; r
a~~~~~~~~~~~~~~~~~~~~~.Fj
m L r A lFigure5.Screenof healthy individuals for theability
ofserum E27 toprecipitateLFA-1. Radiolabeled PBMsfrom 18 healthy individualswere
immunopre-cipitated withserumE27andrun on 10%SDS-PAGE andautoradiographed. Thepositionof theLFA-1 a-chain is markedattheright.
cipitateLFA-1 from the control cells(lane 3)but did not pre-cipitateLFA-l fromautologouscells(lane 4).TheLFA-l family
ofmolecules is presentonthepatient'scellsasdemonstratedby
theirimmunoprecipitation byMAbtoOKM1(lane 2)and LFA-1 (lane 6). Although the f3-chainof LFA-1 isnotseen on this
reproductionof thegel (lanes 5 and6)it is presentandmore readilyseenwithlongerexposure.Thus,itisclear that the lack ofautoreactivity byE27wasnot due to modulation ofLFA-1
byautoantibodiesorlack ofexpressionof these moleculeson
E27'scells.ThefindingthatserumE27 reacted withlymphocytes
obtained from several donorsbut not withautologousLFA-l
molecules suggested the patient expressed a variant form of LFA-1.
LFA-iphenotypic variation in thepopulation. PatientE27 couldmanifestararemutation of theLFA-1 moleculesorexpress
anallotypicvariant alsoexpressed byother individuals.Inorder to testthesetwopossibilities,PBMs from40healthyindividuals
werescreened for reactivitywith E27by immunoprecipitation
andSDS-PAGE. 2 of the 40 individuals(5%)hadnoapparent LFA- molecules immunoprecipitable by serum E27 (Fig. 5,
lanes6and 14).Sinceanimmunodeficiency syndrome involving
the absenceof the three related moleculesconstitutingthe
LFA-1 family has been described(15-23), the cellsfrom thetwo
in-dividuals who didnot reactwithserum E27were reexamined todetermine ifthey expressed LFA-l molecules on their cell surfaces. Both LFA-l a-and LFA-1 (3-chains could be
immu-noprecipitated fromcells ofbothindividualsby specific MAb
(Fig. 6, lanes 4, 5, 7, 8) but again,asintheprevious experiment,
their LFA-l molecules did notreact withserum E27 (Fig. 6,
lanes 6, 9). 5% ofindividuals from the normal population
ex-aminedthusexpressedaphenotypic variant ofLFA- Ithatdoes
not reactwithserumE27.
Physicochemical propertiesofallotypic LFA-Imolecules. The size andcharge characteristics of the LFA-1 molecules isolated from one of thenonreactive individualswere compared with thoseof themorecommonLFA- I phenotype by O'Farrell
2D-PAGE(Fig. 7). By this analysis the ,3-chain prepared fromthe E27-nonreactive individuals appeared to be identical to the
LFA-1 13-chain of the E27-reactive individuals. The positions of
the a-chains were also identical and although the more basic
spotsarefainterinthisfigure, other gels showed identical
inten-sitiesaswellaspositions for the a-chains. This result indicated
12 3 4 5 6
7
89MW(KD)
200-92-
s
-*-F*AFAI3
'."". .' s '' ..
45-W Nm t
PPT AB:
a8E?7
E2
,a
E27 a38 E27
DONOR:
B
CE27-
REACTIVE
NONREACTIVE
Figure 6. LFA-1 precipitation fromserumE27-nonreactive
individu-als.LymphocyteswerepreparedfromindividualA,previouslyshown tohave LFA-1 precipitated byserumE27(lanes 1-3),and individuals B(lanes 4-6) and C (lanes 7-9) previously shownnot tohaveLFA-1 immunoprecipitated byserumE27. The cellswereradioiodinated and immunoprecipitated either withMAbspecificforLFA-1 a-subunit
(lanes1,4, 7), LFA-1#subunit(lanes 2, 5, 8),or serumE27(lanes 3, 6, 9). Sampleswerereduced andrun on8%SDS-PAGE and
autora-diographed.
1610 Pischel, Marlin, Springer, Woods,and Bluestein
MW(KD)
200-*
92
-PPT
AB:
IE27
IEF
--a
---
-|
200-92- mm
..s...e. X..
*~~~~~~~
. ;;g... .-a
Figure 7.Comparison ofLFA-l moleculesfrom E27-reactiveand nonreactiveindividualsbytwo-dimensionalgelelectrophoresis. Lym-phocyteswerepreparedfrom individualA,previouslyshown to have LFA-1 precipitatedbyserum E27, and individualB, previouslyshown not tohaveLFA-l precipitatedby serum E27. The cells were radioio-dinated andLFA-l immunoprecipitated fromeachwithMAb TS1/22 and samples reduced andrunonO'Farrell two-dimensionalgels.
washed inNaCl-Tris-NP-40 solution
(pH 8.0).
The chainsre-mainingboundtotheantibodyandSepharose proteinAwere
denatured andrun onSDS-PAGE(Fig. 8). Aftertreatmentat
pH
11.2,
complete disappearance of the LFA-1 fl-chain andpreferentialretention of the a-chainswasobserved. Since after
treatmentswithpH 11.2 and 11.5 the E27antibody-precipitate partially retained the LFA-1 a-chain without anyapparent
(3-chain present (even upon prolonged film exposures) the anti-LFA-1 reactivityobserved islikely specifictothe a-chain itself andnotdirected towards either thefl-chain orana-fl combi-natorial determinant. WhenLFA-l samples
immunoprecipitated
with thefl-chain specific MAb TS1/18 were treated with
pH
11.2both thea-andf-chain completely disappeared (datanot
shown). Thus, the pattern of a-chain retention observed with E27isnotthe result of nonspecific retention of the a-chain.
Additional evidencesupportingthe a-chainspecificityofthe E27serum comesfromimmunoprecipitation experiments
using
other celltypes. Inextractsof PBMs there existsa mixture of LFA-1 molecules with Mac-l molecules. If the E27 antibodies
werespecific for the f-chain, the immunoprecipitates would be
predictedtocontaintwotypesofa-chains. Thiswasexamined
by preparing peripheral blood mononuclear cells containing
lymphocytes and monocytes, radioiodinating them and
im-pm
Co
an
pH
3
4
1 23 4
567
8
thatlarge alterations of thea-orfl-chain structures, suchas a
sizable peptidedeletion orlackofcarbohydrate do notexist. Instead, a minor difference ofthe LFA-1 chains, such as an aminoacidsubstitution, which isnotdetectableby the method of O'Farrell 2D-PAGEanalysis,probablyaccountsforthe
pres-ence orabsenceofreactivitywithserumE27.
Chainspecificity ofserum E27.Itwasof interestto determine the portion ofthe LFA-1 molecules recognizedby serum E27 since thoseantibodiesprobablyrecognizethe alteredportionof the LFA-1 molecules. Various approaches have been used to
determinethis. FirstwhenLFA-1 molecules were precipitated
byserumE27 and theintensity ofthe a-chain band compared with that of thef-chain,severalexperiments demonstrated that thea-chain band was moreintensethan thefl-chainband (e.g.,
Fig. 1).This patternofintensities is very similar to that produced
bythe LFA- 1 a-chainspecificMAb TS1/22 and contrasts to the nearlyevenintensity ofthe a- and
f-chains
usuallyobserved inimmunoprecipitatesproducedwith theLFA-1
fl-chain
specific MAbTSI/18.Asecond approach to theidentificationof the chain
recog-nizedby serum E27 has been to prepare LFA-1
immunoprecip-itates containingboththe LFA-1a-and
f-chains
andsubjecting theseimmunoprecipitates
toincubation conditions withvarious pHstopartially dissociatethe LFA-Isubunits.Similar treatments have been used to partially denature and dissociate the a- and f-chains ofLFA-Itoshow thepreferentialretentionofonechainorthe otherdependingupontheepitope specificity ofthe MAb
(7).E27andLFA-l immunoprecipitatespreparedfrom iodinated
lymphocytes were treatedfor30min at
40C
with solutionscom-posed of0.5 MNaCl,
0.5%
NP-40, 100mMdiethylaminead-justedtopH 7.2, 10.7, 11.2, or 11.5,and thencentrifugedand
MW(KD)
200--9:
PPT
Al
..t
.-X;
,f...
,4,...
*....'....°
B:
IE27
1
aTS1/22
munoprecipitating with MAb to either the LFA- 1 a-chain (Fig. 9,lane 1) or 3-chain (lane 2), or with serum E27 (lane 3). The immunoprecipitates were run on a 6%SDS-PAGEgel capable
ofseparating the Mac-l (OKM 1)a-chain(165,000mol wt) from the LFA-1 a-chain (177,000 molwt). LFA-1 ,8-chain specific MAbTSl/18 immunoprecipitated bothofthose a-chains,but serum E27,like thea-chainspecificMAbTSI/22only
precip-itatedthe LFA- 1 and not the Mac- 1 a-chain.This supports the
contentionthat E27 reactswiththe LFA-l at-chain andnotthe shared 1-chain.
Analysisof theLFA-i chainspecificity ofserumE27 using
heterohybridoma cells. MouseXHuman
heterohybridoma
cells have been developed thatretainthe humanchromosomescoding for either the human a- or human 13-chain of LFA-1 (9). Oneofthoselines(MBXBW5+) expresses hybridLFA-l molecules composedof humana-subunitassociatedwith murine 1-chain.
Anotherline
(MB1l33)
expresses human LFA-1 1-chain inas-sociationwithmurinea-chain,anddoesnotexpress human
a-chains.SublinesMBXBW5-andMB13-werederived fromthese two lines and have lost the previously retained human chro-mosomesandnolonger express the humanLFA-1a-or1-chains,
respectively. These cell lineswere surface radioiodinated and reactedwith serum E27 orwithMAbsTSI/18,orTS1/22, and
immunoprecipitates prepared and run on SDS-PAGE. As
ex-pected, human LFA-I a-and1-chainswereimmunoprecipitated fromhumanphytohemagglutinin(PHA) blast cells(Fig.10, lanes
1 2
3
MW(KD)
200-175-
^-czLFA-1
165-
aa
92-PPT A.B:
aL
E27
Figuwre
9.Analysisof alphachainsimmunoprecipitatedfrom PBMsbyserumE27.Radiolabeled PBMswereimmunoprecipitatedwith either
anti-LFA-lIa
MAbTSl1/22
(lane1),anti-LFA-113f
MAbTSl1/22
(lane 2),or serumE27(lane3)andrun onlow percent(6%)SDS-PAGE andautoradiographed.1
2 3
4 5 6
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me~~~~~~~~~~~~~~~~~Z
OS
LO,
UPPT
AB:
a3E
a,8ECELLS:
PHA
BW5147
a/3
E
a+
a,
E
a-
'+
Figure 10. SerumE27immunoprecipitation of LFA-I frommouse Xhumanheterohybridoma cells. Human PHA cells (lanes 1-3), mu-rineBW5147cells (lanes 4-6), and three mouseXhuman
heterohy-bridoma celltypes: human a+-MBXB5+ (lanes 7-9), human a-nega-tive MBXB5- (lanes 10-12), and human 1-posia-nega-tive
MB#33+
(lanes13-15), were radioiodinatedand reactedwith eitherMAb to human LFA-1 a-chain (TSLFA-1/22, lanes LFA-1, 4, 7, LFA-10, LFA-13), MAb to human LFA-lI3-chain(TSI/18,lanes2, 5, 8, 11, 15),orserumE27(lanes3, 6, 9,12,
15). Immunoprecipitateswerepreparedandrun onreduced8% SDS-PAGEandautoradiographed.
1-3),butnoLFA-l wasimmunoprecipitated from the BW5 147
mousecells(lanes 4-6). The human a-chain specific MAbTSl/
22 precipitated LFA-l from the human
a'-hybrid
(MBXBW5+,lane 7) butnonewas seenwith the derivative human LFA-l
a-negative (MBXBW5-) cells (lane 10) orfrom the human
3+-hybrid (lane 13). The 13-chain specific MAb TSI/18precipitated
adetectable LFA-1 a-chain from the human LFA-l 1-positive
cells(MB133, lane 14) and a less intense 13-chain (seen more
clearlyon alonger exposure). MAb TS 1/18 didnotprecipitate
LFA-l fromthe humana+-hybrid(lane 8) or from the human LFA-1 13-negative(MB13- cell line, datanotshown).These
re-sultsareconsistent with previous observations and indicate the cells were expressing the LFA-I subunits in the anticipated fashion.
Serum E27precipitatedLFA-l from the humanLFA-l
a-chain positive cells(MBXBW5+,lane 9), but notfrom the de-rivative human a-negative (MBXBW5- cells, lane 12).
Further-more serum E27 did not precipitate LFA-l from the human 13-positivehybrid cells(MB133+,lane 15). The serum E27
anti-LFA- 1 reactivity thus is related to determinants present upon
the human a-chain.
Discussion
We haveidentified two phenotypes of the humanLFA-l mol-eculesbyadifferential abilityto reactornot reactwithserum E27. This serumimmunoprecipitates LFA-1 from most indi-viduals (95% of those tested). Its reactivity with an epitope on
the LFA-1 a-chain has been supported by several lines of
evi-dence: preferential a-chainimmunoprecipitation; lackof
reac-tivity with Mac-l; dissociation experiments showing preferential retention of the a-chain; and most convincingly by experiments
showing precipitationfrom mouseXhumanhybrids expressing humanLFA- I a-chain andnotwithhybrids expressinghuman LFA- 1 13-chain. Since the immune reactive epitope should
co-incide with the site ofallotypicvariation,theLFA- I variability detected by thisserum mostlikely residesinthe a-chain.
Attempts toshow that E27 reacts withdissociatedLFA-l a-chains using the technique of Westernblottinghave been un-successful (data not shown). The lack of reactivity with disso-ciated LFA-1 a-chain on the blots might indicate that the im-munoreactive sitesareperturbedby the conditionsused, orthat they are dependent upon a-and
#-chain
interactions for theirimmunoreactive conformation. Since human a- and mouse p-chains were precipitated from the human a-chain bearing human
Xmouseheterohybridoma cells, any chain interactions necessary for the molecule'santigenicityare not apparentlyinfluenced by substitution with murine
#-chain.
The mostlikely explanation for the failure of E27 toimmunoblottodissociated LFA-l chains isthat theblottingconditions alter theantigenicsite on the a-chains. Studies of some LFA-1 specific monoclonal antibodies have revealedsimilardifficultiesinblotting (Marlin, S. D., and T.A. Springer, personal observation).To the bestof our knowledge this is the first description of
anantigenicvariationofthe human LFA-I molecules. However,
anallotypic variationof murineLFA-l has been described (4). The E27-reactive epitope is distinct from the TS1/22-reactive
epitopeof LFA-I since'the MAbrecognizedbothforms ofthe
LFA- 1 molecules. No other anti-LFA-I MAbhavebeen shown to react with onlya subset ofhuman LFA-1 molecules. Ifas
suspected, thedifferencebetween the two phenotypes of LFA-1 moleculesarises from alteration ofthea-chainstructure,this could be the result ofamino acid substitutions, multiple amino
acid changes orvarious postranslational modifications. As in-dicated by the identicaltwo-dimensional O'Farrell gel
electro-phoresispatterns ofthe normal and variant LFA-l a-chains, the variations do not have a large effect upon the molecular weight or the pI of the a-chain. For the molecules to have such similar two-dimensional electrophoretic patterns, the changes
between themareprobablyminor andmostlikely involveone or afewamino acid substitutions.
This LFA-l variation contrasts with the inherited immu-nodeficiency syndrome involving thepartial orcomplete defi-ciency ofthe Mac- 1, LFA-I family ofmolecules(15-23).Those
patientssynthesize normala-chainprecursor molecules but
ap-parentlydon't
synthesize
normal,-chainsandas aconsequence fewdimersareformed (24, 25). Invitrotestsshowtheir gran-ulocyte and monocytes havedefectsinmotility, adherence, andphagocytosis. Inaddition, they havediminished antibody
de-pendentcell-mediated
cytotoxicity
(ADCC),and natural killer (NK) cellactivity, and have subtle T cellabnormalities (26).MAbthatbind
specifically
totheLFA- Iglycoproteins
have established the involvement of these molecules in severalim-munologicalevents. Monoclonal antibodies directedatLFA-1
block Tcell-mediated
killing
(1, 3, 5, 27-34),NKcelllysis
(5, 31-33, 35), ADCC(36, 37), mitogen and antigen-inducedre-sponses(5)and
neutrophil
adherence processes(38).
Anti-LFA-1 MAbsinterfere with the
Mg"2-dependent
adherence step of killer cell function (4, 27, 31, 34). Thesefindingsimplicate theLFA- 1moleculesasmoleculesimportantinadhesionprocesses betweencellsof theimmunesystem.
The LFA-1phenotypicvariantswehavedetectedbylackof
reactivitywithserumE27mightalsobecandidatestohave subtle
alterationsof their immune responses. Ofthevariant individuals we haveidentified,onepatient (E27) has the autoimmune disease lupus. The othertwoindividualsareclinicallywell, and prelim-inaryscreeningof their cellsabilitytostimulateandrespondin
mixed leukocyte reactions andto PHA has
provided
normal results (data not shown). More detailed studies will be neededbefore anyconclusionscan bedrawn
regarding
effectsonim-munefunctionrelatedtothe variant form ofLFA-1.
The LFA-1 variant described here may be oneofseveral
LFA-1 variants. Since detection ofthis variant relied upon a serumcontaining alloantibodiestoLFA-1, other LFA-l variants
maybe detected byexamining serumfromotheralloimmunized
individuals. Separate experimentsdesignedtoinvestigate surface
molecules reactive with serum from lupus patients, some of
whom had beenpreviouslytransfusedorbeenpregnantdidnot
revealantibodiestoLFA-1
although
manydid have antibodiestoHLA(unpublishedobservations).Similarly, normal individ-ualswho hadpreviouslybeenpregnant havenotbeenobserved
tohave antibodiestothe LFA-1 molecules. Thismayindicate thatthehigh level of antibodiestoLFA-Iobserved in this
patient
arerelativelyuncommon.Alternatively repeatedimmunization
orthe alteredimmuneregulationoflupusmayhavecontributed
tothehighlevels. Futureanalysis ofthepresence of these
an-tibodies in individuals ofknown LFA-l phenotype who have receivedtransfusionsorbeenpregnantwill help
clarify
thefre-quency, amount ofLFA-l antibodies and thediversity of the
LFA-1 molecules.
Acknowledgments
Wethank Ms. Esther Avery for expert technical assistance.
These studies have been supported by grants AI-24624, AM-30036, AM-07062, and AM-09174; and the Lupus Foundation of America. Ken D. Pischel is a recipient of an Arthritis Foundation Investigator Award.
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