De novo synthesis and secretion of a 36-kD
protein by cells that form lupus inclusions in
response to alpha-interferon.
S A Rich
J Clin Invest.
1995;
95(1)
:219-226.
https://doi.org/10.1172/JCI117643
.
In response to the pure recombinant human alpha-IFN, IFLrA, Raji and Daudi were the only
two cell lines among 19 human lymphoblastoid cell lines tested that formed the human
lupus inclusions (LI) to a high frequency. Raji, Daudi, and five other cell lines were
examined for protein changes that might accompany LI formation. Their selection was
based upon T or B origin, association with Epstein-Barr virus, and ability to form LI. A trace
protein of an estimated molecular mass of 36 kD (p36) and an isoelectric point of 5.6 was
detected on two-dimensional gels only of alpha-IFN-treated Raji and Daudi cells.
Gamma-IFN did not induce p36 or LI in any of these seven cell lines. In Daudi cells p36 and LI
formed simultaneously in response to IFLrA, and persisted until the alpha-IFN-induced
death of the culture. In Raji cells, p36 and LI appearance and disappearance coincided with
the addition and removal of alpha-IFN. Fractionation of Raji cells with nonionic-detergent
buffer placed p36 with the inclusions in the cytoplasmic supernatant. With detergent-free
buffer p36 and LI were distributed evenly between the nuclear and cytoplasmic fractions.
Pulse-chase experiments revealed that p36 was secreted. The de novo synthesis of p36
with alpha-IFN treatment was shown by labeling the cell proteins with [35S] methionine
before and after the addition of alpha-IFN. These results along […]
Research Article
De Novo
Synthesis and Secretion
of
a
36-kD Protein
by
Cells That Form
Lupus
Inclusions
in
Response
to
a-Interferon
Steven A. Rich
Laboratory ofPathology, Wadsworth Center forLaboratories andResearch,New York StateDepartment of Health, and SchoolofPublic
Health, State University ofNew York, The UniversityatAlbany, Albany,New York 12201-0509
Abstract
Inresponsetothepurerecombinant humana-IFN,IFLrA,
Raji and Daudiweretheonlytwocell linesamong19 human
lymphoblastoid cell lines tested that formed the human
lu-pusinclusions (LI) to a high frequency. Raji, Daudi,and
five other cell lineswereexamined forprotein changes that
might accompanyLIformation. Their selection wasbased
uponTorBorigin,association withEpstein-Barr virus,and
abilitytoformLI.A traceproteinofanestimated molecular
mass of 36 kD (p36) and an isoelectric point of 5.6 was
detectedontwo-dimensionalgels onlyofa-IFN-treatedRaji
and Daudi cells. Gamma-IFN didnot induce p36 or LIin
any of these seven cell lines. In Daudi cells p36 and LI formedsimultaneouslyinresponsetoIFLrA,andpersisted
until the a-IFN-induced death of the culture. InRaji cells, p36and LI appearance and disappearance coincided with the addition and removal of a-IFN. Fractionation ofRaji cells withnonionic-detergent bufferplaced p36with the
in-clusions in the cytoplasmic supernatant. With detergent-free bufferp36and LIweredistributed evenly between the
nuclear andcytoplasmicfractions.Pulse-chase experiments
revealed thatp36 was secreted. The de novo synthesis of
p36 with a-IFN treatmentwas shown by labelingthe cell
proteins with [35S]methioninebeforeand afterthe addition
of a-IFN. These resultsalong with previous results onthe denovosynthesisof LI in theendoplasmicreticulum(which
is involved in theprocessing and secretion of proteins)
sug-gestarole for LI in the synthesis and secretion of p36.(J.
Clin. Invest. 199595:219-226.) Key words: cell
differentia-tion*inclusion bodies *electrophoresis * lupus
erythemato-sus* acquired immunodeficiency syndrome.
Introduction
Humanlupusinclusions(LI)' (1)areabnormalcytoplasmic
structuresthat form in thecytoplasmofreticuloendothelial cells
AddresscorrespondencetoDr. Steven A.Rich, Wadsworth Centerfor
Laboratories andResearch,New YorkState Department of Health,
Em-pireStatePlaza, PO Box 509, Albany, NY 12201-0509. Phone:
518-474-6389;FAX: 518-474-7992.
Receivedfor publication 28 April 1994 andinrevised form 21July
1994.
1.Abbreviations usedinthispaper:IFLrA, human recombinanta-IFN
A; LI, lupusinclusion;OAS, oligoadenylate synthetase; p36, 36-kD
a-IFN-inducedprotein.
TheJournal of Clinical Investigation, Inc. Volume95, January 1995,219-226
of individuals with SLE (2) and AIDS (3). Virus-like
struc-tures, tubuloreticular inclusions and tubuloreticular structures areothernamesusedintheliterature to refer to LI. Such
inclu-sionsare notdetected in the cells ofhealthyindividuals. These
inclusions areinduced both in vitro andin vivo by type I(a and/; 1) butnottype1 (y)IFN(4).The inclusions are known to beproducts of normal cells abnormally stimulated with a-IFN asshownbytheirinductioninperipheral blood mononu-clear cells prepared from adult Red Cross blood donors (4),
andfrom umbilical cord bloods from routine deliveries (5). The formation ofLI in SLE and AIDS is the consequence of chronichighlevels of endogenous IFNin the circulationof
individuals with these diseases (6, 7). Persistently high levels
ofIFN contrast sharply with the typical 24-h burst of IFNthat
isassociated withavirus infection. The IFN in SLE and AIDS isan unusual a-IFN that is acid-labile like y-IFN, butthat is
neutralizedby antibodies to a-IFNs(6, 8). This unusual a-IFN
inthe seraof individuals with SLE and AIDS hasan
extraordi-nary abilitytoinduce the inclusions (8).
LIarereported to be a prognostic marker for disease pro-gression in individuals with AIDS (3, 9). Induction ofLI by SLE sera corresponds to the disease activities of the serum
donorsattheextremesof disease activity (8). Individuals with the greatestserumconcentrationsof IFN induce LI to the
great-est frequencies, and these patients have the greatest disease
activities. Individuals in remission donothave measurable
lev-els of IFN and theirseradonotinducetheinclusions.
Undetect-ableserumlevels ofIFN aretypical of healthy individuals (8). Thusfor both AIDS andSLEthe chronic presenceof IFN and formation ofLIis associated with diseaseactivity.
LIaredenovosynthesizedcomplexes of ribonucleoprotein and membrane with carbohydrate andno DNA(10). Theyform
inarestrictedregion of the endoplasmic reticulum that makes
contactwithadjacent regions of theouternuclearenvelope and
theGolgi apparatus (11). Theseresults suggest thatLImight
function in membrane biogenesis, the trafficking of proteinsto
the plasma membrane orto cytoplasmic vesicles, or the pro-cessing of proteins for secretion.
Little else is known about the origin, cell function, and
systemic significance of these inclusions because they have
neverbeenpurified and their molecular components have never been identified and characterized (12).An inabilitytoreadily purify these inclusions is consistent with their localization in
the endoplasmic reticulum, a membrane system not enclosed withinadoublemembrane andthereforenotabletobe isolated
in intact form (10).
Results from the current study show that an IFN-induced protein ofamolecular mass of 36-kD(p36)and anisoelectric point of 5.6 is associated withLI.This protein providesa new avenue for investigation into the possible significance ofthe
roles ofLI andIFNin the immune-based diseases ofSLE and
Methods
IFNpreparations and titers. The purified recombinant human IFNs IFLrA (Hoffmann-La Roche, Nutley, NJ) andgamma(Genentech Inc.,
South SanFrancisco, CA)wereassayed for antiviral activityonWISH
cells challengedwithvesicularstomatitis virus (Indiana strain) (13).
Their titers are reported relative to the National Institutes of Health
human IFN ca- and y- standards (G023-901-527 and Gg23-901-530,
respectively; kindly provided bytheAntiviral Substances Program of theNational Institutes ofAllergy and InfectiousDiseases). 1U/ml of
IFN provides50%protectionof cellmonolayers.
Celllines,culture and IFN induction. 19 humanlymphoblastoid cell
lines(seeTableI)weregrowninsuspension in RPMI 1640 medium/
10% fetal calfserum(GIBCO BRL,Gaithersburg,MD)/100 U/ml of penicillin and 100/ig/ml ofstreptomycin (14). Tissueculture flasks (Bellco Glass, Inc., Vineland,NJ)werekeptinahumidity-controlled incubator(Wedco,SilverSprings,MD)witha5% CO2 atmosphereat 370C. The cellswere maintained inexponential growth andat <5%
nonviability by resuspension every 3 or 4 d in fresh medium. Cell
densitiesweredetermined induplicateonamodel F cytometer(Coulter
Cytometry, Hialeah, FL), and the percentage of nonviable cells was
determinedbytrypan blue exclusion. Cellsweretaken fromculturesat
indicated times of 100U/mlofIFLrAory-IFNtreatmentforanalysis ofLI, p36,andoligo-isoadenylatesynthetase.
Epstein-Barrnuclearantigenassay. All 19 of the human
lympho-blastoidcell lineswereevaluated forEpstein-Barrnuclearantigen (15).
Multi-well slideswerepreparedfrom PBS-washed cells thatwerefixed
for5 mininice-cold methanol andacetone( 1:1, vol/vol),and air-dried.
Epstein-Barr positiveandnegativehumansera(virus testingprogram,
Wadsworth Center for Laboratories and Research) were decomple-mented and added toduplicatewells of cells. The slideswereprocessed
as described (15), mounted in pH 9 mounting fluid, and read on a
fluorescencelightmicroscopeusingaX40dry objectiveand X1O
eye-pieces.
[35S]Methionine radiolabeling ofcell and secretedproteins. Cells were washed in methionine-free RPMI 1640 media (GIBCO BRL)/
10%dialyzedFCS. 20 x 106 cellswereincubated for 40minat37°C in8 mlofmediumsupplementedwith 25ACi/mlof[35S}methionine
(ICNBiomedicalsInc., Irvine, CA).For the detection of secreted
pro-teins, half of these radiolabeled cellswerewashed andresuspendedin 4ml of RPMI1640/0.75%FCS,incubated for2h,and thesupernatant
wascentrifugedat100,000gfor 60min (type50rotor,Beckman Instrs.
Inc.,Fullerton,CA)beforeprocessingfortwo-dimensionalgel analysis.
Forthedetection of cellproteins,theremainingcellswerewashed and
suspended in 2 ml of RPMI 1640/10% FCS, incubated for 5 min, then washed andsuspendedin 2 ml of serum-free RPMI 1640before
processingfortwo-dimensionalgelanalysis.Incorporated radioactivity
was determined from portions ofthe two-dimensional gel samples. IFLrAandy-IFNwereaddedtothecorrespondingwash and incubation
mediaasrequired.
Cellfractionation. 50 x 106IFLrA-inducedRajicellswere
resus-pendedin4°C reticulocytestandard buffer/0.ImM phenylmethylsulfo-nylfluoridewith and without0.1%Tween 40 (10, 16). After 10min
onice the cellsweregentlyvortexed untillysed. Nucleiwerepelleted at1500gfor 10min.LIwerepelletedfrom the nuclearsupernatantby
a15-min centrifugationat8,000g. Nuclei and LIpelletswereoverlaid withglutaraldehydefor electronmicroscopy,andprecipitatedwith TCA for two-dimensionalgel analysis.
Electronmicroscopeenumerationof LU.Cellsampleswerefixedin 3%glutaraldehyde,postfixedin osmiumtetroxide, dehydratedingraded ethanol, and embedded in Epon (1). Thin (0.25-jim) sections were stained with uranyl acetate and lead citrate, and examined on a 301
electronmicroscope(Philips Electronic Instrs.Co.,Mahwah, NJ). LI frequencyestimates arebasedon abinomialmodel(1).Theratio of
themeandiameter of inclusions to that of whole cells is theprobability (d) ofobserving an LI in arandom thin section ofagiven cell that containsanLI.Factoringin theprobabilitythatanLI exists in the cell
Table I. LI FormationinHumanTandBLymphoblastoid
Cell Lines
Cell line LIfirquency Cellorigin EBNAstatust
Daudi 15 B +
Raji 12 B +
Raji-R§
<2 B +U698 <2 B
NC 37 <2 B +
Jiyoye <2 B +
EB-3 <2 B +
Namalwa <2 B +
SB <2 B +
HSB2 <2 T
-MOLT <2 T
-CEM 0 T
-BJAB11 0 B _
BJAB/HRIKII
0 B +BJAB/B95-811 0 B +
RAMOS1 0 B
AWRAMOS1 0 B +
LUKII** 0 B
8866.7** 0 B
*Percentage of400independentcell sections examined that contained
anLI(seeEM enumeration ofLI; Methods).Cellswerecultured for 72 h in the presence of 100 U/ml of IFLrA. t The measured presence
(+)orabsence(-)ofEpstein-BarrVirus NuclearAntigenwas deter-minedbythecomplement-fixationfluorescence assay(seeEBNAAssay,
Methods). §Raji-Rwasderived fromRajicellsbycontinuousgrowth in the presence ofbromodeoxyuridinetoselect cells thatwereresistant tothe toxic effects ofbromodeoxyuridine. 11BJAB/HRlK and BJAB/ B95-8werederived from theEBV-negativecellline BJABby transfor-mation in vitro with the Epstein-Barr viruses HRlK and B95-8, respec-tively. IAW RAMOS wasderived from the EBV-negative cell line
RAMOSbyinfection in culture with theEpstein-BarrvirusAW. ** LUK II and 8866.7 produceda-IFNconstitutively.
(P) gives aprobability of observing LIinacellpopulation (P*), or
P* = dP.Foreachsample 400 independentcell sectionswere
exam-ined. Thin sectionsseparated bygreater than the
20-/im
diameter of the cells were used to approximate random sampling. A 10% frequency (P*) (40outof 400independentcellsections)is consistent withone2.0-jim diameter LI (P= 1) per cell of a20-,umdiameter (d = 2.0 jim/20tim= 10%). The corresponding 95% confidence interval of LI
frequenciesfor400 independent cell sections is 7-13%(1).
Two-dimensional polyacrylamide gel electrophoresis. Aliquots of 107 cells (1 mg ofprotein) werewashedin serum-free RPMI 1640, precipitatedwith 10% TCA (wt/vol), and extracted twice withethyl ether, dried, and processed for isoelectric focusing (17). Two-dimen-sional polyacrylamide gel protein maps wereobtained (17, 18) with 200
til
of samplerunon3-mm inner diameter and 12-cm longfocusing cylinders. Cylinderswerefocusedat400 V for 16 h, and thenat 800 V for 1 h inaPolyanalyst gelelectrophoresis apparatus (HAAKE,Inc.,Paramus, NJ) using 0.1 M phosphoric acid (anode) and 0.1 M sodium
hydroxide(cathode). Equilibrated cylinders (5 ml of 3% (wt/vol)SDS,
6.2% (wt/vol)/3-mercaptoethanol, 62.5 mM Tris, pH 6.8 for 15 min
Figure 1. Electron micrographs of lupus inclusions (LI) in thin sections(0.1 ym) of (a)Daudi and (b) Raji cells that were cultured for 72 h with 100U/ml ofIFLrA. N,nucleus;L,lipid granule. LI are seen to be complexes of membrane with tubular structures of 20-28 nm diameter. The sections were stained with uranyl acetate and lead citrate, and examined in a Philips 301 electron microscope. Bar= 1
ym.
cylinders equilibrated in 1 ml ofwater. The vertical slab apparatus (SE500; Hoefer Sci. Instrs., San Francisco, CA) was run at 100Cand
110 Vfor 30min,followed by 220 V until the bromphenol blue dye marker reached the bottom of the slab (- 3 h). Slab gels were stained with either Coomassie brilliant blue or silver (Bio Rad Laboratories, Richmond, CA). For fluorography slab gels were fixed for 45 min in 50%methanol/ 10% aceticacid,soaked in water for 30min,and equili-bratedin 1 Msodium salicylate for an hour (19), before drying and processing (20).
Oligoadenylate synthetase (OAS)assay. Cellpelletswashed inPBS were overlaid with 500
Ml
of Triton X-100 lysis buffer (0.5% wt/vol Triton X-100, 20 mM Hepes, 120 mM KC1, 5 mM magnesium acetate, 7mM,6-mercaptoethanol, 10% wt/vol glycerol) and vortexed (5, 21). Supernatant (10041)
after 2 min of centrifugation at 15,600 g wasadsorbed topoly(rI) (rC)-agarose beads (Pharmacia LKB Biotechnol-ogy Inc., Piscataway, NJ). The picomoles of ATP incorporated into isoadenylate oligomers were calculated from the cpm in the eluted prod-uctsfrom the alumina column (Sigma Chemical Co., St. Louis, MO) (5, 21) and the cpm of thea[32P]ATP(Amersham, Arlington Heights, IL) added to the adsorbed agarose beads (5).
Results
IFLrA induction of lupus inclusions in human lymphoblastoid cells. 19 human lymphoblastoid cell lines were evaluated for
theirabilitytoformlupus inclusions inresponse tothe purified recombinant leukocyteIFN, IFLrA(TableI). BeforeIFN treat-ment no inclusions were seen in anyof thecelllines. With a-IFN treatment two of the celllines, Daudi (Fig. 1 a) andRaji (Fig. 1 b), formed inclusionsat acell frequency of> 10% of
cell sections, nine at afrequency <2%, and in the remaining eight lines no inclusions were detected. LUK II and 8866.7 constitutively producea-IFN. Neitherofthesecell linesformed
LIeither beforeorafter the addition of IFLrA. Both Daudi and Raji cells formedLIwhengrownin media conditioned by these cells, which showedthatthe IFN theyproduced had the
biologi-cal activity to induceLI. LIformation was notrestricted to B orT cellorigin, orinfection with Epstein-Barrvirus (Table I,
EBNAstatus).
Two-dimensional protein gel analyses of IFN-induced cells. ThehighLIfrequencies in Daudi and Raji cells suggested that LI-associated IFLrA-inducedproteins would also be elevated. Comparison of two-dimensional protein gels revealed onlyone
IFLrA-induced protein thatwascommon toboth IFLrA-treated cells (Fig. 2, aand c;Daudi and Raji, respectively) that was not present inthe untreated cells (Fig. 2, b andd; Daudi and
Raji, respectively). This protein, termedp36,has an estimated
size of 36 kD andan isoelectric point of 5.6.
Five additional cell lines (Table II) chosen from those listed
inTableI werealsoexamined bytwo-dimensional gels for p36 protein. Selection of these cell lineswasbasedontheirB (SB, RAMOS, and AW RAMOS) and T (HSB2 and MOLT) cell origins, their association with Epstein-Barr virus (SB and AW
RAMOS) and their formation of LI, although at thelow fre-quencies of<2%(SB,HSB2, and MOLT). However, p36was not detected in any of these other cell lines either before or
afterIFN treatmentwith 100U/ml ofIFLrA.
Gamma-IFN did not induce p36 orLI in any of the cell linesexamined, including Daudi and Raji cells. OAS activation indicated thatall seven of these cell lines were responsive to
botha- and y-IFNs. Forall of these cell linesOAS activation
was lesswith y-IFN thanwithIFLrA. Daudi and Raji didnot
always show activation with y-IFN, and when activation
oc-curred, itwas always < twofold incontrast with a >twofold andup tofivefold (RAMOS) activationinthe other five lines. OAS activation withIFLrA was shown to betransient, rising
rapidly by 18-24 h and falling to almost untreated levels by
144 h.
TimecourseofIFLrAinductionof lupus inclusions and p36 protein inDaudiand Raji cells. In Daudi cellsa-IFN induced
a
94-
67-
43-
30-0-%
L.g
-(A
I1
Am.
..
A_
e^...
:.:g-~iM~
.:."''.'.,,,~~~~~~',4*!
.4O A= sa
20.!-144a
94-
67-
43-
30-
20.1-
144-4 .0 l.
4.0
*~~~~~~~~~3
* *-4h
5.0
5.5
6.0
7u
Isoelectric pH
value
Figure2.Two-dimensionalpolyacrylamide gel electrophoresis mapsofDaudi(ab)andRaji (c,d)cells after(a,c)andbefore(bd) culturing for
72 hwith 100 U/ml ofIFLrA. p36wasdetectedonlyafter IFLrA treatment (ac). Each sample consistsof 400
jig
of total cellprotein.Molecular massesandisoelectricpointsarenoted on the vertical and horizontalaxes,respectively.Silverstainingwasusedbecause it is moresensitive than Coomassie brilliantblue,and itpreferentially enhances p36 relativetothesurrounding proteins.todieby 48 h, which increasedto100%by 120 h. Both the LI
andp36 proteinwereapparentthroughoutthe120 h timeperiod
ofcontinued presence of a-IFN. In Raji cells
a-FEN
inducedthep36 proteinby 12 h,and the LIby24 h (Fig. 3 b). They
werepresentthroughoutthe 168 h of a-IFNtreatment.Unlike Daudicells, Raji cellgrowthandviability wereunaffectedby
IFLrA.By48hafter the removal of a-IFN the p36 proteinwas nolonger detected, and the inclusionswerereducedtothe very
lowfrequencyof 1.25%.Theessentially sametimecoursesof
appearance ofp36andLIupona-TFNstimulationof Daudi and
Raji cells,anddisappearanceupon removal of a-IFN withRaji
cells, provide additional evidence for the association ofp36
with LI.
Localizationof lupus inclusions and thep36proteinincell
fractions. Purified nuclei from IFLrA-induced Raji cells were
prepared with nonionic detergent buffer (Fig.4a). TheLIfrom
these cellswereidentified only in thecytoplasmic supernatant
(Fig. 4b). Fractionation in the absence of nonionic detergents
produced nuclei that contained alargeamountofcytoplasmic
membrane andLI(Fig. 4c). TheLI were also located in the
corresponding cytoplasmic fraction from these nuclei (Fig. 4
d). Two-dimensional gelsshowedthe presence ofp36 protein
in theonenuclear(Fig.4c)andtwocytoplasmic (Fig.4b and d) fractions that contained LI. No p36 was apparent in the nuclearpellet withoutLIthatwas prepared with the nonionic
detergent (Fig.4a).
Secretionofp36protein. Two-dimensionalgelsof radiola-beled proteins released into the media by Raji cells revealed
thatp36protein was secretedby theIFLrA-treatedcells
(Fig.
5 a), but notbytheuntreated cells (Fig. 5 b). Specificity for
theproteins beingsecreted ( 16.1% ofincorporated
[3H]-amino
acids)wasshownby comparisonwith
[3H]thymidine
and[3H]-.%A. M -.
-IA-:: A ins NOWh ^E~~
Table II. Formationofp36 inSelectedCell Lines
p36 Protein* Oligoadenylate synthetaset Cell lines IFLrA y 0 IFLrA y 0 Daudi + - - 6177 1428 888 Raji + - - 4157 1252 721
SB - - - 2147 1472 375
HSB2 - - - 1065 185 90
MOLT - - - 1052 456 233
RAMOS - - - 2378 1023 203 AWRAMOS - - - 1687 843 188
*The presence (+) or absence (-) ofp36wasdetermined on
silver-stainedtwo-dimensional gels run with 400,gof each cell line before (0),and afterculturingfor 72 h with 100 U/mlof IFLrA(e.g.,Fig.2, aand b for Daudi andRaji, respectively)ory-LFN. tOligoadenylate synthetase assay values are reported as pmolofATPhydrolyzedper 1 X 105 cells(see Oligoadenylate synthetase assay, Methods) before(0)
andafter culturing for 72 h with 100 U/ml of IFLrA or y-IFN.
uridine (2.6 and 1.6%, respectively). No differences were ob-tained for theamountof secreted radiolabel between the
IFLrA-induced and untreated cells.
The chase mediumwasalso examined ontwo-dimensional gels from the other six cell lines listed in Table II. Daudi, like Raji, secreted p36 in chase medium only after IFLrA treatment. None of the other five cell lines showed the presence of p36 either withorwithout IFLrA treatment.
De novosynthesis ofp36 protein. Raji cells were radiola-beled with [35S] methionine for 18h(onepopulation doubling), chasedfor 6 h, and cultured for an additional 18 h in the
pres-enceof 100 U/mlof IFLrA. However, p36 was not detected in
an autoradiograph of these cells (Fig. 6 a), which indicated
thatp36wasnotformed fromproteinssynthesized before IFLrA addition. Yetp36was detected in autoradiographs of aliquots of these same cells whenpulsed atthe times of2 (Fig. 6 b) and 18 (Fig. 6 c) h after the addition ofIFLrA. This showed that thesecellswereactively synthesizing p36atthis time. Thus p36 was synthesized de novo in response to IFLrA, and not frommodificationstoproteins that were synthesized before the addition ofIFLrA.
Discussion
This study demonstrated that in Raji and Daudi cells a-IFN
induces LI inassociation witha36-kDproteinwith an isoelec-tricpoint of 5.6. Only these2celllines, among 19tested(Table I), formedLI tohighfrequencies and p36todetectable levels (Fig. 1, Table H). The association of p36 proteinwith LI was
further demonstrated by cellline responses to a- and y-IFNs
(Table II), by timecourse studies withthe addition and removal
of a-IFNtoRaji and Daudi cell cultures (Fig. 3),andbytheir
codistribution in nuclearandcytoplasmicfractions ofRajicells
(Fig. 4). Raji cellswerealsoshowntosynthesizep36denovo
(Fig. 6), like LI (10).
p36was notdetected in cell lines with low LIfrequencies. Thiswasprobably duetothetwo-dimensionalgel assay's rela-tively low sensitivity.LIfrequency is the ratio of theLI tocell diameter (1).AnLIfrequencyof 10% describes
2.0-,um
LIin 20-,um cells (2.0tm/20
um), and LI of 0.4 um in the samediameter cell gives a 2% frequency (0.4 ,.sm/20 ,um).
Two-dimensionalgelsmeasurethe relative concentration of cell pro-teins, a volumetric relationship. Assuming that p36 is an LI
component, its tracepresence inRaji and Daudi cells (at 10%
orgreater LIfrequency) indicates thatat2%LI orless(Table
I)p36 wouldnotbe detected[1/125th theamount(0.4,um/2.0
Mm)3].
This analysis of other cell lines that didn't formLI above
a2% frequency concurred with nop36 detected in Raji cells with a< 2%frequency ofLI at48haftertheremoval ofIFLrA
(Fig. 3 b).Incontrast, the addition of IFLrA to both Raji and
Daudi cellsinduced p36 before the formation ofLI (Fig. 4 a at 6 h, and Fig. 4 b at 12 h). The subsequent exponential increase in LIfrequency suggested the accumulation of a critical
amountof LI precursors before the formation of the uniqueLI
structure, andthatp36 might be suchaprecursor.
LI are knownto be disparate from an antiviral state since they do not form in WISH and MDBK cells witha- and y-IFNtreatment(22). P36 differs frommostotherreported
IFN-induced proteins (23, 24) since it is not induced by y-IFN (Table II, 4), and it is undetectable without a-IFN treatment (Figs. 2 b and d, 5 b, and 6 a).
Rapid secretion of p36 could explain its absence from cell lineswithoutLI. Incells with LI, p36 might indicate an inhibi-tion of this rapid secretion. However, p36 was not secreted
18
a
o0--
Figure3. Timecourseof IFLrAsC a (100U/mi) inductionofp136(4-)
15 o8o\ and LI (o) in Daudi (a) andRaji
>-
12 (b) cells.p36 is indicatedasdetect-O ableorabsent fromtwo-dimensional
Z 0 6
Uj
/ ol
\gels
ofcorresponding
timepoint
0X
O / 4 I t samples. LI frequency was theper-E 6 / t \ centageof 400
independent
cellsec-3 3 / 36kD protein detected 0 2 36 kD protein detected tions examined that contained an LI (see EM enumeration of LI; Meth-ods). Incontrast tothecomplete
0 612 24 48 72 96 120 224 48 72 96 120 144 168 -24 -48 -2 -6 IFLrAkillingoftheDaudicellculture 4 +IFLrA * * +IFLrA < -IFLrA_p- by 120 h (% nonviable cells of 9, 54, H H 86, and 100 at 48, 72, 96, and 120 h, respectively), Rajicells were
unaf-fected.Thetimecourseofdisappearanceofp36 and LI began at 168 h with the removal of IFLrA. The asterisk in (a) denotes the detection, but
w'..
I ..2
Figure4. Fractionation of p36with LI. Nuclear (a,c) and cytoplasmic (b,d) fractions of 72 h IFLrA-induced (100 U/ml)Rajicells were prepared with the low ionic strength buffer reticulocyte standard buffer in the presence (ab) and absence (c,d) of the nonionic detergent Tween 40 (0.1%, wt/vol). N, nucleus. LI distribution was estimated from the average number of LI per grid square in the nuclear, and cytoplasmic (8,000 g for 20
min),pellets. With Tween40 allLI wereinthe cytoplasmic fraction (b; e.g.,no LI weredetected in the corresponding nuclear pellet, a). Without Tween40 LI distribution was approximately equal between the nuclear (c) and cytoplasmic (d) fractions. p36 was identified only in the three fractions in which the LI were present (b-d). Electron micrographsareof thin sections (0.1 [m)that werestained with uranyl acetate and lead citrate,and examined inaPhilips 301 electron microscope. Bar = 1
ytm.
eitherinthe absenceor presence of a-IFNby cell lineswithout The a-IFN-activated intracellular pathway consists of the
LI, and it was among Raji and Daudi cell-secreted proteins nuclear translocation of threecytoplasmic proteins (p84, p91,
upon a-IFN treatment (Fig. 5). The < 3% ofthymidine and andp113) aftertheirphosphorylation atspecific tyrosine resi-uridinedetected in aliquots ofthesesamples in contrast to the dues, their combining with a nuclear resident protein (p48),
16% of3H amino acids indicates specificity forprotein secre- andtranscriptional activation of a-IFN-specificgenes(25). Cell
tion, and shows that LI did not inhibit it. LI formation in a activation by y-IFN involvesthephosphorylation of the 91-kD restricted endoplasmicreticulumregionthat makes contact with cytoplasmicresidentprotein,whichformsadimer,translocates
the adjacentouter nuclear envelope and Golgi apparatus (11) tothenucleus,and activatesy-IFN-specificgenes(26).
LA
I
r
-Afi 4w wamF
_m
0 ., g
r
0 OF~~
I, .. I
5.0 5.5 6.0
b
7.0
4.05.0:
5.5 6.0 7.0Isoelectric
pH
Value
Figure 5. Two-dimensionalgel autoradiographsofproteinssecretedby Rajicells treated with 100 U/mlofIFLrA for 72 h(a),andnottreated withIFLrA(b). p36 was secreted only by Raji cells afterLFLrAtreatment(a), but not in the proteins secreted by untreatedRajicells(b).Inboth
samples the amounts of3H-aminoacids(16.1% ofthe totalincorporated duringthepulse), or[3H]thymidine(2.6%)or [3H]uridine(1.6%)
measured in thesecretorymedium were the same. Theapproximatelyeightfoldgreater release of proteinoverDNA and RNAdemonstratesa
selective secretionof protein,andp36 bya-IFN-treated cells.
LIandp36 couldberelevanttoa-IFN'simmunemodulatory
effects (27-29). Two-dimensionalgelsofa-IFN-induced
hu-manlymphoblastoid (30,31 )orperipheralbloodmononuclear
(32, 33) cells revealaproteinwithp36's approximatemolecular mass and isoelectric point. LI, y-IFN, cell fractionation and
secretion of proteins were not investigated in these studies. Daudi but notRaji cells wereincluded in the lymphoblastoid
studies (30, 31), and therefore it appeared that the
a-IFN-inducedprotein changes wereassociatedwith Daudicell's se-veregrowthinhibitionand death (Fig. 3a).
97
~67-'A 43 '
30 ' @ ' k
(U-With silver staining p36 is visible in whole cell samples (Fig.2aandc), and withCoomassiebrilliant blueupon
enrich-ment.Thisindicatedthatp36was acandidateproteinfor
purifi-cation andfurther characterization (17, 34, 35). Tryptic peptide microsequence of purified p36 did not share any significant sequenceidentitywithproteinslisted in theSWISS-PROT (Ge-neva,Switzerland)database, suggestingthatit isa newprotein
(Rich,S.A., unpublished results). Anti-peptide antiserum
pre-pared against p36 synthetic peptides has detected p36protein
in Western blots ofRajicells only after IFLrA induction, and
IsoelectricpHValue
Figure6. Two-dimensionalgel autoradiographs ofRajicellproteins. Rajicellswereradiolabeled with [35S ]methionine for 18 h(one population doubling),chased for 6h,and cultured for 18 h with 100U/mlof IFLrA(a). Aliquotsofthesesamecellswereradiolabeled for 40minafter
culturingfor 2(b)and 18(c)hwith IFLrA.Togethertheseresults showed thatp36wasnotformed fromproteinssynthesized (radiolabeled)
before the addition of IFLrA(a),and itssynthesisafter 2(b)and 18(c)h of IFLrAtreatmentwasadenovoresponsetoIFLrA. a
94
67_ht
_. 43 0
a.-5 30
U
0
20.1-12OA
4.0
I.
at anestimated>400-foldinduction level. Confirmation ofthe
microsequence results will require amplification, cloning, and sequencing of p36 mRNA cDNA. In situ immunoelectron mi-croscopy with a suitable antibody preparation should reveal the cellular location of p36 and its relationship with LI.
Bcellproduction and secretion of p36 suggests the possibil-ity for interleukin activpossibil-ity. Another possibilpossibil-ity is that secreted p36 induces autoantibodies. Thus a-IFN in individuals with SLEand AIDS could stimulate LI formation and p36 production andsecretion. Also, p36 could modulate the immune system by interleukin or autoantigen activities. Further characterization of p36 and LI should provide additional understanding of a-IFN's roles in SLE and AIDS immunopathology.
Acknowledgments
The expert technical assistance of William E. Gibbons is greatly ac-knowledged. Iamindebted to Drs. Himangshu and Mahuya Bose for reading the manuscript, to Dr. Sidney Pestka (Robert Wood Johnson Medical School) for providing the IFLrA, to Mr. Scott Geromanos
(Sloan-Kettering Institute for Cancer Research, Rockefeller Research Laboratories) for the microsequencing of purified p36, to Dr. Lee H.
Kronenberg (Lee Biomolecular Research Laboratories\ Inc.) for the LUKII and 8866.7 celllines, and to Dr. Julius Youngner (School of
Medicine, UniversityofPittsburgh) forthe RAMOS and AW RAMOS
celllines.
Thiswork wassupportedbyagrant from theNational Institutes of Health (AR41619toS. A. Rich).
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