SERUM FACTORS IN LUPUS ERYTHEMATOSUS …

SERUMFACTORSIN LUPUS ERYTHEMATOSUSANDOTHERDISEASESREACTINGWITH CELL NUCLEI AND NUCLEOPROTEINEXTRACTS: ELECTROPHORETIC,ULTRACENTRIFUGAL

ANDCHROMATOGRAPHICSTUDIES

BY HOWARDC. GOODMAN,JOHNL. FAHEYAND RICHARDA. MALMGREN

(From the Laboratory of Cellular Physiology and Metabolism, National Heart Institute, andthe Metabolismii Service and Laboratory of Pathology, National Cancer Institute,

Bethesda, Md.)

(Submitted for publication April 12, 1960; accepted June 30, 1960)

In the past decade much interest has been fo-cused on the factors in human serum which inmany ways resemble antibodies against compo-nents of cell nuclei. The lupus erythematosus(LE) cell, discovered by Hargraves, Richmondand Morton in 1948 (1), is now known to resultfrom the interaction of leukocyte nuclei and afactor(s) most frequently found in the serum ofpatients with lupus erythematosus. Miescher (2)employed the y-globulin consumption test to dem-onstrate a reaction between factors in LE serumand isolated cell nuclei. Since this time, reactionsbetween factors in LE serum and isolated cellnuclei (3, 4), intact nuclei in tissue sections (5-7), extracts containing nucleoprotein (8-10),nucleoprotein precipitates (11), preparations ofdesoxyribonucleic acid [DNA (4, 12-14)] andhistone (15) have been demonstrated. The avail-able information on the anti-DNA and other re-active factors in LE sera has been recently re-viewed ( 16).

As more information concerning the serum-cellnucleus interactions has become available, thenature and the significance of the phenomena haveappeared more complex. Anti-cell nucleus factorshave been found in the sera of patients with dis-eases other than lupus erythematosus (7, 11), insera which do not contain detectable LE cell fac-tor. It appears, therefore, that not all serum-cellnucleus interactions are of the kind that bringabout the changes necessary for the production ofLE cells. In addition, recent studies (17, 18) in-dicate that the source of the tissue used in makingnucleoprotein extracts is an additional variableaffecting the detection of serum anti-nucleus fac-tors. Certain sera were found to react with a livernucleoprotein component which was heat-stableand was destroyed by desoxyribonuclease, whereas

other sera reacted with a heat-labile component ofnucleoprotein extracts made from thymus tissue.The present study was undertaken, therefore, tocharacterize in physicochemical terms several ofthe serum anti-nucleus factors, to determinewhether the serum factors reacting with what ap-pear to be different nucleoprotein materials mightdiffer also in other properties, and to determinethe relationship of the anti-nucleoprotein factorsto the LE cell factor and other anti-nucleus fac-tors.

Studies of the 'v-globulins in man have empha-sized that they are composed of two groups ofproteins with ultracentrifugal sedimentation coeffi-cients of 6.6S (molecular weight about 160,000)or of 18S (molecular weight about 1,000,000)(19). These groups contain a range of differingmolecules (20). Anion-exchange cellulose chro-matography is a useful means of separating the18S v-globulins from most of the 6.6S moleculesand also provides a means for subdividing the 6.6Sav-globulin group (20). By chromatographicmeans, antibody activities associated with 18S pro-tein molecules have been separated from smaller6.6S antibody molecules (21-24).

In a preliminary report (24) on the anti-nucleusactivities of LE sera, it was shown that certainanti-nucleus factors, presumably 18S 'v-globulins,could be separated by anion-exchange cellulosechromatography from the LE cell factor whichappeared in this and other (3, 25, 26) studies tobe within the 6.6S y-globulin group. The presentreport describes the electrophoretic, ultracentrif-ugal and chromatographic characterization of thefactors in sera from patients with lupus erythe-matosus and other diseases which react with hu-man leukocytes to form LE cells, with mouseliver cell nuclei as detected by fluorescein-labeled

1595

GOODMAN,FAHEY AND MALMGREN

TABLE I

Results of anti-cell nucleus and anti-nucleoprotein extract tests in sera from patientswith systemic lupus erythematosus and other diseases

No. of patients with positiveanti-nucleoprotein tanned-cellhemagglutiniation test

-- No. of patienitsNo. of p)atients Human Calf withl positive

with positive liver thymus fluorescein aniti-No. of LE cell test siucleoproteini nucleoprotein liver nuclei

Diagnosis patients (human leukocyte) extract extract (imiouse) test

*Systemic lupus erythematosus 27 25 15 23 26Rheumatoid arthritis 30 2 6 3 6Scleroderma 4 0 0 1 2Other diseases* 53 0 0 0 6

* Includes idiopathic nephrotic syndrome, 17; dysgammaglobulinemias (principally macroglobulinemiia), 13; con-valescent mumps, 3.

antibody techniques, and with liver or thymus nu-cleoprotein extracts in tanned-cell hemagglutina-tion tests.

METHODS

The tannic acid hemagglutination test was performedessentially as described by Boyden (27). Human type 0Rh negative red cells or sheep red blood cells were used.The washed cells were "tanned" by mixing with a 1: 25,000solution of tannic acid in pH 7.2 buffered saline for 30minutes at room temperature. The tanned red cells, af-ter washing, were coated by mixing with the nucleo-protein extracts for 30 minutes at room temperature.Nucleoprotein extracts were made from human liverobtained at autopsy and from calf thymus; the methodfor extraction of nucleoproteins with solutions of lowionic strength, described by Chargaff (28), was followedclosely. All extracts were clarified by centrifugation inthe Spinco model L preparative ultracentrifuge andstored frozen until used. The extracts were diluted from1: 10 to 1: 400 with 0.15 M NaCl solution buffered atpH 7.2 before coating the tanned red cells; the con-centration chosen was the highest that would not pro-duce spontaneous agglutination of the red cells. Fordetermination of anti-thyroglobulin titers, tanned redcells were coated with a 0.02 per cent solution of athyroglobulin extract. The thyroglobulin was 86 percent pure.

Quantitative total serum protein measurements, ana-lytic paper electrophoresis and preparative electropho-retic procedures utilizing polyvinyl chloride particleblocks have been described (20). Ultracentrifugal frac-tionation was carried out in the manner of Robbins,Petermann, and Rall (29) utilizing a Spinco model Lpreparative centrifuge. Sera were diluted 1: 10 with0.15 M NaCl solution and serum fractions were dia-lyzed thoroughly against the same solution prior toultracentrifugation in the Spinco 40 rotor at 39,000 rpmfor 150 minutes. At the end of this time successive sam-ples were collected from the top to the bottom of theultracentrifuge tube by using a sampling syringe andneedle. Determinations were made of the anti-nucleo-

protein extract activity, and the results wvere plotted interms of the cumulative activity with increasing depthof the tube.

Anion-exchange cellulose chromatography was per-formed on columns of diethylaminoethyl (DEAE) cel-lulose (30), employing 2 g of adsorbent an(d 1 ml ofserum. The starting and equilibrating potassiuml bufferwas 0.02 M phosphate, pH 8.0. A 150 ml gradlient elutionsystem, providing a progressive rise in phosphate mo-larity to 0.25 M while maintaining pH 8, was usedl toelute the adsorbed proteins from the coluini (20).Effluent from the column was collected in about 45 frac-tionis and the protein content estimated by measuring theoptical density at 280 m,l in a Beckman spectrophotom-eter. The effluent was then combined into 10 or morepools which were tested for anti-nucleoprotein extractactivity after dialysis against 0.15 M NaCl and \vithoutconcentration.

For the fluorescent antibody technique, a 1 X 1 X 0.5cm piece of mouse liver was placed in a dry ice ethaniolbath at - 700 C as quickly as possible after removal fromthe animal. When frozen, the tissue was transferred toa cryostat and sectioned at 5 A at - 120 C. The sec-tions were air-dried and then fixed in acetone for 10 min-utes. Various dilutions of the patient's serum to be testedwere put on the mouse liver sections and allowed tostand for 20 minutes at room temperature. The serumwas then washed off with buffered saline, pH 7.4, with3 changes of the saline at 5-minute intervals. Fluorescein-labeled anti-human globulin 1 was then placed oni the sec-tion and allowed to react for 20 minutes at room tem-perature. The sections were again washed as beforewith buffered saline, mounted in 25 per cent glycerin inbuffered saline and examined microscopically under ultra-violet light at a wave length of 390 to 440 m,u. Thegreatest dilution of the serum that produced fluorescenceof the mouse liver cell nuclei was considered the titer ofthat serum.

1 The fluorescein-conjugated horse anti-human globu-lin was obtained from the Progressive Laboratories,Baltimore, Md. This serum had been previously ad-sorbed with liver powder to remove nonspecific fluores-cence.

159)6

CHARACTERIZATIONOF SERUMFACTORSREACTING WITH CELL NUCLEI

RESULTS

Serological studies. Sera from 114 patientswith systemic lupus erythematosus (SLE), rheu-matoid arthritis (RA), scleroderma, idiopathicnephrotic syndrome and other diseases were ex-

amined. The results of LE cell tests, the titer ofreactivity of the sera with nucleoprotein extractsmade from human liver and calf thymus, and thetiter of reactivity with mouse liver cell nuclei as

determined by fluorescein-labeled anti-human glob-ulin tests are listed in Table I. The LE cell testwas positive in most cases of SLE and rarelypositive in other diseases. Anti-human liver nu-

cleoprotein extract activity was detected in 56 per

cent of the patients with SLE and 20 per cent ofa selected series of patients with RA. Anti-calfthymus nucleoprotein extract activity was presentcent of the patients with SLE and in 20 per cent ofRA sera and 25 per cent of the scleroderma sera.

The fluorescein-labeled antibody to human t-glob-ulin revealed anti-cell nucleus activity in 96 per

cent of the SLE sera, 20 per cent of the RA sera,

50 per cent of the scleroderma sera and 11 per centof the other sera tested. The six positive sera inthe last group included four patients with idio-pathic nephrotic syndrome.

In view of this diversity of results when a va-

riety of serological tests for anti-nuclei and anti-nucleoprotein extract activity was employed, a

group of six sera was selected for further study(Table II).

The sera of the second group of three subj ects(P.N., S.R. and M.F.) were selected becausethey reacted very strongly with the nucleoproteinextract made from calf thymus but not at all, or

practically not at all, with the human liver nucleo-protein extract. All three sera also reacted withcell nuclei in the fluorescein antibody test, al-though the titer of serum S.R. seemed lower thanthe others. Serum M. F., from a patient withscleroderma, did not contain detectable LE cellfactor.

The sera of the first group of three subjectsjects (A.N., J.B. and H.L.) were chosen becausethey reacted primarily with the human liver nu-

cleoprotein extract. In addition, the three sera

reacted with cell nuclei in the fluorescein antibodytest at high titer. Serum H. L. was also foundto contain a moderately high titer of anti-thyro-globulin activity, and was the only one of the 15LE sera tested that contained anti-thyroglobulinactivity. A comparison of the results obtainedwith these six sera shows that it would not bepossible to predict, on the basis of the results inone test system, what the behavior of the serum

would be in the other four test systems. This sug-

gests that multiple factors were involved in theanti-nucleus and anti-nucleoprotein extract ac-

tivity detected in these six sera.

Electrophoretic studies. Sera from SubjectsA.N., J.B. and P.N. were fractionated by zone

(block) electrophoresis. The fractions of theA.N. and J.B. sera were tested for reactivity with

TABLE II

Serological characteristics of six sera with anti-nucleus and anti-nucleoproteinextract activity chosen for further study

Anti-nucleoprotein extract;tanned cell hemagglutination

Anti-liverHuman Calf nuclei (mouse); Anti-thyroglobulin

liver thymus fluorescein extract;LE cell test nucleoprotein nucleoprotein antibody tanned cell

Patient Diagnosis (human leukocyte) extract extract test hemagglutination

Group I1. P.N. SLE* + 80t 60,000t > 128t < lot2. S.R. SLE + <10 100,000 16 <103. M.F. Scleroderma 0 <10 3,000,000 > 128 <10

Group II4. A.N. SLE + 1,280 160 > 200 105. J.B. SLE + 1,280 80 > 128 <106. H.L. SLE + 1.280 <10 64 6,250

* Systemic lupus erythematosus.t Reciprocal of the greatest serial diltftion of serum which gave a positive test.

1597

GOODMAN,FAHEYAND MALMGREN

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FIG. 1. DISTRIBUTION OF REACTIVITY WITH HUMANLIVER NUCLEOPROTEIN

EXTRACT AFTER ZONE (BLOCK) ELECTROPHORETIC FRACTIONATION OF A.N.SERUM. The shaded area represents the distribution of anti-nucleoproteinextract activity. The distribution of serum proteins was roughly determinedby the location of the pigment bands, pink in the region, blue in the a2

region, and pale yellow in the albumin region, and was confirmed by analyti-cal electrophoresis of the block sections.

human liver nucleoprotein extract and the frac-tions of serum P.N. were tested with calf thymusnucleoprotein extract. In each instance, serologi-cal activity was associated with the y-globulinelectrophoretic region as illustrated for serum

A.N. in Figure 1. This serum contained an in-creased concentration of y-globulins, 4.5 g per

100 ml.Ultracentrifugal studies. Four sera were frac-

tionated in a Spinco preparative ultracentrifuge.Ultracentrifugation was conducted in such a man-

ner that most of the 18S y-globulins, but little ofthe 6.6S proteins, would be concentrated at thebottom of the tube. The results are presentedgraphically in Figure 2.

In this figure, the cumulative percentage of sero-

logical reactivity is plotted against the distancefrom the top of the tube. The shaded area repre-

sents the behavior of the 6.6S y-globulin anti-thy-roglobulin activity in five sera of patients withchronic thyroiditis which were studied in the same

way (23). The sedimentation properties of theanti-calf thymus extract activity in serum P.N.closely resembled those of the 6.6S anti-thyro-globulin activities. Very little of the activity hadsettled to the bottom of the centrifuge tube.

Anti-human liver nucleoprotein activity, on theother hand, sedimented more rapidly. A largeproportion of this activity in sera A.N., J.B. andH.L., the sera of Group II, was found at the bot-tom of the centrifuge tube, in contrast to the anti-thyroglobulin activity which was also present inH.L. serum, and in contrast to the anti-thymus ex-

tract activity of serum P.N. This greater sedi-mentation of anti-liver nucleoprotein activity inwhole serum could be due either to the associationof this activity with a larger molecule or the dis-tribution of this activity between both 6.6S andlarger molecules. Data to be presented below sug-

gest that the latter explanation is correct.Chromatographic studies. Whole sera of y-glob-

ulins obtained by preparative (block) electro-phoresis from each serum were subjected to anion-exchange cellulose chromatography and the chro-matographic fractions were tested for anti-nucleo-protein extract activity, anti-cell nucleus activity,and for capacity to form LE cells. Representa-tive results are illustrated in Figures 3, 4 and 5.Consistent differences were observed between thesera of Group I, reacting primarily with thymusnucleoprotein extracts (Figures 3 and 5), and thesera of Group II, reacting with human liver nu-

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1598

CHARACTERIZATIONOF SERUMFACTORSREACTINGWITH CELL NUCLEI

CUMULATIVE 60_REACTIVITY _..(% OF TOTAL) ....

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0 20 40 60 80 100%OF DEPTH OF TUBE

FIG. 2. SEDIMENTATION CHARACTERISTICS OF ANTI-NUCLEOPROTEIN EX-TRACT ACTIVITIES IN WHOLESERUM. Ultracentrifugation was conducted ina preparative centrifuge (39,000 rpm, 150 minutes, 0.15 M NaCi). Aftercentrifugation, successive samples were removed from each tube starting atthe top. The distribution of serological reactivity is expressed in terms ofcumulative per cent of the total in each tube. The shaded area is the dis-tribution of anti-thyroglobulin activities found in a concurrent study of fivesera from patients with chronic thyroiditis (23).

cleoprotein extracts (Figures 4 and 5). In theGroup I (anti-thymus) sera, almost all reactivitywas found between 5 and 15 per cent of the effluentvolume and all reactivity was found within 40 percent of the effluent volume, while in the Group II(anti-liver nucleoprotein) sera the activity (ex-cept for LE cell factor) was found in both the 5to 15 per cent and the 50 to 80 per cent effluentregions.

A comparison of the chromatographic elutionpatterns for whole serum and for y-globulins pre-pared from whole serum (Figures 3 and 4) dem-onstrates that most of the y-globulins are eluted inthe region of 5 to 15 per cent of the effluent volume.Under the conditions of chromatography em-ployed here, only y-globulins with sedimentationcoefficients of 6.6S are eluted in this region (20).In the Group I sera (P.N., S.R. and M.F.), anti-calf thymus nucleoprotein extract activity wasfound only in the initial portion of the chromato-gram (Figure 3). In these sera, anti-cell nu-cleus activity was also found only in the 5 to 15

per cent effluent region. These findings, and thesedimentation of the anti-calf thymus extract ac-tivity when whole serum was ulfracentrifuged, in-dicate that this activity and the anti-mouse livercell nucleus activity in these sera are propertiesof y-globulins with sedimentation coefficients of6.6S.

In the Group II sera (A.N., J.B. and H.L.) thefactors reacting with liver nucleoprotein extractswere eluted in the region of 5 to 15 per cent and50 to 80 per cent of the effluent volume. As isseen in Figure 4, the distribution of activity issimilar when whole serum or y-globulin is chro-matographed, an observation consistent with the re-sults of electrophoretic fractionation showing theactivity to be within the y-globulin group. The find-ing of two chromatographic regions with activityindicates that in these sera the anti-nucleoproteinactivity was associated with two different groupsof y-globulin molecules.

Ultracentrifugal studies of these two regionsfrom the LE sera chromatograms were undertaken

1599


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FIG. 3. CHROMATOGRAPHICDISTRIBUTION OF SEROLOGI-CALLY DETERMINED ANTI-CALF TH1YMUIUS NUCLEOPROTEINREACTIVITY IS SHOWN(TOP) IN WHOLESERUM OF PA-TIENT P.N., A.ND (MIDDLE) IN THE -y-GLOBULIN OBTAINEDBY PREPARATIVE ELECTROPHORESISOF SERUM P.N. Alsoshown (bottom) is the anti-cell nucleus activity in they-globulin as determined by the fluorescenit antibody tech-nique. Chromatography was conducted by standard pro-cedures on diethylaminoethyl-cellulose columns. Pro-teins were eluted by means of increasing phosphate con-ceintrationi from 0.02 to 0.25 M, while pH was maintainiedat pH 8.0.

to determiiine whether the anti-nucleoprotein ac-tivities of these regions vere associated with pro-teins of different sedimentation properties.

Previous studies (20) had shown that they-globulin eluted in the 50 to 80 per cent regionof the chromiiatogram was composed primarily of18S y-globutlins in contrast to the 5 to 15 per centeffluent region which contained only 6.6S -y-globu-lins. In Figure 6 the ultracentrifugal propertiesof the proteins contained in the 5 to 15 per centand the 60 to 80 per cent region of the chromato-

gram of L.B. y-globulins is shown. The proteinseluted first are seen to sediment less rapidly(sO20, w -18S) than those eluted in the 60 to 80per cent effluent region (s020, w

- 18S). Confir-muation that the anti-nucleoprotein activity wassimilarly divided between faster and slower sedi-menting y-globulin components was obtained by

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FIG. 4. CHROMATOGRAPHICDISTRIBUTION OF SEROLOGI-CAL REACTIVITY WITH HUMANLIVER NUCLEOPROTEINEX-TRACT IN WHOLESERUMOF PATIENT J.B. AND IN ly-GLOBU-LINS PREPARED FROM SERUM J.B. Activity is found intwo chromatogram regions. Below, the anti-nucleus ac-tivity (by the fluorescent antibody technique) is shownin the two chromatogram regions of the -y-globulins, and(bottom) lupus erythematosus (LE) cell activity isfound in only one chromatogram region of the -y-globulins.Chromatography procedure was the same as that describedfor Figure 3.

1600


preparative ultracentrifugal fractionation. In Fig-ure 7 is illustrated the rapid sedimentation of theanti-human liver nucleoprotein activity in the 60to 80 per cent effluent fraction which contrastswith the slower sedimentation behavior of the ac-tivity in the 5 to 15 per cent chromatogram efflu-ent region. The agreement between the sedimen-tation behavior of the serological activities andthe behavior of the proteins in these two regionssupports the conclusions that the anti-liver nu-cleoprotein activity is associated with two groupsof y-globulin molecules, those with sedimentation

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FIG. 5. CHROMATOGRAPHICDISTRIBUTION OF SEROLOGI-CAL REACTIVITY WITH HUMANLIVER EXTRACT IN WHOLESERUMH.L. WITH ACTIVITY FOUNDIN TWOCHROMATOGRAMREGIONS; BELOW IS SHOWNTHE CHROMATOGRAPHICDIS-TRIBUTION OF REACTIVITY WITH CALF THYMUSEXTRACT INWHOLESERA S.R. AND M.F., WITH REACTIVITY IN ONLYONE REGION. Chromatography procedures were the sameas described for Figure 3.

Direction ofSedimentation

FIG. 6. ULTRACENTRIFUGALANALYSIS OF THE 5 TO 15PER CENT REGION (UPPER) AND IN THE 60 TO 80 PER CENTEFFLUENT REGION (LOWER) FROM AN ANION-EXCHANGECELLULOSE CHROMATOGRAMOF ELECTROPHORETICALLYPRE-PARED -y-GLOBULIN FROM L.B. SERUM. The photographswere obtained 26 and 24 minutes, respectively, after reach-ing rotor speed of 59,780. The direction of sedimentationis to the right. Samples had been dialyzed against 0.15 MNaCl prior to analysis. The sedimentation coefficient wasapproximately 6.6S for the component in the upper, and18S in the lower photographs.

coefficients of 6.6S and 18S, and that these twogroups have been separated by anion-exchangecellulose chromatography.

If the results of the preparative ultracentrifugalanalysis of the two J.B. y-globulin components il-lustrated in Figure 7 are compared with the re-sults when J.B. whole serum was similarly ana-lyzed (Figure 2), it is seen that the whole serumresults fall between the findings for the individualcomponents. This observation is consistent withthe interpretation that the pattern of distributionof anti-nucleoprotein extract activity of this type(anti-human liver) after centrifugation of wholeserum (Figure 2) is due to the presence of twocomponents. The existence of two ultracentrif-ugally different components may be suspectedwhen the whole serum results do not conform to

1601

GOODMAN,FAHEYAND MALMGREN

CUMULATIVE 60REACTIVITY(/o OF TOTAL) /

40

20

0 20 40 60 8o 100%OF DEPTH OF TUBE

FIG. 7. DIFFERENCES IN THE SEDIMENTATION CHARAC-TERISTICS OF TWO ANTI-HUMAN LIVER EXTRACT COMPO-NENTS (5 TO 15 PER CENT AND 60 TO 80 PER CENT) OB-

TAINED BY CHROMATOGRAPHICFRACTIONATION OF SERUMJ.B. Ultracentrifugation was carried out as in Figure 2.

the expected distribution of activity for 6.6S or18S components alone. Whole serum preparativeultracentrifugation, however, is a relatively crudetechnique, and small amounts of one type of y-glob-ulin activity would be difficult to detect in the pres-ence of large amounts of the other type of y-globu-lin activity. Anion-exchange cellulose chroma-tography provided a better means of separatingand detecting the presence of both 6.6S and 18Sy-globulin activities.

Fluorescein-labeled anti-human globulin tests.The y-globulins of the patient's sera which com-bined with mouse liver nuclei were detected bytheir reaction with fluorescein-labeled anti-humanglobulin. Examples of positive and negative re-sults are illustrated in Figure 8. In the Group Isera (reacting with calf thymus nucleoprotein ex-tract) the anti-cell nucleus factors were found onlyin the 5 to 15 per cent chromatogram effluent re-gion (Figure 3), whereas in the Group II sera(reacting serologically with liver nucleoproteinextract) the positive fluorescent anti-globulin testswere obtained in both the 5 to 15 per cent and 50to 80 per cent effluent regions (Figure 4). Thisshows that at least two physicochemically differenty-globulin components in these latter sera givepositive fluorescein antiglobulin reactions and, onthe basis of the observations noted above, it seemsprobable that the two components are 6.6S and18S y-globulins.

Some of the activities detected by the fluores-cein anti-globulin test may be the same as thosedetected by the serological anti-nucleoprotein ex-tract tests. Very likely several activities are in-volved, for inspection of the data in Table II re-veals that the activities detected by these tWo typesof tests were not increased equally in LE sera.

LE cell tests. The y-globulin components re-sponsible for LE cell formation were present onlyin the 5 to 15 per cent chromatogramii effluent re-gion [Figure 4 and Patient H.L. (22) ]. As only6.6S y-globulins are found in this chromatogramregion, the factor(s) responsible for LE cell for-mation must be 6.6S y-globulins. The two serafractionated for detailed study of the factors re-sponsible for LE cell formation were chosen be-cause they had both 6.6S and 18S y-globulinswhich reacted with nuclear components by sero-logical and histochemical tests (Group II sera).In view of the absence of LE cell factor in ma-

FIG. 8. MOUSELIVER SECTIONS EXPOSED TO HUMANSERUM, WASHED, EXPOSEDTO FLUORESCEIN-LABELED ANTI-BODY TO HUMANy-GLOBULIN, AND WASHEDAGAIN. A.Lack of fluorescence of the cell nuclei after exposure tonormal serum (cell nuclei appear as dark holes) whenobserved with an ultraviolet light source. B. Brightfluorescence of the nuclei after exposure to lupus ervthe-matosus serum. 150 X magnification.

1602

CHARACTERIZATIONOF SERUMFACTORSREACTING WITH CELL NUCLEI

terial from the 50 to 80 per cent effluent regionwhich contained factors reacting with nucleopro-tein extract and cell nuclei, it is clear that the LEcell factor has been separated chromatographicallyfrom some of the other anti-nucleus factors inthese sera.

DISCUSSION

The discussion of the multiple serum factorsreacting with tissue components in diseases suchas lupus erythematosus is complicated by the lackof exact knowledge of the tissue substances withwhich the sera react. The crude nucleoproteinextracts employed could well contain cytoplasmiccontaminants, and in fact, Deicher, Holman andKunkel (31) have recently reported complementfixation reactions with liver cell mitochondrialpreparations in the 19S globulins of one of eightSLE sera tested. Although in the present ex-perinments no definitive studies were made of thenature of the active substances extracted fromcalf thymus because of their lability, the substancesin human liver extracts with which the sera re-acted were found to be very heat-stable and were(lestroyed by incubation with desoxyribonuclease(17); they were assumed to be nucleoproteins.In any case, whatever reactions with cytoplasmicconstituents may have been present, all six of thesera studied here in detail reacted with compo-nents of cell nuclei as shown by the fluorescein-anti-globulin test. Each differed from the others,however, in some aspect of its reactions with cellnucleus components. The sera were consideredin two groups. Three of the sera were in GroupI, reacting with nucleoprotein extracts of calf thy-mus and showing activity only within the 6.6Sy-globulins. The three sera of Group II reactedprimarily with nucleoprotein extracts from humanliver and contained this activity in both 6.6S and18S y-globulins. This latter group of sera alsocontained two types of y-globulin reacting withthe mouse liver cell nucleus.

These observations could be explained eitheron the basis of a fundamental difference in im-mune response between the two groups of patients(i.e., those in Group I are limited to the manu-facture of 6.6S antibodies, whereas those in GroupII can also make 18S antibodies) or on the basisthat the two patient groups were subjected to dif-ferent kinds of immune challenge. Although we

cannot yet distinguish between these alternativeexplanations, there is some evidence that thesepatients did not have a strict limitation of theircapacity to manufacture 6.6S or 18S antibody.Serum M.F., which contained only 6.6S y-globu-lins reacting with components of cell nuclei, alsocontained a high titer of rheumatoid factor whichhas been shown to be a -y-macroglobulin (32).On the other hand, Patient H.L., who was ca-pable of producing 18S globulins which reactedwith human liver nucleoprotein extracts, manu-factures only 6.6S y-globulins reacting with thyro-globulin. Antibodies to thyroglobulin extracts,however, need not be only 6.6S y-globulins and,indeed, some patients produce largely 18S reac-tive globulins (23). These observations suggestthat the different activities found in LE sera maybe related to different tissue substances and thatthe tissue substances themselves might help todetermine whether the antibodies to them are tobe 6.6S or 18S y-globulins.

The finding that the LE cell factor can be sepa-rated from some of the other anti-nucleus factorsin serum is consistent with the concept of the ex-istence of multiple anti-nucleus factors in the se-rum of patients with lupus erythematosus (4, 13,15, 24). It is clear, therefore, that some serum-cell nucleus interactions are of a type which donot lead to the production of LE cells; the reasonsfor this are not evident. The data showing thatother anti-nucleus factors are more widely dis-tributed than is the LE cell factor is consistentwith previous reports (7, 11). It will be neces-sary to test large numbers of sera from each ofmany disease categories, as well as a large numberof normal sera, for the presence of anti-nucleusfactors in order to determine their actual distri-bution.

Although the y-globulins which react with nu-clei in the manner required for the production ofLE cells have so far been found only among the6.6S y-globulins (3, 25, 26), it is now apparentthat 18S y-globulin components may react withcell nucleus components in lupus erythematosus.Anti-cell nucleus reactivity can be added to thelist of antibody-like activities already found inthis group of macroglobulins: the isohemagglu-tinins (22, 33), rheumatoid factor (32) and theanti-thyroglobulin "antibodies" found in the serumof certain patients with thyroiditis (23). The

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nomenclature of these macroglobulins is not uni-form, and the proteins known as 18S or 19Sy-globulins or y,-macroglobulins in the Americanliterature are classified as 132 M-globulins in theimmunoelectrophoretic studies of Burtin and co-workers (34). However, immunochemical stud-ies of the macroglobulins which react with nucleo-protein extracts were not done.

It will be interesting to determine whether anycorrelations can be made between clinical find-ings and the serological differences described inthe present study. So far as can be determined ina preliminary study (35), no obvious differencesin clinical state can be correlated with the differ-ent patterns of anti-nucleus factors. The signifi-cance of the appearance of these antibody-like pro-teins in the serum of patients with lupus erythema-tosus and other diseases is not known. The pro-duction in rabbits of what may be somewhat simi-lar auto-antibody-like anti-nucleus factors, afterimmunization with foreign cell nucleus materials(17, 36), at least suggests the possibility thatbacterial or viral infection might result, in cer-tain susceptible individuals, in the production ofantibodies to nucleoproteins or other materialswhich cross react with similar components in hu-man cell nuclei.

The ease with which DEAEcellulose chroma-tography allows recognition and separation of the6.6S and 18S reactive factors should facilitate in-vestigation of the mechanism of their productionas well as the role, if any, played by these serumfactors in the initiation or maintenance of diseaseprocesses.

SUMMARY

Multiple factors in sera from patients with lupuserythematosus (LE) and other diseases werefound to react with components of cell nuclei.These anti-cell nucleus factors were detected byserological tests employing calf thymus and hu-man liver nucleoprotein extracts and by fluores-cent anti-globulin tests with mouse liver tissue andby LE cell tests.

All of the serum factors were in the y-globulinsseparated by zone electrophoresis. The anti-nu-cleus factors were further characterized by anion-exchange chromatography and in some cases byultracentrifugal studies,

The LE cell factor and the anti-thymus nucleo-protein activity were found only among 6.6Sy-globulins. In contrast, anti-humiian liv-er nii-cleoprotein extract activity was found largely withthe 18S y-globulins. although somne activ-it-y wasalso found with 6.6S y-globulins. The anti-cellnuclei activity detected by the fluiorescent anti-globulin test was found with the 6.6S y-globulinsin LE sera reacting with calf thymus nticleopro-tein extract and was found with 18S anid 6.6Sy-globulins in LE sera reacting primarily withhuman liver nucleoprotein extract.

The qualitative and quantitative variety of se-rum factors in lupus erythematosus was notable,although the reasons for their appearanlce andtheir role in diseases remains obscure. Anion-exchange cellulose chromatography provided anexcellent means for separating and identifying thereactive 6.6S and 18S y-globulins.

ACKNOWLEDGMENTS

We wish to thank Drs. John Nasou and Kurt Blochfor making available many of the sera utilized in thesestudies, and Dr. George Brecher and Miss Minna Feldfor performing the LE cell tests. We are also in-debted to Mr. Robert Bowser for help in anti-nucleo-protein extract assays and ult:acentrifugal procedures,Miss Anne P. Horbett for zone electrophoretic proce-dures, Miss Mary E. Lawrence for assistance with thechromatography, and Mr. Willie Mills for assistance inthe fluorescent antibody tests.

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