-
A SPECIFIC INHIBITOR FORHUMANDESOXYRIBONUCLEASEANDAN INHIBITOR
OF THE LUPUSERYTHEMATOSUS
CELL PHENOMENONFROMLEUCOCYTES1
By NATHANIEL B. KURNICK, LAWRENCEI.
SCHWARTZ,SANFORDPARISER,'ANDSTANLEYL. LEE
(From the Department of Medicine, Tulane University School of
Medicine, New Orleans,Louisiana; and the Hematology and Pathology
Laboratories of the Mount Sinai
Hospital, New York, N. Y.)
(Submitted for publication September 29, 1952; accepted December
3, 1952)
In speculating upon the probable role of desoxy-ribonuclease
(DNase) in the depolymerization ofdesoxyribonucleic acid (DNA),
which character-izes (1) the "lupus erythematosus (L.E.) cell"(2),
we have suggested (3), as have Klempererand his associates (4),
that an increased serum en-zyme activity, potentiation of an
intracellularDNase, or destruction of an intracellular inhibitorof
DNase by a serum factor, might be responsible.In a previous
communication (5) we reported evi-dence which excludes the serum
DNase from re-sponsibility for the phenomenon. Studies on
theinteraction of leucocytes and serum, in an ap-proach to the
alternative possibilities, disclosed thepresence of a specific
intracellular inhibitor of hu-man serum DNase and an inhibitor of
the "L.E.cell" phenomenon (6, 7). In the present paper,we shall
report upon some of the characteristics ofthese factors.
I. SERUMDNAsE INHIBITION
A. Preparation of leucocyte extractsLeucocytes (WBC) were
prepared with the aid of
Bovine fraction I from heparinized blood as previouslydescribed
(5) or by centrifugation at 0 to 2°C of fresh"ACD" bloods (120 ml.
of 1.47 per cent dextrose, 1.32per cent sodium citrate, 0.48 per
cent citric acid plus 480
ILThis work was aided by grants to Tulane UniversitySchool of
Medicine (N. B. K.) from the National HeartInstitute, U.S.P.H.S.
(No. H-714), the American HeartAssociation, American Cancer Society
(recommended bythe Committee on Growth, National Research
Council)and the Life Insurance Medical Research Fund, and tothe
Mount Sinai Hospital from the Life Insurance Medi-cal Research Fund
and A. A. List
2Anna Ruth Lowenberg Fellow in Hematology, MountSinai
Hospital.
' Weare indebted to Dr. J. W. Davenport and Mr. JohnGavey of the
Medical Department of the New OrleansRegional Blood Center,
American Red Cross, for generoussupplies of human blood.
ml. whole blood) followed by collection of the WBClayer and
repeated washing at 0 to 2°C with 0.14 MNaCIuntil a good separation
of WBCfrom erythrocytes wasobtained. The latter technique was used
with large vol-umes of human blood (2500-3000 ml.).
Extracts of WBCwere prepared by either of twomethods:
(a) Suspensions of 24 X 10, or more WBCper ml. in0.14 M NaCl or
heparinized plasma were lysed by re-peated freezing in
acetone-solid CO, mixtures followedby thawing by immersion in a
37°C water bath. Com-plete. disintegration of cells was
accomplished by freezingand thawing four times. An aliquot was
clarified by cen-trifugation at 2700 X G (measured at center of
tube)for 30 minutes in a Sorvall angle centrifuge. The ma-terial
was stored at - 20'C.
(b) Approximately 12 X 10' fresh WBCwere sus-pended in 30 ml.
0.14 MNaCl at 0 to 2°C and blended ina micro Waring Blendor
equipped with an ice jacket (8),slightly modified by the addition
of a rubber diaphragmseal for the ice jacket, to permit pouring
from the Blendorwithout contamination of the material by the
ice-salinemixture in the jacket, for 10 minutes (sufficient for
com-plete fragmentation of the cells, as demonstrated bymicroscopic
examination). The homogenate was cen-trifuged for 15 minutes at 430
X G. The supernatant,representing the bulk of the cytoplasmic
material andsome soluble nuclear components, was labelled
"cyto-plasmic fraction." This was separated by centrifugationfor 40
minutes at 2700 X G into a "cytoplasmic super-natant" and
"cytoplasmic sediment." The sediment ob-tained from the whole
homogenate at 430 X Gwas washedrepeatedly with saline in order to
obtain a clean "chro-mosomal fraction" and fractionated into
nucleohistone(DNH) and "residual chromosomes" after Mirsky andRis
(9, 10). From DNH, purified desoxyribonucleic acid(DNA) was
prepared according to Mirsky and Pollister(11). All fractions were
resuspended in 0.14 MNaa toreconstitute, approximately, the
original concentration(12 X 10' WBCin 30 ml.). All procedures were
car-ried out at 0 to 2°C. Upon completion of the
fractionation,sodium ethyl mercuri-thiosalicylate was added to a
finalconcentration of 1: 100,000, and the material was storedat -
20'C. A pale pink color was imparted to the cyto-plasmic fraction
and the supernatant obtained therefrom
193
-
N. B. KURNICK, L. I. SCHWARTZ,S. PARISER, AND S. L. LEE
by the hemoglobin of the RBCwhich remained entrappedin the
WBClayers.
B. Inhibition of serum DNase
Each of the WBCextracts prepared by thefreezing-thawing method
was tested for DNaseactivity by the methyl green method (3) as
modi-fied for serum (12). In no case was any DNaseactivity detected
under these conditions of ionicstrength and pH. Tests for DNase
activities underother conditions (13-17) were not made, sincethese
would not interfere with our experiments.Tests for DNase inhibition
against human serumwere performed by substituting the fraction to
betested for part or all of the 3 ml. saline used in di-luting the
substrate (12). Simultaneous observa-tions on the inhibition of the
"L.E. cell" phe-nomenon by each of the fractions are reported
be-
E.520 . B
.480 a\''c,440 \-
.400 -\
.360 *\ \'
.320 \\ \
.280 \ >>.E
.240 A
.200
o 30 60 90 120 150 180 210 840MINUITES
FIG. 1. HUMANSERUMDNASEINHIBMON BY HUMANLEucocvT (WBC)
EXTRACTS
Each point represents optical density of 3 ml.
sub-strate-inhibitor-serum mixture added to 0.5 ml. 0.33 Msodium
citrate after incubation for intervals indicatedat 37°C. Each 19
ml. of the incubated mixture contains1 ml. serum and 1 ml. of the
following saline WBCextracts: A. None (1 ml. 0.14 M saline), B.
Supernatantfrom 6 X 10' frozen and thawed cells, C.
"Cytoplasmicsupernatant," D. Unwashed "cytoplasmic sediment,"
E.Chromosome fraction.
low. The observations on the two types of inhibi-tion parallel
each other closely. Rarely, prepara-tions which had previously been
found to containinhibitor, failed to inhibit the "L.E. cell"
phe-nomenon and only weakly inhibited human serumDNase. As will be
seen, these failures were prob-ably due to proteolysis (autolysis),
whereas the dis-crepancies result from the greater sensitivity of
theDNase system.
Inhibition of human serum DNase was observedwith the frozen and
thawed extracts, both beforeand after centrifugation, with the
total "cyto-plasmic fraction," and the "cytoplasmic super-natant"
prepared from blended WBC. Neitherthe "cytoplasmic sediment" nor
the "chromosomalfraction" (nor fractions prepared
therefrom)demonstrated inhibition (Fig. 1).
The relationship between the amount of in-hibitor present and
the degree of inhibition is ap-parently not linear (Fig. 2), and
resembles thecurve reported by Zamenhof and Chargaff ( 13) fortheir
yeast DNase inhibitor.
C. Physico-chemical properties of inhibitorProlonged dialysis
against frequent changes of
0.14 M saline at 0°C did not alter the inhibitoryactivity of
extract fractions. No loss of inhibitionresulted from heating at
56°C for 2 hours (bothunbuffered and buffered at pH 7.5 with
tri-methylol-amino-methane buffer [18]). Heatingto 100°C in a
boiling water bath for 30 minutescompletely destroyed all
inhibitory effect. Theaddition of citric acid to bring the final
concentra-tion to 1 per cent, followed by dialysis againstsaline at
0°C (to remove citrate), resulted in lossof inhibitory activity by
"cytoplasmic supernatant."This was probably a pH effect, since
treatment ofinhibitors prepared by freezing and thawing cellsinto
saline with 0.1 N NaOHor 0.06 N HCl alsodestroyed their inhibitory
effect. However,0.00025 N HCl (as used in the pepsin
digestiondescribed below) did not affect inhibition.
Since the citrate ion is a potent inhibitor ofDNase, it seemed
possible that traces of this ionfrom the ACDsolution might have
been responsi-ble for the inhibition of serum DNase by the
"cyto-plasmic supernatant." The following observations,previously
noted, militate against this possibility:
(a) Potent inhibitors were prepared fromheparinized blood, (b)
100°C destroyed the in-
194
-
SPECIFIC INHIBITOR OF DESOXYRIBONUCLEASEAND "L. E. CELL"
0coE 60
Z 400-
20
o
a e4xioiwec/mI,fro3 swthaud tnp(s-avs serunrO 'is n 0 1I
serulnro t N) Ul} of " serurnTIA -go X io*wc/ml. fro3Wnr thcAed in
saLuits sVSmwe !
0 Q15 0.25 0.5 IVOLUMEINHIBITOR IN ML.(Finalvl,9ml.)
FIG. 2. DEGREEOF INHIBITION OF SERUMDNASEVS.
VOLUME(CONCENTRATION)OF INHIBITOR (19 ML. FINAL VOLUMEOF
SUBSTRATE-INHIBITOR-SERUM MIXTURECONTAINING 1 ML. SERUM)
hibition, (c) 1 per cent citric acid, NaOH, andHCI destroyed
inhibition, and (d) dialysis did notreduce inhibition. This
possibility was furtherexcluded by a direct test:
To 20 ml. of a "whole chromosome" suspension, and ofa
"cytoplasmic sediment" suspension, 5 ml. of "ACD"solution were
added. After an hour at room temperature,the material was dialyzed
against 0.14 M NaCl at 0-2Cfor 24 hours, with frequent changes of
the bath. No in-hibition of human serum DNase was detected with
eithermaterial before or after the treatment with "ACD."
D. Effects of enzymes"Cytoplasmic supernatant" inhibitors were
in-
cubated for 2 hours at 37°C with an equal volumeof solutions
containing 3 mg. per ml. of the follow-ing enzymes 4 in 0.01 MpH
7.5 trimethylol-amino-methane buffer (18): ribonuclease, trypsin,
chymo-trypsin, papain, and yeast protease. Ribonucleasewas also
used at 56°C for 2 hours. Pepsin wassimilarly used, but dissolved
in water (final pH4.7) or in 0.0005 N HCG. Two ml. of each mix-ture
was tested for inhibitory effect against humanserum DNase (final
volume 19 ml. as in [12]).Controls consisted of the inhibitor
incubated withthe solvent (i.e., without enzyme) and of the en-zyme
incubated with saline (i.e., without inhibi-tor). The inhibitor
controls showed unimpairedinhibitory action and the enzyme controls
had noeffect on serum DNase activity except in the case
4 Crystalline enzymes obtained from General Biochemi-cals, Inc.,
Chagrin Falls, Ohio.
of yeast protease, which, alone, revealed DNaseactivity, and
produced an additive DNase activitywith sera. All of the
proteolytic enzymes com-pletely destroyed the inhibition of serum
DNase,while ribonuclease was without effect upon theinhibitor.
E. Permeability of cell membrane to inhibitorSince WBCwere found
to contain an inhibitor
for human serum DNase, it appeared possiblethat the very low
serum DNase values commonlyencountered in normal sera (12) might
resultfrom exposure of the serum to the WBCof theblood before
separation, particularly if separationwas not carried out promptly.
Freshly clottedblood was allowed to stand at room temperatureand at
37°C. Samples of sera were removed atintervals from 15 minutes to 6
hours after col-lection. Similar experiments were performedwith
heparinized blood. No change in DNase ac-tivity was observed over
this period. After 24hours at 37°C, there was moderate reduction
ofDNase activity, but this was no greater than wasnoted when
separated serum was allowed to standfor the same interval. The fact
that no significantamount of inhibitor "leaked"' into autologous
se-rum or plasma was also supported by the observa-tion that in no
case was inactive serum found toinhibit the DNase of an active
serum. However,0.14 Msaline, allowed to stand in contact with
iso-lated WBCat 0 to 2°C for 24 to 48 hours, ex-
195
-
N. B. KURNICK, L. I. SCHWARTZ, S. PARISER, AND S. L. LEE
TABLE I
Inhibition of srum desoxyribonucease by varsous ceU types
Approx. No. cells X 100 per ml.No. of ceLlsper ml. of Lympho-
Granulo- Mono- Blast Inhibition.
Diagnosis saline cytes cytes cytes cels per cent
1) Lymph. Leukemia 20 X 10' 14 6 0 0 322) Normal 35 X 10 1S 18 2
0 393) Monocytosis 170 X 106 17 68 8S 0 414) Normal 174 X 10 75 84
1S 0 665) Ac. Myel. Leukemia 240 X 10" 5 35 0 200 566) Chronic
Myel. Leukemia 300 X 10 2 290 0 8 36
tracted a significant amount of inhibitor fromthem, as
demonstrated by inhibition of serumDNase by the supernatant.
Furthermore, 60 x10' WBCwashed four times with 0.14 M NaCl,then
frozen and thawed into 1 ml. of the test se-rum, produced only
slight inhibition of DNase,while unwashed WBC, similarly treated,
producedcomplete inhibition.
-420
0 50 60 9 12D 15080 21 240 0 30 60 9O120 150 80aIO 240MINUTES
MAINJUTES
FIG. 3. INHImON OF VAnous DESOXYRIONUcASESBY
HUMANWBC"CYToPLAsMIc SUPENATANT"
Solid line represents activity of substrate-serum alone;dotted
line represents activity of substrate-serum mix-ture plus 1 ml.
"cytoplasmic supernatant" (final volume19 ml.). 1, 2. Dog serum, 3,
4. Rabbit serum, 5, 6. Guineapig serum, 7, 8. Cat serum, 9, 10. Rat
serum, 11, 12. Ham-ster serum, 13,14. Human serum, 15,16. Bovine
pancreaticDNase.
F. Inhibitor preparations from different cell typesThe presence
of inhibitor in different types of
cells was studied by freezing and thawing in0.14 M saline,
WBCisolated from a normal per-son, a patient with post-splenectomy
monocytosis(40,000 WBCper cmm. capillary blood, 50%smonocytes), a
patient with chronic myelogenousleukemia, one with lymphosarcoma
and lymphaticleukemia (707% lymphocytes), and one with
acutemyelogenous leukemia. The effect of 0.5 ml. ofthe supernatant
from each was tested against 1 ml.of a single serum sample (Table
I).
The data suggest that lymphocytes may be richerin inhibitor than
granulocytes (cf. 1 and 6) andthat monocytes may be poor in
inhibitor (cf. 3 and4). The relationship found between the
inhibitionby 35 x 10' and by 174 x 10' normal WBCap-pears
consistent with the data in Fig. 2. Imma-ture leucocytes appear to
be richer in inhibitorthan mature WBC(cf. 5 and 6). Caution
isnecessary in interpreting these observations, how-ever, since
completeness of inhibitor extractionmay vary among the cell
types.
G. Species specificity of inhibitorNine different inhibitors
prepared from human
WBCwere tested against the sera of dog (diluted3: 4 with
saline), cat (diluted 3:100), rabbit(diluted 1: 2), hamster
(undiluted), guinea pig(diluted 3:4), rat (undiluted), and mouse
(un-diluted). Dilutions were selected so that 1 ml.of the diluted
serum approximated the DNase ac-tivity of active human serum (about
140 x 10-'units per ml. serum [see (3) for the definition ofDNase
unit]). Bovine pancreatic DNase (19)'was similarly tested in a
final concentration of0.005 Ig. per ml. The results of a typical
experi-
5 Worthington Laboratories, Freehold, N. J.
196
-
SPECIFIC INHIBITOR OF DESOXYRIBONUCLEASEAND "L. E. CELL
TABLE IS
Enhanced susceptibility of washed eucocytes to "L.E. ceW'
transformaion
Unwashed WBC Washed (4 X) WBCL.E. serum
dilution "L.E. "L.E.(initial) "Globs" cel" Rosettes Total
"Globs" cels" Rosettes Total
Undil. 2 2 1 5 2 3 3 81:2 0 1 1 2 2 2 3 71:4 0 0 0 0 2 2 2 61:8
0 0 0 0 1 0 1 21:16 0 0 0 0 0 0 0 0
ment are shown in Fig. 3. As indicated by thecurves, only the
human and rabbit DNase werecompletely inhibited. There was moderate
inhibi-tion of guinea pig and hamster serum DNase,questionable
inhibition of canine DNase, and noinhibition of rat, cat, and mouse
serum and bovinepancreatic DNase.
II. "L.E. CELL" PHENOMENONINHIBITOR
A. Quantitation of "L.E. cell" phenomenonSera with "L.E.
phenomenon activity" (L.E. serum)
were the same as those reported previously (5). Leuco-cyte (WBC)
"buttons" of 10-15 X 10. WBCwere pre-pared as in that paper (5).
These WBC"buttons" wereused as substrate for the L.E. phenomenon
without fur-ther treatment or after washing four times by
resuspensionin about 7 ml. isotonic saline and re-centrifugation.
The"buttons" were suspended in 0.5 ml. saline (or inhibitorto be
tested), 0.5 ml. of L.E. serum (diluted with normalserum) was
added, and the mixture incubated at 37°C for1 hour. The WBCwere
then sedimented by centrifuga-tion for 5 minutes at low speed,
smeared upon glass slides,fixed in absolute methyl alcohol for 5
minutes, andstained in dilute Giemsa solution for 30 minutes.
Thestained smears of a given experiment (with appropriatecontrols)
were examined by a single observer who (toinsure objectivity) did
not know the labelling code.Each of the three morphological
manifestations of the
LE. phenomenon (a. classical "L.E. cell" with singlelarge
inclusion, or "droplet cell" with several small in-clusions, b.
rosettes, c. "globs" [5]) was graded from 0to 4 on the basis of
frequency of occurrence.
B. Increased susceptibility of washed WBCto"L.E. cell"
transformation
It was regularly found that washed WBCweremore susceptible to
"L.E. cell" transformation thanwere unwashed WBC, as measured both
by theintensity of the "L.E. cell" phenomenon at anygiven dilution
of L.E. serum and by the titer towhich the L.E. serum would
tolerate dilution be-fore loss of demonstrable L.E. activity (Table
II).To exclude the possibilities (a) that the removalof the native
plasma or the heparin and fibrinogenincluded therein and (b) that
the traumatizationof the WBCby repeated centrifugation had
causedtheir greater susceptibility to the "L.E. cell"
trans-formation, two controls were added. In one, thewashed WBCwere
resuspended in their originalplasma, re-centrifuged, and tested. No
diminu-tion in the enhancement of the "L.E. cell" phe-nomenon
produced by washing was observed.In the second control, unwashed
WBCwere re-suspended in the original plasma and re-centrifuged
TABLE III
Inhibon of "L.E. ceU" phenomenon by kucocyte homogenat*
Control (normal plasma) Inhibited (WBChomogenate)"L.E. serum
dilution "L.E. "L.E.(initial) "Globs" cells" Rosetts Total
"Globs" cedls" Rosettes Total
Undil. 2 3 3 8 2 1 1 41:2 1 2 3 6 1 1 1 31:4 1 2 1 4 1 1 1 31:8
1 1 1 3 0 0 0 01:16 1 1 0 2 0 0 0 01:32 1 0 0 1 0 0 0 01:64 0 0 0 0
0 0 0 0
Washed WBC(15 X 106) plus 0.5 ml. normal plasma or WBChomogenate
plus 0.5 ml. L.E. serum (diluted withnormal serum).
** WBChomogenate consists of 40 X 10 WBCper ml. frozen and
thawed in normal plasma.
197
-
N. B. -KURNICK, L. I. SCHWARTZ,S. PARISER, AND S. L. LEE
'and resuspended four times. Such unwashed, butrepeatedly
resuspended and re-centrifuged WBCrevealed no increased
susceptibility to "L.E. cell"transformation. Controls in which
normal sera,with and without DNase activity, were substitutedfor
active L.E. sera revealed no "L.E. cell" induc-tion in either
washed or unwashed WBC.
C. "L.E. cell" phenomenon inhibitionThe probability that the
increased susceptibility
of washed WBCto "L.E. cell" transformation wasdue to the partial
removal from the WBCof in-hibitor was confirmed by replacing the
inhibitor.
8
7
6
4
3
\.---\ ---GUI; ----.
\I
'N~
v-.ashed oaLl tLreanwashQd ceLl tiirev,ashodce11
tf'nhibibtThLan,shed call +*inhibitortitim
1:2 1:4 1:8 1:16 1:3. 164 1:128 1:256 1:512F1NALvi tLUTxC*Jor LE
SERCUM
FIG. 4. EFFECT OF SUPERNATANTFROM24 x 10' WBCnL. FROZEN AND
THAWEDIN NORMALSERUMON
INTENSITY OF "L.E. CELL" PHENOMENONAT SEVERALDILUTIONS OF L.E.
SERUM(IN NORMALSERUM)
The washed WBCwere resuspended in a WBChomogenate or a
WBCfraction (see Part I, A fordescription of fractions). The whole
homogenates,and each of the fractions which contained
DNaseinhibitor ("cytoplasmic fraction," "cytoplasmicsupernatant"),
but not those without DNase in-hibitor activity (chromosomal
fraction, cytoplas-mic sediment, DNH, DNA, histone,
"residualchromosomes"), reduced the intensity of the "L.E.cell"
phenomenon in washed WBC(Table III).Slight inhibition of "L.E.
cell" transformation ofunwashed WBCwas also demonstrated (Fig.
4).
In order to test the relationship between inhi-bition of the
"L.E. cell" phenomenon and the con-
- a contro[- - inhibitor 1:8
6 ' V4
undlkuLed
-3~ ~ ~ ~~N
156 l12 124 IAIBLE. SEWI DIXUlmON (Fial)
FIG. 5. EFcEr OF CONCENTRATIONOF INHIBrIOR ONTHE INTENSITY OF
THE "L.E. CELL" PHENOMENONPRO-DUCEDIN WASHEDWBCBY SEVERAL DILUTIONS
OF L.E.SERUM
centration of the inhibitor, an experiment was per-formed in
which the inhibitor, a homogenate of24 x 106 WBC per ml. plasma
(frozen andthawed), was serially diluted with normal plasma.The
control contained normal plasma in place ofthe inhibitor. The data
are presented in Fig. 5,and the percentage inhibition in Fig. 6.
The simi-larity between Fig. 6 and Fig. 2 is apparent. Itshould be
borne in mind that the percentage inhibi-tions shown in Fig. 6
represent the ratios of thetotal grading units of the inhibited and
uninhibitedexperiments. This is only a very rough estimateof the
degree of inhibition, therefore. Since the
l00O
80
o 60
1 40
20
o
a
0
* 1:4 LE: serrn1:8 " of
A 1:16 " .,D 1: X .
0 .1 .2 .3 .4 .5\ULUME ilRlUBITOP. (in frbl VoL 1.5 ml.)
FIG. 6. DEGRmOF INHIBMON OF "L.E. CELI' PHE-NOMENONVS.
VOLUME(CONCENTRATION) OF INHIBITOR(FROM DATA IN FIG. 5)
Z
0
ID
-i
1-
198
I
-
SPECIFIC INHIBITOR OF DESOXYRIBONUCLEASEAND "L. E. CELL 19
final volume is approximately 1.5 ml. in the "L.E.cell"
experiments, whereas it is 19 ml. in theDNase experiments, the
greater sensitivity of theDNase system to the inhibitor is
obvious.
D. Physico-chemical properties of "L.E. cell"phenomenon
inhibitor
Like the DNase inhibitor, the "L.E. cell" phe-nomenon inhibitor
was found to be unaffected bya temperature of 56°C for 30 minutes,
but wasdestroyed at 100°C for 15 minutes. Similarly,dialysis did
not impair the inhibition of the "L.E.cell" phenomenon. After
repeated use over a pe-riod of weeks, which required bringing the
materialto room temperature for short intervals and re-freezing,
inhibition of both the serum DNase andof the "L.E. cell" phenomenon
became progres-sively less effective. This was presumed to be
TABLE IV
Effect of anions on "L.E. ceU" phenomenon
Result*Concen-
Salt tration Undil.* 1:4 1:16 1:64
Sodium Chloride 0.14M 10 6 3 1Sodium Arsenate 0.2 M 10 4 0
0Potassium Oxalate 0.2 M 6 1 0 0Sodium Citrate 0.2 M 5 2 1 0
* Total grading score. Maximum is 12 (4 each for "L.E.cells,"
rosettes, "globs")
** Dilutions indicated are those of the L.E. serum(diluted with
normal serum)
due to proteolysis, since, under the conditions de-scribed in
Part I, D, the "L.E. cell" inhibitor wasalso destroyed by
proteolytic enzymes.
E. Effect of magnesium ion binding anions on"L.E. cell"
phenomenon
Since intracellular DNase is thought to playa part in the
production of the "L.E. cell" phe-nomenon, anionic DNase inhibitors
were tested.These ions are thought to act by binding Mg++,which is
required for optimal DNase activity. TheWBCbuttons were suspended
in 0.5 ml. of theelectrolyte solution, followed by 0.5 ml. L.E.
se-rum. Thus, the final concentration of the salt wasapproximately
one-third that indicated in TableIV, which presents the observed
results. Definiteinhibition by citrate, oxalate, and arsenate, in
orderof decreasing effect was apparent.
DISCUSSION
The experimental data demonstrate the presencein human
leucocytes of an inhibitor for humanserum DNase, and an inhibitor
of the "L.E. cell"phenomenon.
These inhibitors do not permeate a cellophanemembrane, are
destroyed by heating to 100°C butare stable at 56°C, are stable at
- 20°C for sev-eral months and at 0-2oC for at least several
days.They are destroyed by a number of proteolytic en-zymes, but
not nucleolytic enzymes. Extremesof pH also destroy the DNase
inhibitor. Effortsat purification and further physico-chemical
char-acterization of the factor or factors are in progress.
The DNase inhibitor reveals species specificityto some extent
(only in the rabbit is the same de-gree of inhibition obtained as
in the human). Itmay be suggested that the specificity is due to
therequirement for a hypothetical serum co-factor.However,
inhibitors prepared by freezing andthawing WBCin their own plasma,
which wouldcontain the hypothetical co-factor, reveal the
samespecificity as do saline extracts. Thus, the speci-ficity is
probably a function of differences in DNasefrom various species.
Immunologic differences inDNases have already been demonstrated by
Mc-Carty (20).
That the inhibitors are present in the leucocytesis apparent
from the method of preparation. Theonly other elements regularly
present are throm-bocytes. However, potent inhibitors were
obtainedfrom the WBCof a patient with severe thrombo-cytopenia. The
inhibitors are found in the super-natant upon rupturing the cells
in saline. Al-though this fraction is ordinarily regarded as
cyto-plasmic, it undoubtedly includes some proteins ofnuclear
origin. The cytological localization of theinhibitors is,
therefore, unsettled.
Desoxyribonuclease inhibition by cell extractshas also been
reported by others. The inhibitorof yeast DNase which Zamenhof and
Chargaff(13), obtained from yeast, resembles the inhibitorin
WBCdescribed in this paper in its species speci-ficity (for yeast),
in its instantaneous effect uponthe enzyme activity, and in its
protein nature, butis less stable (destroyed at 56°C in 5
minutes).Dabrowska, Cooper and Laskowski (21) andCooper, Trautmann
and Laskowski (22) pre-pared an inhibitor of bovine DNase from
several
199
-
N. B. KURNICK,- L. I. SCHWARTZ, S. PARISER, AND S. L. LEE
avian and mammalian organs. These inhibitorswere also proteins
and were very unstable. Thisinstability interferes with the
separation of excessDNase activity from the inhibitor content of
cellextracts since both DNase activity and inhibitorare destroyed
at 56°C. Thus, these inhibitorsdiffer from ours both in lack of
species specificityand in stability.
Henstell and Freedman (23, 24) have recentlyreported on the
preparation of an inhibitor of bo-vine DNase from leucocytes by the
dilution ofwhole blood 1: 100 or 1: 1000 with water. Theirinhibitor
appears to be very similar to that of Las-kowski's group in its
non-specificity and instability.It differs from that reported by
us, because it ispotent in very great dilution and is less,
ratherthan more, abundant in immature cells. It is ap-parent that
we failed to observe the inhibitor re-ported by these authors
because of its instability,since several hours were required for
the prepara-tion of the inhibitor in leucocytes, and it was
nottested on the day of preparation.
Bernheimer and Ruffier (25) isolated a specificDNase inhibitor
from streptococci. This inhibitor,however, differs from that
described here in be-ing destroyed by ribonuclease but not by
proteoly-tic enzymes, thus indicating its non-protein na-ture. It
appears, then, that we have observed anew inhibitor from mammalian
tissues, most closelyresembling that described by Zamenhof and
Char-gaff (13) in yeast.
Close parallelism between the inhibitor of serumDNase described
by us and the inhibitor of the"L.E. cell" phenomenon suggest their
identity.The two activities are located in the same WBCfraction.
They show the same heat stability andare non-dialyzable. Complete
correspondence isnot observed in the intensity of the effect, in
thatmore nearly complete inhibition of serum DNasethan of the "L.E.
cell" phenomenon is usually ob-tained. This, however, may not
indicate lack ofidentity of the inhibitors, since the
intracellularDNase is probably less accessible to inhibitoradded!
in vitro, and the intra- and extracellularDNases may not be
identical in susceptibility tothe inhibitor. Proof of the identity
of the inhibi-tors must await their further purification.
Since the evidence suggests the identity of theintracellular
DNase inhibitor and the inhibitor ofthe "L.E. cell" phenomenon, it
is probable that
the depolymerization of DNAwhich characterizesthe "L.E. cell" is
secondary to a derangement ofthe intracellular DNase-DNase
inhibitor system.This abnormality is not primary in the cell,
sinceit can be induced in leucocytes from a normal in-dividual (26)
by serum from a patient with sys-temic lupus erythematosus.
Wesuggest that theL.E. plasma factor (27), which itself has noDNase
activity (5), causes the release of the in-tracellular DNase from
inhibition. The inhibitionof the "L.E. cell" phenomenon by anionic
DNaseinhibitors, as well as by an extract which containsa naturally
occurring DNase inhibitor, supportsthis hypothesis.
This hypothesis led to the development of a"sensitized" "L.E.
cell" test, using WBC, whichhad been washed to remove inhibitor.
False posi-tive tests are not produced, perhaps because un-broken
WBCcontain some inhibitor even afterrepeated washing. This was
demonstrated for theDNase inhibitor. Thus, the L.E. serum factor
isstill required to elicit the phenomenon.
The fact that mature granulocytes show greatersusceptibility to
the "L.E. cell" transformationthan lymphocytes (28) may be due to
the apparentgreater inhibitor content of the latter noted in
ourstudies (Table I). Likewise, the lesser suscepti-bility of
immature granulocytes may be attributedto their greater DNase
inhibitor content as com-pared with mature leucocytes.
Observations on the DNase inhibitor content ofvarious tissues,
normal and pathological, and atvarious stages of development are in
progress.Some observations on lymphocytes, blasts, andgranulocytes
have already been referred to above.The possible role of a specific
inhibitor of DNasein cellular division, growth, senescence, and
thedevelopment of tumors, which suggests itself, isunder further
investigation.
SUMMARY
1. An inhibitor for human serum DNase hasbeen observed in human
leucocytes. It is a pro-tein, soluble in saline, stable at 560C.
Testedagainst several species, it is equally effective onlyagainst
rabbit serum DNase and has little or noinfluence on some others. In
its stability andspecificity it differs from other DNase
inhibitorspreviously reported.
200
-
SPECIFIC INHIBITOR OF DESOXYRIBONUCLEASEAND "L. E. CELL 201
2. An inhibitor for the "L.E. cell" phenomenonhas been
demonstrated in leucocytes. Evidencewhich suggests its identity
with the DNase in-hibitor is presented.
3. A sensitive "L.E. cell" test using washedWBCis described.
4. A mechanism for the "L.E. cell" phenomenonis discussed.
ACKNOWLEDGMENTS
The technical assistance of Mrs. Mary Eason is grate-fully
acknowledged.
The encouragement of Dr. Paul Klemperer throughoutthese studies
is deeply appreciated.
REFERENCES
1. Lee, S. L., Michael, S. R., and Vural, I. L., The L.E.(Lupus
erythematosus) cell. Clinical and chemi-cal studies. Am. J. Med.,
195.1, 10, 446.
2. Hargraves, M. M., Richmond, H., and Morton, R.,Presentation
of two bone marrow elements: the"tart" cell and the "L.E." cell.
Proc. Staff Meet.,Mayo Clin., 1948, 23, 25.
3. Kurnick, N. B., The determination of desoxyribonu-clease
activity by methyl green; Application to se-rum. Arch. Biochem.,
1950, 29, 41.
4. Klemperer, P., Gueft, B., Lee, S. L., Leuchtenberger,C., and
Pollister, A. W., Cytochemical changes ofacute lupus erythematosus.
Arch. Path., 1950, 49,503.
5. Kurnick, N. B., Pariser, S., Schwartz, L. J., Lee, S. L.,and
Irvine, W., Studies on desoxyribonuclease insystemic lupus
erythematosus: Non-participationof serum desoxyribonuclease in the
L.E. phe-nomenon. J. Clin. Invest., 1952, 31, 1036.
6. Kurnick, N. B., discussion of paper by Sparrow, A. H.,Moses,
M. J., and Dubow, R. J., Relationships be-tween ionizing radiation,
chromosome breakageand certain other nuclear disturbances. Exptl.
CellRes., 1952, 2, 266.
7. Kurnick, N. B., Schwartz, L. J., Pariser, S., Lee, S.,and
Irvine, W., The role of desoxyribonuclease anda nuclease inhibitor
from leucocytes in the lupuserythematosus cell phenomenon. J. Clin.
Invest.,1952, 31, 645.
8. Sorof, S., and Cohen, P. P., Modified semi-micro-and
micro-Waring blendors for low temperatureuse. Exptl. Cell Res.,
1951, 2, 299.
9. Mirsky, A. E., and Ris, H., Isolated chromosomes.J. Gen.
Physiol., 1947, 31, 1.
10. Mirsky, A. E., and Ris, H., The chemical compositionof
isolated chromosomes. J. Gen. Physiol., 1947,31, 7.
11. Mirsky, A. E., and Pollister, A. W., Chromosin,
adesoxyribose nucleoprotein complex of the cell nu-cleus. J. Gen.
Physiol., 1946, 30, 117.
12. Kurnick, N. B., Desoxyribonuclease activity of seraof man
and some other species. Arch. Biochem.and Biophys., in press.
13. Zamenhof, S., and Chargaff, E., Studies on the
desoxy-pentose-nuclease of yeast and its specific
cellularregulation. J. Biol. Chem., 1949, 180, 727.
14. Catcheside, D. G., and Holmes, B., The action of en-zymes on
chromosomes. Symp. Soc. Exptl. Biol.,Cambridge, 1947, 1, Nucleic
Acid, 225.
15. Maver, M. E., and Greco, A. E., The nuclease activi-ties of
cathepsin preparations from calf spleen andthymus. J. Biol. Chem.,
1949, 181, 861.
16. Brown, K. D., Jacobs, G., and Laskowski, M., Thedistribution
of nucleodepolymerases in calf thy-mus fractions. J. Biol. Chem.,
1952, 194, 445.
17. Webb, M., Use of desoxyribonuclease inhibitors in
theisolation of desoxyribonucleic acids. Nature, 1952,169, 417.
18. Gomori, G., Buffers in the range of pH 6.5 to 9.6.Proc. Soc.
Exper. Biol. & Med., 1946, 62, 33.
19. Kunitz, M., Isolation of crystalline desoxyribonu-clease
from beef pancreas. Science, 1948, 108, 19.
20. McCarty, M., Purification and properties of
desoxyri-bonuclease isolated from beef pancreas. J. Gen.Physiol.,
1946, 29, 123.
21. Dabrowska, W., Cooper, E. J., and Laskowski, M.,A specific
inhibitor for desoxyribonuclease. J. Biol.Chem., 1949, 177,
991.
22. Cooper, E. J., Trautmann, M. L., and Laskowski,
M.,Occurrence and distribution of an inhibitor for
de-soxyribonuclease in animal tissues. Proc. Soc.Exper. Biol. &
Med., 1950, 73, 219.
23. Henstell, H. H., and Freedman, R. I., The viscosi-metric
determination of desoxyribonuclease inhibi-tion. Cancer Res., 1952,
12, 341.
24. Henstell, H. H., Freedman, R. I., and Ginsburg, B.,An
inhibitor of desoxyribonuclease in human whiteblood cells and bone
marrow cells and its relation-ship to cellular maturity. Cancer
Res., 1952, 12,346.
25. Bernheimer, A. W., and Ruffier, N. K., Elaborationof
desoxyribonuclease by streptococci in the rest-ing state and
inhibition of the enzyme by a sub-stance extractable from the
cocci. J. Exp. Med.,1951, 93, 399.
26. Berman, L., Axelrod, A. R., Goodman, H. L., andMcClaughry,
R. I., So-called "lupus erythematosusinclusion phenomenon" of bone
marrow and blood.Morphologic and serologic studies. Am. J.
Chin.Path., 1950, 20, 403.
27. Haserick, J. R., Lewis, L. A., and Bortz, D. W.,Blood factor
in acute disseminated lupus erythemato-sus; I. Determination of
gammaglobulin as specificplasma fraction. Am. J. M. Sc., 1950, 219,
660.
28. Rohn, R. J., and Bond, W. H., Some supravital ob-servations
on the "L.E." phenomenon. Am. J.Med., 1952, 12, 422.
201