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Immunologic and clinical observations of granulocyte-specific antinuclear antibodies.

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Arthritis and Rheumatism
official Journal of the ARA Secfion of fheArfhrifisFoundafion
JUNE, 1969
Immunologic and Clinical Observations of Granulocyte-Specific
Antinuclear Antibodies
The presence of granulocyte-specific antinucleoprotein antibodies was established
by absorption tests in 31 sera and unmasked in three additional sera. The majority of patients demonstrated rheumatoid arthritis, but 2 patients had lupoid
hepatitis. Differences in fluorescent patterns of staining and degrees of heterologous reactivity delineated two separate
granulocyte-specific antinuclear antibodies, one of which showed a significant
association with granulocytopenia.
FACTORS associated with
systemic lupus in general show diffuse
reactivity by immunofluorescence with
nuclei of various mammalian tissues. Sera
from patients with rheumatoid arthritis,
however, tend to show variable reactivity
with nuclei from different sources although
peripheral blood leukocytes frequently provide the required nuclear antigens.l Faber
and associates identified an antibody which
gave evidence of reactivity only with granulocytic nuclei and occurred primarily in
sera of rheumatoid arthritis patient^.^^^
To establish the specificity of granulocyte-reactive antinuclear antibodies, absorption tests have been found necessary.
Elling demonstrated that the granulocyte
reactivity in occasional sera was decreased
or abolished by absorption with nongranulocytic c e k 4 Although it was suggested
that these results might reflect contamination of the absorbent with granulocytes,
similar experiments in this laboratory have
indicated that apparent granulocyte specifk
reactivity may be abolished subsequent to
absorption with calf thymus nucleoprotein
in the absence of granulocytes. Accordingly, in the following studies this absorptive
technic was employed to substantiate the
occurrence of granulocyte specifh (GS) antinuclear antibodies in 31 sera. Immunologic characteristics of the GS antibodies
and associated clinical aspects were investigated. Based on differences in fluorescent patterns of staining and degrees of
heterologous nuclear reactivity, evidence is
From the Department of Medicine, Western
Reserve University School of Medicine, and University Hospitals, Cleveland, Ohio.
Supported b y the Hankins Foundation and the
Arthritis Foundation of Northeast Ohio.
KATHRYN H. SVEC,M.D.: Assistant Professor of
Medicine, Western Reserve University School of
Medicine, and University Hospitals, Cleveland,
Ohio 44106.
Reprint requests should be addressed to Dr.
Svec at University Hospitals, 2065 Adelbert Road,
Cleveland, Ohio 44106.
RHEUMATISM, VOL. 12, No. 3 (JUNE 1969)
presented for the occurrence of two GS from fresh calf thymus at low ionic strength by
antibodies, one of which has a significant the method of Chargaff.8 Sera were absorbed with
an equal volume of nucleoprotein for 1 hour at
association with granulocytopenia.
37OC and 18 hours at 4OC. Control sera were
diluted 1/2 with physiologic saline solution.
Normal human granulocytes obtained in heparinized peripheral blood were sedimented in 5 per
Clinical m a t e d . Sera were obtained from staff
patients in the University Hospitals Arthritis Clinic cent dextran, molecular weight 232,000, and sepaand from private patients of rheumatologists in the rated by differential centrifugation.9 Only final
Arthritis Unit. Complete clinical and laboratory preparations consisting of 90 per cent or more
granulocytes were used. Neutrophils were also obdata were available. All sera were stored at -22OC.
The American Rheumatism Association's revised tained from synovial fluid collected by aspiration
criteria5 were used for the diagnosis of rheumatoid and open drainage from a patient with arthritis
arthritis. Six patients who showed 2 to 10 LE cells and ulcerative colitis. The fluid contained over
on two cover glass smears were included in the 60,000 leukocytes per cubic millimeter, 98 to 100
rheumatoid arthritis group because they exhibited per cent of which were polymorphonuclear leukodeforming arthritis with rheumatoid nodules and cytes. Cultures and gram and acid-fast stains of the
no evidence of rash, serositis, vasculitis, or renal fluid were negative.
A 100 per cent pure preparation of normal huinvolvement. No clinical differences were apparent
between these patients and the rheumatoid arthri- man lymphocytes was provided in chyle obtained
tis patients who had negative LE cell preparations. from thoracic duct cannulation by Dr. William
Systemic lupus was diagnosed on the basis of typi- Falor, Akron, Ohio,lo and made available by Dr.
cal multisystem involvement and positive tests for Rune Stjernholm, Cleveland. Prior to storage, the
antinuclear factors, frequently including LE cells granulocytes from synovial fluid and the lymphoand/or anti-DNA. Lupoid hepatitis was defined as cytes from chyle were washed three and five times,
chronic liver disease with associated antinuclear respectively, with physiologic saline solution.
Washed granulocytes from synovial fluid were
factors and variable lupus-like extrahepatic sympemployed to prepare subcellular fractions. Nuclei
Zmmunofluorescent methods. Antinuclear factors were isolated with 0.2 per cent citric acid by the
were detected by the indirect fluorescent antibody method of Nathan and Snapper.11 Staining of the
technic with nuclei provided by normal human resulting preparation with Wright's stain revealed
spleen imprints. The preparation of the imprints that 95 per cent of the nuclei were devoid of
and the sensitivity of these nuclei have been previ- cytoplasm. Nucleoprotein was extracted at low
ously described.6 For studies of heterologous re- ionic strength.8 In contrast to the nucleoprotein
activity, imprints were similarly prepared from prepared from calf thymus, this product was less
rat, rabbit, cow, and dog spleens. When human readily soluble in distilled water and was stored
liver and kidney sections were employed, tis- as a suspension.
Final preparations of human cells and cell fracsues were frozen and cut in the cryostat at 4 microns. Unfixed air-dried imprints or sections were tions were packed by centrifugation as small
washed with phosphate-buffered saline, 0.01 M, measured aliquots in Lusteroid tubes and stored
pH 7.2,for 10 min. and incubated for 60 min. at at -22OC. In absorption tests, equal volumes of abroom temperature with sera and fluorescent anti- sorbent and serum at 1/2 dilution were incubated.
sera, respectively. The fluorescent conjugates spe- In control tests, sera were diluted 1/4 with phoscific for y globulin and for yG, yM, and yA globu- phate-buffered saline.
Treatment of nuclei with enzymes. Serum nulins have been previously described.7 yA antinuclear factors were tested in a triple layer system. clear reactivity was tested with human spleen imTwofold serum dilutions were employed for prints which had been treated with DNA-ase or
quantitation of reactivity. Prior to studies of trypsin for 60 min. at room temperature and
species specificity and immunoglobulin composition washed twice with buffered saline. DNA-ase ( 1x
of antinuclear factors, sera were routinely ab- recrystallized),* 0.25 mg. per milliliter of 0.1 M
sorbed with calf thymus nucleoprotein. Fluorescent KH,PO,, pH 7, with 1 &M MgCl, per 0.1 mg.
microscopy was performed with a Leitz micro- DNA-ase and trypsin ( 2 x recrystallized)," 0.01
scope, used with an Osram HBO 200 watt mer- mg. per milliliter of phosphate-buffered saline,
cury lamp, and fitted with a UG-1 exciter filter 0.01 M, pH 7.8, were employed.
and a colorless ultraviolet absorbing bamer filter.
Absorption tests. Nucleoprotein was isolated
*Worthington Biochemicals, Freehold, N. J.
Definition of study sera containing granulocyte specific ( G S ) antibodies. In the
course of routine immunofluorescent testing of over 5000 sera for antinuclear activity with spleen imprints, 41 sera were encountered which gave prominent staining
of the nuclei of granulocytes and lesser or
undetectable reactivity with the majority of
cell nuclei, i.e., lymphocytes. In 31 sera,
the occurrence of GS antibodies was demonstrated when absorption with calf
thymus nucleoprotein resulted in the abolition of lymphocyte staining while reactivity
with granulocytic nuclei persisted. Subsequent to absorption with calf thymus nucleoprotein, however, all nuclear reactivity
was abolished from 10 sera. The presence
of GS antibodies was most often verified in
sera which showed a marked difference between granulocyte and lymphocyte nuclear
reactivity prior to absorption (Table 1,
Fig. 1).
Fluorescent patterns of GS reactiuity. In
25 of the study sera, GS nuclear staining
was homogeneous (type I staining, Fig. 2 ) .
Six sera reacted with granulocyte nuclei
to give a bizarre pattern of staining consisting of irregular fluorescent streaks and
globules which appeared to be intranuclear
although nuclear outlines were indistinct
(type I1 staining, Figs. 3A and B). The
same two types of reactivity were demonstrable with granulocyte nuclei when present in human liver and kidney sections,
while lesser or undetectable reactivity occurred with parenchymal nuclei. Absorption with calf thymus nucleoprotein did not
alter either pattern of GS staining.
Further tests of antigenic specificities. Absorption of selected sera with human peripheral cell preparations gave additional
evidence that both types of reactivity were
granulocyte specific. When six type I and
four type I1 sera were absorbed with
human lymphocytes, no diminution in
granulocytic nuclear reactivity occurred.
Absorption of the same sera with either
whole granulocytes or isolated nuclei of
granulocytes completely abolished all nuclear reactivity. The diffuse nuclear reactivity in two lupus sera with both lymphocytes and granulocytes was abolished
following incubation with any of the above
Species specificities. When tested with
heterologous spleen imprints, all 25 sera
containing type I antibodies demonstrated
Table 1.-Effect of Absorption of Sera with Calf Thymus Nucleoprotein on
Fluorescent Nuclear Reactivity with Spleen Imprints
After Absorption
of Sera
3 f
Nuclear Reactivity*
GS Anbbody
Number of
Sera Showiq no
Nuclear Reachvlty t
'Graded 1 to 44- on the basis of intensity of fluorescence.
f Staining of granulocytic nuclei unchanged in intensity but reaction with lymphocytic nuclei
4 No staining of either lymphocytic or granulocytic nuclei.
Fig. 1.-Intense homogeneous nuclear reactivity with granulocytes in human spleen
imprint demonstrated by immunofluorescence. Nuclei of lymphocytes show only faint
staining. X 430.
rabbit spleen. The six sera containing type
I1 antibodies showed no reactivity with any
of the heterologous spleen nuclei. The presence of granulocytes in imprints from all
species was verified with Wright’s stain.
Immunoglobulin composition. Type I GS
factors were consistently found present in
the yG immunoglobulin class with titers of
reactivity ranging from 1/16 to 11256. In
only five sera were low titers of yA and/or
yM reactivity detected. Some of these sera
also contained rheumatoid factor, and it is
recognized that apparent macroglobulin
antinuclear activity may occasionally be the
Fig. 2.-Homogeneous
type I immunofluo- result of interaction between rheumatoid
rescent staining pattern with nucleus of granu- factor and yG antinuclear factors.12 Type
locyte. Smaller rounded nuclei of lymphocytes I1 GS antinuclear antibodies were restricted
in background show no fluorescence and are to the yG immunoglobulin class and were
barely discernible. X 1250.
detected at serum dilutions of 118 to 11256.
Clinical obseruations. As shown in Table
reactivity with granulocyte nuclei in dog 2, the majority of patients with either type
spleen imprints, 10 sera reacted with gran- I or I1 antibodies had rheumatoid arthritis.
ulocyte nuclei in both dog and cow spleen, One patient with type I1 antibody demonbut no serum reacted with nuclei of rat or strated an ill-defined connective tissue dis-
Fig. 3.-(A)
and (B): Irregular nonhomogeneous type I1 reactivity with granulocyte
X 1250.
ease diagnosed as possible systemic lupus,
characterized by chronic motor neuropathy
and rare episodes of arthralgias. Statistically signscant clinical differences between patients exhibiting the two types of
GS antibodies are summarized in Table 3.
Group I patients more frequently had definite or classical rheumatoid arthritis and
were on the average 16 years older than
group I1 patients. These differences were
not a reflection of duration of disease since
rheumatic symptoms had been present an
average of 12 years in both groups.
The two groups also differed in that patients with type I1 GS antibodies more
commonly showed leukopenia, 4500 white
blood cellslcu. mm. or less, with granulocytes decreased to 2300/cu. mm. or less.
Both group I granulocytopenic patients
also exhibited splenomegaly, while granulocytopenia occurred as an isolated finding
in the group I1 patients. There was no
correlation between leukopenia and the administration of drugs, including chloroquine.
No significant differences were observed
between patients with type I or I1 antibodies with respect to sex or race, presence of nodules, or occurrence of rheuma-
Table 2.4linical Diagnoses of Patients
Exhibiting Granulocyte-Specific
Antinuclear Antibodies
Rheumatoid arthritis:
Possible systemic lupus
* Numbers of patients.
10 *
Type II
toid factor. LE cells were detected in 6 of
16 (37.5 per cent) group I patients and in
none of the 4 group I1 patients tested
( p > 0.05).
Serial studies. In 14 group I patients,
follow-up sera were obtained at 4 to 6
month intervals for 1 to 4 years. Homogeneous reactivity restricted to granulocytic
nuclei persisted without significant changes
in immunoglobulin composition. Fluctuaations in titers of reactivity occasionally occurred but did not appear to parallel
rheumatic disease activity. Serial sera obtained from 2 group I1 patients over 1 and
2 year periods, respectively, continued to
show the same irregular pattern of staining
Table 3.-Signihant
Clinical Differences Between Patients with
Types I or II Granulocyte-Specific Antinuclear Antibodies
Clinical Feature
Definite or classical rheumatoid arthritis
Average age
Leukopenia with granulocytopenia $
Patieats. 25
23 pts. (92%)
55.7 years
2 pts. (8%)
2 pts. (33%)
39 years
5 pts. (83%)
< 0.01 *
< 0.01 t
< 0.001 *
Fisher’s exact test.
+ T test for groups of unequal distribution.
$4500 leukocytes or less and 2300 granulocytes or less per cu. mm.
of granulocytic nuclei without change in
either titer of reactivity or immunoglobulin
composition. Granulocytopenia which was
present in both patients also persisted. No
patient in either group showed any increase
in nongranulocytic nuclear reactivity.
Detection of masked GS antibodies. Sera
showing strong reactivity with all nuclei of
human spleen imprints were absorbed with
calf thymus nucleoprotein to determine
whether GS antibodies were present but
obscured by other antinuclear antibodies.
Sera from 15 rheumatoid arthritis patients,
55 systemic lupus patients, and 5 lupoid
hepatitis patients were studied. In three instances, absorption revealed GS antinuclear
antibodies which were verified by additional absorption tests with human granulocytes and lymphocytes. The GS antibody in
one serum from a patient with definite
rheumatoid arthritis showed the pattern of
staining and species cross-reactivity characteristic of type I antibodies. The other
two sera were obtained from young lupoid
hepatitis patients, who exhibited splenomegaly and pancytopenia including granulocytopenia. GS antibodies in these sera
gave a pattern of nuclear staining identical
to type I1 reactivity and did not react with
nuclei in the four heterologous spleens
Although many sera prior to absorption
appeared to react with slightly increased
intensity with granulocytes, absorption
with calf thymus nucleoprotein resulted in
complete abolition of all nuclear staining
with the exception of occasional sera from
lupus patients which demonstrated persistence of fine speckling in nuclei, presumably antibody to buffer extract antigen.13 It was considered unlikely that this
residual reactivity could obscure the more
obvious staining given by GS antibodies.
The present report confirms the existence
of nuclear reactivity restricted to granulocytes and presents evidence for two GS
antibodies which can be differentiated on
the basis of patterns of nuclear staining and
degree of species specificity. Although GS
antibodies may occasionally be masked and
the true frequency of these antibodies is
not known, evidence of selective reactivity
with granulocytic nuclei is not commonly
encountered in the course of routine testing
for antinuclear antibodies. On the other
hand, GS antibodies do not appear to be a
rare occurrence in established rheumatoid
arthritis. Among a group of 40 patients with
stage I1 to IV rheumatoid arthritis who
were selected for rehabilitation studies,14
type I GS reactivity was detected and verified by absorption tests in 8 (20 per cent)
of these patients.
In addition to the high degree of antigenic specificity of GS antinuclear antibodies, certain other characteristics are
worthy of note. Although rheumatoid arthritis was by far the most common diagnosis for patients exhibiting either type I or
I1 antibodies, both antibodies were con-
sistently yG globulins, and only occasional
type I antibodies demonstrated additional
weak reactivity in the yM and yA immunoglobulin classes. Faber and Elling similarly
observed that GS antibodies were not sensitive to penicillamine.KThese findings are
at variance with the general prevalence of
high molecular weight antinuclear factors
in rheumatoid arthritis as established by
the use of specific immunofluorescent antitests of mercaptoethanol sensitivity,lK
and studies of antinuclear activity in serum
fractions separated by chromatography and
The reactivity of both GS antibodies
with nucleoprotein was demonstrated by
enzyme and absorption tests. The irregular
pattern of staining given by the type I1 antibody is in contrast to the homogeneous
pattern which has been previously related
to antibody to nucleoprotein.l’J8 The recent report of Ritchie, however, indicates
that homogeneous patterns of staining in
some instances may actually represent composite reactivities of antibodies which individually give nonhomogeneous patterns
of staining. Two yG antibodies which gave
“nodular” or “reticular” staining, respectively, with the 1 M NaCl extractable fraction of rat liver nuclei were detected in
sera of patients with connective tissue disease~.~~
In contrast to the commonly observed
serologic overlap among connective tissue
diseases, type I GS antibodies occurred
only in rheumatoid arthritis patients. Although the disease was usually well established in these patients (average, stage II-
I11) , associated clinical and laboratory findings did not suggest any obvious features
that would set this group apart from the
general population of rheumatoid patients
with comparably advanced disease.
A frequent occurrence of leukopenia,
often in association with Felty’s syndrome,
was previously observed in a group of patients whose sera contained both GS and
granulocyte reactive antibodies? In the
present study, the relationship between
type I1 GS antibody and granulocytopenia
was striking. This was further emphasized
by the unmasking of morphologically and
immunologically identical antibodies in
sera of 2 lupoid hepatitis patients who
demonstrated granulocytopenia. The presence of this antibody was not revealed by
absorption tests in any of the sera of patients with established systemic lupus. It
may be relevant that although leukopenia
is frequent in lupus, white blood cell differentials are usually either normal or show
lymphopenia.20 These observations might
suggest a possible pathogenic role for type
I1 GS antibodies, but it is quite conceivable that these serum factors may be only
reflectors of granulocytic injury produced
by unrelated mechanisms. The currently
available data are descriptive in nature,
and clarification of the actual significance
of GS antinuclear antibodies will depend
on future investigation.
Bruce Veit, Pauline Yanowitz, and Austin
Ray provided excellent technical assistance. Dr.
Robert Kellermeyer kindly reviewed the manuscript.
Le presentia de granulocyto-specific anticorpore anti nucleoproteina esseva establite
per tests de absorption in 31 seros e demascate in 3 seros additional. Le majoritate del
patientes in question demonstrava arthritis rheumatoide, sed 2 de illes habeva hepatitis
lupoide. Le observate differentias in le configurationes fluorescentic del tincturation e
le grados de reactivitate heterologe characterisava duo distincte granulocyto-specific
anticorpores antinucleari, incluse un que monstrava un significative association con
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antibodies, granulocyte, clinical, observations, specific, antinuclear, immunologic
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