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Immunoglobulin G Subclasses of Antinuclear Antibodies and Renal Deposits. Comparison of Systemic Lupus Erythematosus Drug-Induced Lupus and Rheumatoid Arthritis

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Immunoglobulin G Subclasses of Antinuclear Antibodies
and Renal Deposits
Comparison of Systemic Lupus Erythematosus, Druglnduced Lupus
and Rheumatoid Arthritis*
Jovan N. Kacaki, Mary Lou Callerame, Stephen E. Blomgren and
John H. Vaughan
Immunoglobulin G (IgG) subclass representation, and titers, were studied in
antinuclear antibodies (ANA) of 59 sera from patients with systemic lupus
erythematosus (SLE), procainamide-induced lupus syndrome or rheumatoid
arthritis. In all the 3 groups of sera, the highest titers of ANA were found in the
IgG, subclass. Titers of this subclass were significantly higher in the SLE group,
as compared with the RA group. Otherwise, no significant difference was found
in the distribution of ANA among IgG subclasses in the three disorders. In a
comparison of the sera of patients with SLE with and without clinical evidence
of renal disease, no significant difference could be found between the titer or
the distribution of ANA in the IgG subclasses. In renal tissues obtained from 8
patients, however, frequency and intensity of staining for subclasses in the
glomerular deposits roughly paralleled the relative predominance of the subclasses in serum, and the titers of ANA in them. N o instance of selective deposit
of a single subclass was found.
Ever since the subclasses of human IgG
were described (1-3), there has been considerable interest in their antibody activities
(4,5), and in the question of whether they
have differing biologic (6,7) or pathogenetFrom the University of Rochester School of
Medicine and Dentistry, Rochester, NY, and Institute of Rheumatism, Belgrade, Yugoslavia.
JOVAN N. KACAKI, MD: Institute Of Rheumatism,
Belgrade, Yugoslavia. MARY LOU CALLERAME, MD:
University of Rochester, Rochester, NY. STEPHEN E.
BLOMGREN, MD: Scripps Clinic and Research Foundation, La Jolla, Calif. JOHN H . VAUGHAN, MD: Scripps
Clinic and Research Foundation, La Jolla, Calif.
Reprint requests should be addressed to John H.
Vaughan, MD.
Submitted for publication Aug 17, 1970; accepted
Nov 18, 1970.
+Presented at the 1969 Interim Session of the
American Rheumatism Association, Dec 5-6, 1969,
in Tucson, Ariz.
276
ic capabilities. Terry and Fahey were the
first to report the presence of IgG subclasses in an autoantibody. They found that all
three of the subclasses for which they tested
were present in the antithyroglobulins of a
serum which they studied (3).
There have been few studies of the
presence and distribution of IgG subclasses
in the antinuclear antibodies (ANA) of
systemic lupus erythematosus (SLE), or of
their presence in kidney deposits in SLE
nephritis. It has been suggested (8) that a
possibly important determinant in the development of kidney disease in lupus is in
the differences in the IgG subclasses in
which their ANA are contained, especially
those subclasses known to have complement-fixing abilities. Some difference in
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
IMMUNOGLOBULIN G SUBCLASSES
IgG subclasses of ANA has been reported
in a small group of SLE patients with and
without kidney involvement (9), as well as
selective deposition of certain IgG subclasses in the kidneys of patients with nephritis
10).
We have studied the presence and distribution of IgG subclasses in ANA in the
sera of patients with SLE, with and without kidney disease, and have noted the IgG
subclasses present in renal deposits in 8
patients. We have also compared the sera
of patients with SLE with those of patients
with procainamide-induced SLE (P-SLE)
or rheumatoid arthritis (RA), two disorders
which do not characteristically display renal disease.
MATERIALS AND METHODS
Antisera to IgG Subclasses
Antisera to purified human myeloma proteins
IgG,, IgG, and IgG, were raised in rabbits by the
method of Spiegelberg and Weigle (11). With this
method, immune tolerance is induced in rabbits to
one IgG subclass with a mixture of kappa and
lambda myeloma proteins of that class; the
tolerance is then broken by injection of a myeloma
protein of a different subclass. The resulting antibodies recognize H-chain antigens and have lesser
cross-reactivity among the IgG subclasses than do
antisera raised by previous methods. Anti-IgG,
antiserum, received from Dr. John P. Leddy, has
been described (12). After appropriate absorptions,
the antisera were labeled with fluoresceinisothyocyanate (FITC) (13). T h e protein contents,
FITC contents and protein-FITC ratios of the
conjugates are shown in Table 1. The conjugates
were used in indirect immunotluorescent tests for
ANA with smears of human peripheral blood buffy
coats used as substrates (14). and in direct immunofluorescent studies of cryostat sections of renal
biopsy and autopsy tissue sectioned at 5p. Each
conjugate was shown to be specific by its absorption
with myeloma proteins of its respective subclass,
but not with other myeloma proteins.
In Ouchterlony analyses, the undiluted antisera,
on gross examination, appeared similar in antibody
contents when diffused against standard concentrations of their respective subclasses of myeloma
antigens. In order to determine the dilution at
which each antiserum should be used in surveying
unknown sera or tissues, each was tested, in indirect
immunofluorescence, against various dilutions of
known SLE sera that were strongly and specifically
reactive for the ANA of each subclass. T h e dilutions chosen for the surveys were those that were
four times in titer above the endpoints of the
various antiserums and were 1:4, 1:8, 1:2 and 1:4
for anti IgG, through anti IgG, respectively.
Patients' Sera
A group of 59 sera were studied. They were taken
from the serum bank of the Immunology and
Arthritis Units. Twenty-five were from patients
with SLE; 14 of these had evidence of kidney
involvement (proteinuria, erythrocytes and leukocytes and/or casts in the sediment, positive
kidney biopsy, or positive evidence at autopsy).
Seventeen sera were from patients with P-SLE,
randomly selected from the serum bank. T h e third
group consisted of 17 sera from patients with RA,
who were selected for positivity in the test for
ANA. In testing for ANA of the various subclasses,
the patients' sera were titered in fourfold dilutionsfrom undiluted to 1/256. The titers were ranked
from 0 to 5, a negative result being 0, and a titer of
1/256 being 5 .
Table 1. Content of FlTC and FI1C:Protein Ratios in the Conjugates
Final product
Subclass of
antisera
Anti-lgG,
Anti-lgG,
Anti-lgGs
Anti-IgG,
Protein
FlTC used
(mg/ml)
(mg)
FlTC
(pg/ml)
Protein
(mg/ml)
FITC: protein
molecular ratios
50.0
66.7
2.5
5.2
3.35
4.42
4.81
7.0
2.88
2.5
0.7
0.74
1.13
0.61
1.9
2.0
88.5
96.25
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
0.47
0.39
217
KACAKI ET A1
RESULTS
The ranks of ANA titers for the sera of
patients with SLE, P-SLE and RA, using
the various IgG subclasses of antisera, are
shown in Fig. 1. It is clear that in all 3
groups, the highest titers of ANA were in
the IgG, subclass, and the lowest, in the
IgG, subclass. The SLE patients had
somewhat higher mean titers than did either of the other 2 groups; this was statistically significant (15) only for IgG, in the
comparison of SLE and RA patients. No
statistically significant difference was found
between SLE and P-SLE sera in any of the
comparisons.
It can also be seen in the figure that
SLE patients with kidney disease (open
circles) did not differ significantly, in the
distributions of their ANA titers, from the
SLE patients without kidney disease. The
mean titers are shown in Table 2.
The majority of sera showed a homogeneous pattern of staining; the three
disorders, SLE, P-SLE and RA, looked
generally similar in this respect. Some sera
had rim patterns which changed to homogeneous patterns in higher dilutions. Such
change in pattern has been described by
Tan (16). Hijams has noted (17) that the
patterns obtained with leukocytes do not
always correspond with those obtained
with liver slices which are used in substrates.
Two sera from the SLE group consistently manifested a speckled pattern in one
IgG subclass (IgG, or IgG,), while they
exhibited rim or homogeneous patterns
with the other subclass antisera. Both sera
were from patients with kidney involvement.*
Biopsies of the kidneys were obtained
from 5 patients, on 1 of whom an autopsy
was performed subsequently. Specimens
.One of these 2 was Patient Ir in Table 3.
278
from 3 other patients were obtained at
autopsy only. The results are shown in Table 3. Serum and renal tissue from these
patients were obtained less than a month
apart. The most striking finding was that
the 2 patients (Mo and D’A) who had the
highest serum titers and widest distribution
of ANA among the serum IgG subclasses
also had the most striking renal deposits.
The patients with the lowest titers of serum
ANA had the least evidence of deposits in
the kidneys.
The patterns obtained were granular,
either disseminated or focal. IgG, and
IgG, were found in all the positive kidneys. IgG, was found in 5 patients and
IgG,, in 3. In no patient was only one IgG
subclass found to be present. In 1 patient,
two subclasses were found, in 4 patients,
three subclasses were seen, and in 2 patients, all four were found.
However, when the presence or absence
of renal deposits of particular IgG subclasses was compared with titers of the corresponding ANA in each serum, several minor discrepancies were seen. Two instances
were noted (Gr and Pe) in which weak
renal deposits were found without the corresponding subclass of ANA being detected
in the serum; there were two instances (Ma
and Ai) in which ANA of a given subclass
were present in high titer in the serum,
without the corresponding deposits in the
kidney being identifiable.
One of the biopsies (which contained
only a single glomerulus) (Ma) revealed no
deposits, but subsequent autopsy revealed
considerable deposit. In another patient
with negative immunofluorescence (Ai),
there was chronic renal insufficiency and
no active glomerulitis.
DISCUSSION
The most striking finding in comparing
the subclasses of serum ANA in the three
Althritis and Rheumatism, Vol. 14, No. 2 (MarchApril 1971)
Y
N
a
U
z5
F
CI
N
?
t
P
c
3
f
.
I
-0
00.
#
m
2-
1-
OQI.
3-
0..
0
SLE
.
y
m
m
0
I sG1
P-SLE
RA
RA
1
2
I
-
.
SLE
0 l - I - SLE
- 0
-
.
s
3
33
g
.
4
+
P-SLE
lgG4
P-SLE
t
-
.
Y
1
.
M
mJ
m
am
my.
0
Fig 1. Comparison of ranks and titers of ANA in lgG subclasses of 3 groups of sera. Each circle represents 1 patient. Open circles in SLE group represent patients
with kidny involvement. Horizontal lines. means; arrows point at medians.
f3
3
2 -
s4
5-
- 0
r,
E;=
08
ease (Fig 1) was a surprise to us, but when
direct examination of the kidneys by immunofluorescence was compared to ANA
titers among a few sera (Table 3), a better
correlation was seen. It is to be noted that
the best correlations with renal disease
have been with antibody specifically directed to DNA, rather than with the less specific ANA (19,ZO).
I n studying the relation of serum ANA
to kidney involvement, Tojo and Friou (8)
reported that patients with SLE and complicating nephritis had ANA with high
complement-fixing activity. On the other
hand, sera of patients with SLE without
nephritis, but with comparable titers of
ANA, had ANA with lower complementfixing abilities. They suggested that these
differences in complement-fixing ability
might be ascribed to differences in the
representation of ANA in IgG subclasses.
In a follow-up of this suggestion, Friou et
al (9) studied the IgG subclasses of antibody to nucleoprotein from the sera of 8
Table 2. Comparison of Means of Ranks
of Titers in lgG Subclasses of SLE
Patients With and Without Kidney Involvement
Kidneys involved
Kidneys not involved
14 3.93 1.61 1.78 0.78
11 3.72 1.81 2.18 1.00
disorders, SLE, P-SLE and RA was the
overall similarity of the groups to each
other. Nevertheless, a statistically significant difference was found in the mean ranks
of titers of IgG, ANA between the SLE
and RA groups. Higher ANA titers in
SLE, in comparison with RA, have already
been reported (18). Our present finding
enables us to ascribe this difference primarily to IgG,. IgG, ANA manifested a similar tendency to be higher in SLE, but the
difference was not statistically significant.
Failure to find a general correlation between the titer of serum ANA and the
presence of clinical evidence of renal disTable 3.
Comparison of Findings of lgG Subclass Deposits in Kidneys of Patients with SLE
Nephritis with Findings of ANA in lgG Subclasses of Their Sera
~
~~
~
~~~~
~~
~~
Kidney irnmunofl uorescence
Patient
A
Pe
Ma
Gr
Me
Ir
D’A
Mo
Date
IgGi
Oct 4, 1968
July 3, 1968*
July 2, 1969
Aug 20, 1969*
Dec 9, 1968
0
+1
0
+3 Gran
+2 FOC
+1 Gran
FOC
Sept 12, 1968* +1 FOC
Sept 4,1968
April 11, 1968* +4 Gran
Jan 14, 1969
+3 Gran
IgGz
lgGa
0
+1
0
Tr FOC
+1 FOC
0
0
+1 FOC
0
+3 Gran
+4 Gran
+1 Gran
FOC
Tr FOC
+4 Gran
+4 Gran
- =not
IgG4
0
0
+1 Gran
FOC
+1 FOC
O=no detectable immunofluorescence;
* Dates of autopsy
t Ranks of titers
280
Serum ANA
_-
0
0
+1 FOC
Tr FOC
0
0
0
+1 Gran
Date
lgG1 lgGz lgGs IgG,
0
0
2
0
0
0
0
0
0
Nov25,1968 >2 >2
May28,1969
4
4
Sept6,1968
5
2
-
0
0
0
Sept 18, 1968
July3,1968
July28,1969
Jan29,1968
Sept 12, 1968
Mar20,1967
Mar 18, 1968
Jan 14, 1969
3t
4
5
4
4
5
5
5
0
2
4
4
5
3
1
-
1
1
1
4
4
5
4
4
done; Tr=trace; Foc=focal; Gran=granular
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
IMMUNOGLOBULIN G SUBCLASSES
patients with SLE and nephritis, 3 patients
with SLE without nephritis, and 3 patients
with P-SLE. T h e technic used in the study
was that of double diffusion in agar of the
antibodies isolated by immunoprecipitation and DNase digestion against antisera
specific for IgG subclasses. They found that
the antibodies were predominantly identifiable in IgG, and IgG, subclasses, a finding
consistent with the fact that these two
immunoglobulins are in larger quantity in
serum than are the IgG, or IgG, subclasses. Representation of the IgG, and IgG,
subclasses (which are known to have low
complement-fixing ability) was reported to
be somewhat higher in the group of sera
from patients with SLE without nephritis
(3/3 had IgG, and 1/3 had IgG,), than in
the sera from those with nephritis. T h e
number of sera studied, however, was obviously very small.
A more direct way to determine whether
there are significant differences of IgG
subclasses in SLE nephritis is to examine
the types of IgG subclasses deposited in the
kidneys. Schur et al (10) reported a number
of kidney biopsies, including five from patients with SLE nephritis, and 10 from
patients with chronic glomerulonephritis,
in whom histology and immunofluorescence were apparently indistinguishable.
I n both groups, the deposits were granular.
IgG, was seen in varying amount in 11
biopsies. IgG,, the other subclass strong in
complement-fixing ability, was found in
one biopsy to be the only IgG present, but
it was found in significant amounts in nine
others as well. Two biopsies showed only
the presence of the poor complement-fixer,
IgG,. T e n others showed IgG,, together
with other subclasses. T h e other noncomplement-fixer, IgG,, was the predominant
subclass in two biopsies; together with the
other subclasses, it was present in seven.
Schur et al believed that there was a
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
greater degree of inflammation when IgG,
was present.
I n our experience (Table 3), both IgG,
and IgG, were present in all seven kidneys
that stained positively. IgG, was present in
five of seven kidneys, and IgG,, in three of
seven. T h e results are what one might
expect from the relative amounts of these
immunoglobulin subclasses known to be
present in serum, and from the distributions of antibodies among the subclasses
(Fig 1). We did not find any tendency for
serum ANA to be selectively limited to any
one subclass, nor did any of our cases show
any one subclass to be selectively involved
in renal deposits.
From these data we find little to support
the assumption that renal disease in lupus
is related to a selective deposit in the
glomeruli of immunoglobulins capable of
fixing complement. While it is, indeed,
likely that complement-fixing antibodies
are of particular importance in contributing to renal disease, there is no reason to
regard them as the sole means of mediating
antibody-induced renal injury. Indeed, the
report of Schur et a1 (10) of some nephritides with exclusive or predominant staining for IgG, or IgG, may be a testament to
this.
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gamma G globulin. Serum distribution
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KACAKI ET A1
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Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
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