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Serologic Studies In Patients With Systemic Lupus Erythematosus And Central Nervous System Dysfunction.

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ARTHRITIS
289
&
RHEUMATISM
OFFICIAL J O U R N A L OF T H E AMERICAN RHEUMATISM ASSOCIATION
SECTION OF T H E A R T H R I T I S FOUNDATION
SEROLOGIC STUDIES IN PATIENTS
WITH SYSTEMIC LUPUS
ERYTHEMATOSUS AND CENTRAL
NERVOUS SYSTEM DYSFUNCTION
JOHN B. WINFIELD. CAROLYN M BRUNNER, and DAVID KOFFLER
Serologic studies were performed on 25 patients
with systemic lupus erythematosus (SLE) during 29 acute
episodes of central nervous system (CNS) disease. Increased anti-DNA antibody and decreased total serum
hemolytic complement activity were observed only in those
patients with associated extra-CNS disease manifestations. Patients with isolated CNS disease were otherwise
in apparent clinical and serological remission regarding
these two indices. No special association of cold-reactive
IgM antilymphocyte antibodies was demonstrable in patients with ongoing CNS injury. Of special interest was an
increased incidence of anti-Sm antibodies in the patients
with CNS dysfunction relative to that in a large group
From the University of Virginia School of Medicine, Department of Internal Medicine, Charlottesville, Virginia, the Hahnemann
Medical College Department of Pathology, Philadelphia, Pennsylvania, and the Rockefeller University, New York. New York.
Supported in part by U. S. Public Health Service. National
Institutes of Health grant numbers AM 11766, AM 9096. and AM
13721, and grants from The John A. Hartford Foundation, Inc., The
Whitehall Foundation, and The Arthritis Foundation.
John B. Winfield. M. D.: Associate Professor of Medicine,
Division of Rheumatology, University of Virginia School of Medicine,
Senior Investigator for The Arthritis Foundation: Carolyn M. Brunner, M. D.: Associate Professor of Medicine, Division of Rheumatology, University of Virginia School of Medicine: David Koffler. M. D.:
Professor of Pathology, Department of Pathology, Hahnemann Medical College, Philadelphia. Pennsylvania.
Address reprint requests to John B. Winfield, M. D.. Department of Internal Medicine, University of Virginia: School of Medicine, Charlottesville, Virginia 22901.
Submitted for publication October 7, 1977: accepted November I I , 1977.
Arthritis and Rheumatism, Vol. 21,
No. 3 (April 1978)
of patients without neuropsychiatric disease. The incidence
of anti-RNP was not increased.
The data do not support direct involvement in SLE
brain injury of either DNA/anti-DNA complexes or of
lymphocytotoxic antibodies cross-reactive with brain
cells, but do suggest an association of anti-Sm with CNS
disease in this disorder.
The pathogenesis of neuropsychiatric manifestations of systemic lupus erythematosus (SLE) has not
been clearly defined. The enigma of central nervous
system (CNS) injury contrasts with the considerable
evidence implicating immune complexes as mediators of
tissue injury in glomerulonephritis in SLE (1). Previous
investigation of the mechanisms involved in brain injury
has assumed an analogy with glomerulonephritis. Efforts directed toward complement (2-4), immunoglobulin (4,5), and anti-DNA or DNA/anti-DNA immune
complex (4,6,7) determinations in cerebrospinal fluid
have yielded conflicting data and have failed to establish
clearly defined pathogenetic factors. The frequent absence of gross changes in cerebrospinal fluid protein and
of pleocytosis (4,8,9)suggests that any abnormalities, if
present, may be subtle.
Distinctive serologic indices have not been identified, although little information is available in this area.
Of interest is a recent demonstration of free DNA in the
plasma of patients with CNS disease (lo), an observation suggesting participation of unusual antigen excess
DNA/anti-DNA complexes in brain injury. Direct at-
WINFIELD ET AL
290
tack of brain cells by antibody, either specific for neuronal antigens ( 1 1,12) or cross-reactive with lymphocyte
surface determinants (13), has been proposed, but the
pathogenetic relevance of such antibodies remains t o be
established.
T h e present study characterizes the clinical a n d
humoral immune status of patients with SLE during
acute episodes of neuropsychiatric disturbance. T h e
data provide no support for direct involvement of the
DNA/anti-DNA system and lymphocytotoxic antibodies in the pathogenesis of brain dysfunction, but an
association with anti-Sm is indicated.
MATERIALS AND METHODS
Patients and Sera. All patients met the preliminary
criteria for classification of SLE (14) and were receiving medical care at the University of Virginia Hospital or the Rockefeller University Hospital. One of the authors was in attendance for each neuropsychiatric episode that developed during
the period from 1968 to 1976. Central nervous system dysfunction was defined by the presence of unequivocal neurologic or
psychiatric abnormality not attributable to non-SLE related
etiologies. Organic manifestations were associated with objective neurologic signs and/or abnormal laboratory studies, for
example, cerebrospinal fluid examination, electroencephalogram, arteriography. Patients were classified as having extraCNS systemic disease activity when history or physical examination revealed evidence of two or more of the following; rash,
fever, arthralgia or arthritis, serositis, alopecia, stomatitis,
myositis, or glomerulonephritis. Serum was stored frozen
without preservative at -20" and heated at 56°C for 30 minutes prior to use.
DNA and Anti-DNA Antibodies. DNA in serum was
sought by counterimmunoelectrophoresis (1 5) with addition
of 1 M NaCl to the serum well to increase sensitivity by reducing nonspecific protein-DNA interactions (10). Sera lacking
DNA demonstrable by this technique were reexamined after
digestion with pronase, an approach enabling detection of 0.3
pg DNA/ml of serum even in the presence of anti-DNA
antibody i n relative excess (16).
Anti-DNA antibody was quantitated by an ammonium sulfate precipitation radioimmunoassay (Farr assay) using HeLa cell native DNA intrinsically labeled with SH-thymidine as described elsewhere ( 1 7).
Antibodies to Extractable Nuclear Antigens (ENA).
Antibodies to Sm antigen (used here as a generic term denoting all RNAse-resistant ENA) and ribonucleoprotein (RNP)
were determined by hemagglutination using rabbit thymus
extract (Pel-Freeze Biologicals, Rogers, Arkansas) according
to the method of Tan and Peebles (18). A three tube or greater
decrease in titer following digestion of ENA-coated test cells
with RNAase (Sigma) indicated the presence of anti-RNP in
SLE serum.
Antilymphocyte Antibodies. Peripheral blood lymphocytes from 6 control subjects were isolated from heparinized
venous blood by Ficoll-Hypaque flotation (19). Sera from
patients were examined for antilymphocyte antibodies in mi-
crodroplet complement-dependent cytotoxicity assays and by
indirect immunofluorescence (20,21). Antibody titer was estimated as the highest dilution of serum killing 50% of the test
cells in the microcytotoxicity assay. Immunoglobulin class of
the antilymphocyte antibodies was determined by indirect immunofluorescence using rhoadamine-conjugated F(ab'), fragments of rabbit antibodies monospecific for p and y.
RESULTS
Twenty-five patients were studied during 29 episodes of acute neuropsychiatric illness. Clinical evidence
for S L E disease activity was restricted to the central
nervous system in 16 patients; both C N S and extra-CNS
manifestations were present in the remaining 13 patients. Clinical a n d serologic data for these two groups
of patients are summarized in Tables I and 2.
Patients with isolated C N S disease differed from
those with associated systemic activity in several respects (Table 3). Organic brain dysfunction predominated in patients with isolated C N S disease ( I 4 of
16 episodes); whereas seizures and/or psychosis were
more common in patients with extra-CNS disease (8 of
14). Abnormal serologic indices of SLE clinical activity,
that is, increased anti-nDNA and hypocomplementy-iia, were virtually restricted to patients with associated systemic disease (Table 3). In no patient with isolated neuropsychiatric illness were increased n D N A
binding and decreased C'H,, present simultaneously.
T h e two groups differed for mean percent SH-nDNA
binding and C'H,, at the P = 0.002 and P = 0.006 levels,
respectively. Free D N A in serum was demonstrable infrequently, being present in only 2 patients in each group.
Because of published data suggesting a special
relationship between lymphocytotoxic antibody with
neuropsychiatric disease in S L E (13), particular attention was directed toward this association. Although cold
reactive antilymphocyte antibody was present in serum
during 20 of 29 (69%) episodes, the incidence was not
greater than that in 31 other patients lacking C N S manifestations who were studied in parallel (81%). Immunoglobulin class was predominantly IgM in all patients.
Antibody titers, expressed as l/log, using 50% cytotoxicity as a n end point, did not differ in patients with
(2.4 f 1.1) and without (2.2 f 1.1) C N S disease. Serial
lymphocytotoxic antibody determinations in 4 patients
over a 12-month period also failed to demonstrate an
association between antilymphocyte antibody and C N S
disease.
A relatively high incidence of antibody to
RNAse-insensitive antigen (Sm) in rabbit thymus extract was observed in sera of patients with C N S manifes-
SLE AND CNS DYSFUNCTION
29 1
Table 1. Clinical and Serologic Daia for Patients with Isolated CNS Disease
Patient
C N S Manifestation
Ca
Cf
Cp
Co
Co
Di
Do
Gu
Ki
Mo
Mo
Ra
Th
Va
We
We
* Lower
Coma/Hemiparesis
Hemiparesis
Seizure
Transverse myelitis (1/74)
Transverse myelitis (1 1/74)
Cerebellar ataxia
Encephalitis
Psychosis/vestibular ataxia
Transverse myelitis
Ptosis (10/73)
Psychosis/cortical blindness (1 1/73)
Hemiparesis
Optic neuritis
Seizure
Chorea (8/72)
Chorea (1 2/74)
aH-nDNA
% Binding
Serum
DNA
C’H,,
(U/ml)*
16
8
20
2
10
-$
-
35
29
26
42
-
44
5
-
68
38
40
42
38
41
42
-
39
9
+
-
11
86
6
27
8
36
10
9
-
+
-
Lymphocytotoxic
Anti-ENA (titer, log,)
Antibody
(% Dead PBL) Before RNAse After RNAset
95
30
90
< 10
< 10
< 10
100
90
< 10
20
< 10
44
< 10
< 10
24
24
90
95
95
20
7
I1
12
16
4
-
19
18
22
21
12
12
I1
16
-
9
-
6
5
4
19
18
20
I2
7
limit of normal 34 U/ml.
t Anti-Sm.
$ Negative value.
tations (16 of 25, 64%). The incidence and titer of antiSm did not differ in patients with or without associated
extrasystemic disease activity, a situation contrasting
with that for anti-DNA and C’H,, (Table 3). Anti-Sm
was less frequently found in sera from a large group of
other patients with SLE but lacking CNS disease (60 of
155, 39%, P = 0.15). In this latter group, the incidence
of anti-Sm in 107 patients with active SLE was 48%,
roughly that observed in patients with CNS disease and
associated extra-CNS manifestations (55%, P = 0.80).
Anti-Sm was detectable in 8 of 48 (16%) of non-CNS
patients considered to be in clinical and serologic remission (normal C’H,,, low or absent anti-DNA activity).
The four-fold higher incidence of anti-Sm (71%, Table
Table 2. Clinical and Serologic Daia for Paiienis wiih Both C N S and Exira-CNS Disease
Clinical Status
Patient
CNS
Extra-CNS
aH-nDNA
% Binding
Serum
DNA
C’H,,
(U/ml)*
Lymphocytotoxic
Anti-ENA (titer, log,)
Antibody
(W Dead PBL) Before RNAse After RNAset
~~
Ae
AY
Ba
co
De
Fr
Ho
Sa
Sm
Ta
Ta
wo
su
Homonymous, hemianopsia
Ptosis
Seizure/Psychosis
Transverse myelitis (2/71)
Psychosis
Psychosis
Meningitis
Seizure
Seizure
Seizure
Cerebritis
Seizure
Psychosis
* Lower limit of normal,
t Anti-Sm.
3 Negative value.
34U/ml.
Arthritis
Pleuritis
M yositis
Nephritis
Nephritis
Nephritis
Serositis
Nephritis
Nephritis
Nephritis
Arthritis
M yocarditis
Nephritis
79
84
89
99
2
98
27
80
71
14
85
0
17
-
+
22
2
28
11
16
17
95
40
30
20
< 10
85
39
< 10
17
80
70
25
85
35
30
21
36
44
35
38
I2
5
7
12
4
20
7
18
7
8
-
292
WINFIELD ET AL
Table 3. Comparison of Serologic Data in Patients With and Without Extra-CNS Disease
Clinical Group
Isolated CNS
Number of Episodes
Anti-DNA
Number positive
% 'H-nDNA Binding
Serum DNA
Number positive
Serum C'H,,
Number decreased
Hemolytic units/ml
Lymphocytotoxic antibody
Number positive
% PBL killed (pos. only)
Anti-Sm
Number positive
Titer (log, pos. only)
Anti-RNPt
Number positive
Titer (log, pos. only)
CNS + Extra-CNS
16
13
3 (19%)
19 f 21'
9 (69%)
57 f 39
2 (13%)
2 (15%)
4 (25%)
38 f 11*
8 (62%)
2 5 f 13
8 (50%)
78 f 31*
11 (85%)
54 f 29
10/14(71%)
1 I .6 f 6.2*
6/11 (55%)
8.8 f 5.3
5/14 (36%)
6.4 f 2.9*
1 / 1 I(9%)
3.0
0.002
P
=
P
= 0.006
NSS
NS
NS
*
t
Mean f 1 standard deviation.
Calculated by difference: titer of anti-ENA minus titer following RNAse digestion,
$ NS = not significant.
3 ) in patients with isolated CNS disease was highly
significant statistically (P = 0.007; if the missing data for
anti-Sm for patients Ra and Va in Table 1 are considered to be negative, P = 0.01) and was of particular
interest, because with the exception of CNS involvement
these patients were not otherwise distinguishable from
those in complete remission. Although mean titer of
anti-Sm was approximately 3 dilutions greater in the
patients with isolated CNS disease than in the non-CNS
inactive group, this difference was not significant statistically.
A three tube or greater decrease in titer of antiENA after digestion with RNAse was considered to
indicate the presence of anti-RNP in the SLE sera. The
incidence of anti-RNP was 27% in I55 patients lacking
CNS disease and 19% in those with CNS disease. Because the presence of low levels of anti-RNP cannot be
excluded fully in sera not showing a decrease in hemagglutination titer following RNAse digestion, the incidence values for anti-RNP represent minimum estimates.
DISCUSSION
This study demonstrates a nearly complete dissociation between neuropsychiatric disease in SLE and
the DNA/anti-DNA immune complex system. Increased native DNA antibody and hypocomplementemia constitute the major serologic markers of systemic
activity in SLE (1,22), but they were present only exceptionally in patients with organic brain dysfunction in
the absence of extra-CNS manifestations. DNA was
found in only four sera, even though special procedures
were used to increase the sensitivity of the counterimmunoelectrophoresis technique. The failure to demonstrate DNA in most patients after digestion of serum
with pronase to remove antibody potentially masking
DN.A antigenic sites further mitigates against the presence of DNA/anti-DNA complexes. Overwhelming evidence supports a predominant role for DNA and antiDNA in certain types of tissue injury, particularly renal.
The present data argue strongly against a similar immunopathogenesis in the brain.
An ancillary role for DNA/anti-DNA or other
immune complexes in brain injury is not excluded, however. Evidence in human SLE (23) and in New Zealand
black/white F, hybrid mice (24) indicates the choroid
plexus is a site for immune complex deposition. The
choroid plexus functions as a barrier against diffusion of
both large and small molecules directly into the cerebrospinal fluid (CSF). In addition to manufacturing most of
the CSF, the epithelial cells of the choroid plexus are
SLE AND CNS DYSFUNCTION
also metabolically active in a fashion analogous t o that
of the renal tubule (25). T h u s choroid plexus injury
could enhance susceptibility of brain cells t o functional
disturbance by presently undefined mechanism both immunologic and nonimmunologic. Cerebral capillaries
provide the barrier against diffusion from blood to the
brain extracellular space. C N S disease a n d vasculitis
have been associated clinically (26). Vascular injury in
the brain may be a prerequisite, therefore, for factors in
blood, for example antineuronal antibodies, to gain access t o the brain. T h e present findings suggest that if
DNA/anti-DNA immune complex mediated injury to
cerebral vessels or t o the choroid plexus is involved
pathogenetically in C N S dysfunction, such injury need
not be ongoing at the time of the acute neuropsychiatric
episode.
Bluestein a n d Zvaifler have proposed that lymphocytotoxic antibodies cross-reactive with brain cells
in SLE. might participate directly in C N S dysfunction
(13). T h e absence of differences in antilymphocyte antibody incidence and titer in patients with and without
neuropsychiatric manifestations in the present group of
patients provides no support for this mechanism but
does not exclude it. Indeed, more recent evidence from
this group of patients indicates the presence in SLE sera
of an additional IgM antibody that is not completely
crossreactive with lymphocytes and is cytotoxic for neurons in culture (12). By analogy, the observations of
Husby et af. (27) demonstrating a strong relationship
between antibodies specifically directed toward subthalmic and caudate cytoplasmic antigens in patients
with rheumatic fever a n d chorea a r e also pertinent in
this regard. Further characterization of antibodies to
brain cells a n d definition of the pathogenetic relevance
of such antibodies t o C N S manifestations in SLE clearly
represent an important new avenue for study.
Antibody t o RNAse-resistant nuclear antigen
has been shown to be a highly specific serologic finding
in patients with SLE (28). T h e highest incidence of antiSm is found in patients with active disease, but whether
this antibody system contributes importantly t o the natural history of SLE is unknown. Preliminary data suggest associations with vasculitis and leukopenia (29) a n d
with a subset of patients exhibiting hypocomplementemia and mild nonprogressive glomerulonephritis (30).
In the present investigation, anti-Sm was observed in 71% of the patients with isolated C N S disease.
This unexpectedly high incidence may have special significance because the frequency of anti-Sm in unselected
patients has been reported t o be approximately 25%
(28,29), a value in close agreement with that observed
293
for patients without C N S disease in the present study.
The association of anti-Sm with C N S injury demonstrated in this report clearly merits no discussion with
regard t o pathogenetic or etiologic relevance at this
point. Nevertheless, anti-Sm may have diagnositc utility
in certain patients with C N S disease. For example, the
presence of anti-Sm in serum of patient T h (Table 1 )
provided strong evidence favoring SLE rather than multiple sclerosis as the basis for the development of optic
neuritis. I n addition, it will be of particular interest to
determine whether the antibody in patients with C N S
disease, operationally termed anti-Sm in this study, has
any unique antigenic specificities not found in patients
without C N S disease.
ACKNOWLEDGMENTS
The authors are grateful to Ms Lydia LeGrand, M s
Gerda Pirsch, Ms Stephania McGhee, and Ms Virginia
Estabrook for expert technical help, and to Ms Carol Woodward for typing the manuscript.
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