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Serum complement abnormalities in the antinuclear antibody-positive relatives of children with systemic lupus erythematosus.

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954
SERUM COMPLEMENT ABNORMALITIES IN THE
ANTINUCLEAR ANTIBODY-POSITIVE RELATIVES OF
CHILDREN WITH SYSTEMIC LUPUS
ERYTHEMATOSUS
THOMAS J. A. LEHMAN, VIRGIL HANSON, BERNHARD H. SINGSEN, HELEN K. KORNREICH,
BRAM BERNSTEIN, and KAREN K. KING
Serum C3, C4, and total hemolytic complement
(CH50)levels were measured for 21 children with systemic lupus erythematosus (SLE)and 81 first degree
relatives. The mean serum C4 and CH50 levels of the 12
relatives with antinuclear antibodies (ANA) were depressed to levels equal to those of the index cases. A
similar depression was not found for C3,nor was there a
depression of C3,C4,or CH50 in the relatives without
ANA. If preexistent depression of C4 levels can be
documented in the ANA-positive relatives of index
cases, it may provide an explanation for the inherited
predisposition to SLE in some families.
A 1 to 2% incidence of systemic lupus erythemaFrom the Division of Rheumatology and Rehabilitation,
Childrens Hospital of Los Angeles, and the Department of Pediatrics,
University of Southern California School of Medicine.
Supported in part by grant #lT32AM07262-01 from the National Institutes of Health, National Institute of Arthritis, Metabolism
and Digestive Diseases.
Thomas J. A. Lehman, M D Fellow, Pediatric Rheumatology, Trainee, NIAMDD; Virgil Hanson, MD: Professor of Pediatrics,
USC School of Medicine, and Head, Division of Rheumatology and
Rehabilitation, Childrens Hospital; Bernhard H. Singsen, M D Assistant Clinical Professor of Pediatrics, USC School of Medicine, and Division of Rheumatology and Rehabilitation, Childrens Hospital;
Helen K. Kornreich, MD: Associate Professor of Pediatrics, USC
School of Medicine, and Division of Rheumatology and Rehabilitation, Childrens Hospital; Bram H. Bemstein, MD: Associate Clinical
Professor of Pediatrics, USC School of Medicine, and Division of
Rheumatology and Rehabilitation, Childrens Hospital; Karen K.
King, M D Associate Clinical Professor of Pediatrics, USC School of
Medicine, and Division of Rheumatology and Rehabilitation, Childrens Hospital.
Address reprint requests to Virgil Hanson, MD, Division of
Rheumatology and Rehabilitation, Childrens Hospital of Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027.
Submitted for publication October 11, 1978; accepted in revised form April 23, 1979.
Arthritis and Rheumatism, Vol. 22, No.9 (September 1979)
tosus (SLE) has been noted in epidemiologic studies of
first degree relatives of probands with SLE (14). Many
asymptomatic first degree relatives have been reported
to have serologic abnormalities such as hypergammaglobulinemia (1-3), biologic false positive tests
for syphilis (2,3), and, in up to 50%0, antinuclear antibodies (ANA) (5,6). The mechanism by which these abnormalities arise, and whether they are isolated findings
or an expression of “subclinical” SLE, is unknown.
They have been variously interpreted as evidence of either a genetic predisposition (3-5,7), or a combined genetic and environmental etiology for SLE (1,3,6,8,9).
The complement cascade is a genetically influenced system that plays a major role in host defenses
against infectious agents. Children and adults who are
homozygous for an inability to synthesize early complement components have a greatly increased incidence
of SLE-like syndromes (10,ll). This is also true for individuals heterozygous for an inability to synthesize C2
(10- 12). It was therefore questioned whether depressed
serum complement component levels might be associated with the increased frequency of serologic abnormalities observed in the relatives of probands with SLE.
In this study C3, C4, and total hemolytic complement (CH50) were measured in children with SLE
and their first degree relatives. It was felt that these families represented a group with a demonstrated predisposition to early disease onset and, as a result, serologic abnormalities of the first degree relatives might be
more likely to be detected at an early age. An additional
advantage of a pediatric study was that relatives could
be more completely sampled because in most cases the
nuclear family was intact.
SERUM COMPLEMENT ABNORMALITIES
955
Table 1. Mean values for C3, C4, and CH50 in children with systemic lupus erythernatosus, relatives
with ANA, relatives without ANA, and controls
CH50
c4
c3
Index cases*
Relatives
with ANA
Relatives
without ANA
Controls
72.0 f 32.0 NS
11.5 f 46.5 NS
87.4 2 27.7 P < 0.05
77.1 f 11.9 P(0.005
16.7 34.2 Pt0.005
95.1 f 20.9 NS
89.8f 14.1 NS
103.8 f 30.2 NS
99.1 f 18.3 NS
91.8 f 17.4
94.6 f 27.0
95.4 f 14.0
*
* All values are expressed as a percentage of the activity of the pooled normal controls f 1 standard
deviation. P values are for comparison of immediately adjacent columns.
MATERIALS AND METHODS
Twenty-one children with SLE, followed at the Childrens Hospital of Los Angeles, were the index cases. All fulfilled the preliminary criteria of the American Rheumatism
Association for the classification of SLE (13). Eighty-one first
degree relatives of the 21 children were available for study.
This group consisted of 20 mothers, 16 fathers, 27 sisters, and
18 brothers of the index cases. None of the relatives f u m e d
the criteria for the classification of SLE. Controls were 13
male and 28 female medical and paramedical personnel at the
Childrens Hospital of Los Angeles; all were in good health.
Serology. IgG antinuclear antibodies were detected by
conventional indirect fluorescent antibody techniques using
snap frozen mouse liver slices as substrate. Sera were tested at
..
)140.
120
100:
80
-
60
-
40
-
c;
* .f ..
;T
.;.
*I*
':I
:
:
*#:
.
20 0.
PATIENTS
RELATIVES
RELATIVES
WITH
WITHOUT
ANA
ANA
CONTROLS
*Expressed as a percentage of pooled normal control activity
Figure 1. "Scattergram" showing distribution of values for C3.
a n initial dilution of 1 : 10 and, if positive, at serial doubling
dilutions until nuclear fluorescence disappeared. All individuals with ANA at only a 1: 10 dilution were confirmed as positive by duplicate runs of a single specimen. (Fluorescein
isothiocyanate conjugated rabbit antiserum to human IgG, H
and L chain specific was from Hyland Laboratories, Costa
Mesa, California.)
Blood for complement studies was allowed to clot at
room temperature for one haif hour, centrifuged at 4°C for 20
minutes, then immediately placed at -7OOC until utilized.
Serum C3 and C4 levels were determined by single radial immunodiffusion (Behring Diagnostics, Somerville, New Jersey).
Every plate included a control consisting of pooled normal
sera; complement levels were expressed as a percentage of the
activity of this pooled normal control in each plate.
Total hemolytic complement was measured by a modification of the method of Cohen (14) which uses bacto antisheep cell hemolysin (Difco Laboratories, Detroit, Michigan).
A pooled normal sera control was run with each CH50 titration because of the variability of sheep red cell fragility. Individual levels were expressed as a percentage of the pooled
normal control activity in each run.
Antibodies to double-stranded DNA were detected by
utilizing Crithidia luciliae (15), with an initial serum dilution
of 1 : 10, and serial doubling dilutions until the end point of kinetoplast fluorescence.
Statistics. Individuals tested were divided into four
groups: index children, relatives with ANA, relatives without
ANA, and controls. Mean values and standard deviations of
C3, C4, and CH50 were calculated for each group. Group
means were compared utilizing Student's pooled t test with an
alpha value of 0.05. The distribution of ANA and anti-DNA
antibodies among relatives was tested by Chi-square with
Yates' correction. Linear regression analyses were performed
by conventional techniques with a Texas Instruments programmable calculator and a program provided by the manufacturer.
RESULTS
Eighteen of 21 SLE index cases (86%), all of
whom were receiving corticosteroid therapy, had ANA
titers of 1 : 10 or greater (median 1 :40, range 1 : 10-
LEHMAN ET AL
956
.....
.i.
i.
.* *.
8 -
.
..
..
0
.
RELATIVES
WITHOUT
CONTROLS
ANA
“EXP-
os o percentage of p ~ d e dmm~
control octivity.
Figure 2. “Scattergram” showing distribution of values for C4.
1 :640) at the time of this study. The low median titer
and the absence of ANA in 3 patients resulted from the
inclusion of children with long standing disease who
were well controlled on corticosteroid therapy. All children were strongly ANA-positive and manifested typical SLE at the time of initial diagnosis. Twelve of the 81
first degree relatives of these patients (15%) had ANA
(median titer 1: 10, range 1: 10-1 :80). The 12 relatives
included 5 of 20 mothers, 3 of 27 sisters, 3 of 16 fathers,
and 1 of 18 brothers. The distribution of ANA among
the relatives was not significant for relationship or sex.
Two of the 41 normal controls, neither of whom routinely handled SLE sera, also had ANA (1 : 10, 1 :40).
Eight of the 21 SLE index cases also had antibodies directed against double-stranded DNA, but none of the
relatives or controls had these antibodies (Pc 0.005).
Mean values for C3, C4, and CH50 for SLE index cases, relatives with ANA, relatives without ANA,
and controls are illustrated in Table 1. The distribution
of values for each group can be seen in Figures 1,2, and
3. The individual normal controls have a median value
which is lower than the pooled normal control sera
(which was the reference 100% value) because the pooling of sera creates a “supernormal” sera in which any
slight degree of deficiency in a given individual is corrected by other members of the pool. Comparison of the
mean serum complement levels between the relatives
with ANA and those without ANA revealed significant
differences for C4 and CH50 (P < 0.005). The distribution of serum CH50 values of the relatives with ANA is
continuous; however, that for C4 reveals 5 patients with
normal values while the remainder are depressed. This
finding did not occur for serum C3 levels. The relatives
without ANA had serum C4 and CH50 levels which
were not significantly different from those of the controls. Other comparisons which were of statistical significance at the 0.05 level of confidence were decreased C3,
C4, and CH50 in index cases as compared to relatives
without ANA, or to controls; and decreased C4 and
CH50 for relatives with ANA versus controls.
Linear regression analysis was performed to determine the relative importance of C3 and C4 in determining the CH50 of various groups. For the patients
there was a significant correlation between the serum
C3 level and the CH50 (r = 0.825; P c 0.001). There
was also a significant correlation between serum C4 levels and CH50 in the patients (r = 0.487; P < 0.05).
When the relatives with ANA were evaluated, however,
only C4 was significantly correlated with the serum
CH50 of the relatives with ANA (r = 0.614; P < 0.05).
No correlation was found between C3 and the CH5O of
the relatives with ANA (r = 0.080; P > 0.05). This sug-
>I40
.
I00
80
CH!&
60
40
I
..
.*
120
:f
.
.$.
..
.*
0 .
.
20
0
WITHOUT
CONTROLS
*Expressed 0s a percentage of pooled normal contrd activity.
Figure 3. “Scattergram”showing distributionof values for CH50.
SERUM COMPLEMENT ABNORMALITIES
gests that C4 was more of a limiting factor in determining the CH50 values of the relatives with ANA. No relationship was found between degree of ANA positivity
and C4 depression (r = 0.080; P > 0.05).
DISCUSSION
In the present investigation it was demonstrated
that the presence of ANA in the first degree relatives of
children with SLE was correlated with a depression of
the mean serum levels of C4 and CH50, without a concomitant depression of C3. A previous study of C3 and
C4 levels in the first degree relatives of patients with
SLE did not find a statistically significant difference
from the control population, but the relatives with serologic abnormalities were not separately evaluated (8).
The decrease in the mean serum C4 level of the
relatives with ANA in this study is not large; however,
the distribution is discontinuous. The 5 relatives with
ANA whose C4 levels were normal may represent a C4
protein with decreased biologic activity. Alternatively,
other genetic factors which predispose to the development of SLE, including deficiency of other early complement components, may have been important.
Decreased serum complement levels in patients
with SLE have been shown to result from either increased utilization or decreased synthesis (16,17). Increased complement utilization in the first degree relatives with ANA could have occurred via the classical
pathway as the result of antigen-antibody interaction
directly involving ANA or involving other antigen-antibody systems whose interaction was linked to the development of ANA. Evaluation of first degree relatives of
index cases with SLE has not revealed an increased incidence of C3 or C4 deposition at the dermal-epidermal
junction; but, again, the seropositive relatives were not
separately evaluated (8).
If the mechanism of complement depression in
our ANA-positive relatives was direct utilization via the
classical pathway, as usually occurs in patients with
SLE, one would expect to have found significant depressions of both C3 and C4. Conversely, if alternate
pathway activation was a major factor, one would expect depression of C3, but not C4 (18). Neither was the
case in this study. The possibility that differing synthetic
rates allowed C3 production to increase with utilization
to a degree not possible for C4 cannot be excluded, and
it is impossible to fully rule out classical pathway activation at this time.
Decreased synthesis of C4 in the relatives with
ANA could have resulted from either a genetic or a
957
feedback mechanism. Decreased synthesis of C3 has
been documented in some patients with SLE (17). The
mechanism of this decrease has not been determined,
nor is it known whether the decrease preceded or followed the onset of symptoms. The inherited inability to
synthesize various complement components including
Clr, Cls, C4, C3, and C5 has been associated with the
development of SLE-like syndromes in some patients
(10-12). Both of the patients who have been reported
with C4 deficiency had SLE-like syndromes (19,20). In
this study no relative with a very low CH50 was detected, indicating none was homozygous for an inability
to synthesize complement components. Heterozygotes,
however, could have been present.
Determination of specific protein synthetic rates,
including C4, by genes in the H2 region of chromosome
17 has been documented in mice (2 1). These genes seem
to interact with both hormonal levels and the sex of the
individual. Similar genes in the HLA region of the sixth
human chromosome might play a role in the control of
human complement levels. Genetic polymorphism of
C4 with several alleles closely linked to the HLA-B
locus has been documented in humans (22,23). Decreased C4 synthetic rates or a dysfunctional C4 protein
could be inherited characteristics either occurring as
isolated abnormalities or related to the development of
SLE-like disease.
We suggest two possible models relating preexistent depression of C4 levels to the occurrence of
antinuclear antibodies and SLE or SLE-like syndromes.
The first model postulates linkage of a gene controlling
C4 synthetic rates with other genes in the HLA region
which are responsible for increased autoimmune reactivity. The depression of C4 is probably a coincidental
characteristic in this model. The second model accepts
the hypothesis that a low grade viral pathogen or repeated viral infection is an etiologic factor in SLE (24).
Serum C4 plays a major role in the neutralization of
some viruses (25), and some individuals with low serum
levels of C4 have defective viral neutralization (26) and
impaired opsonization (27). Therefore, individuals with
decreased serum C4 levels may inadequately handle infections by low grade viral pathogens, which in turn
might be responsible for the development of serologic
abnormalities.
In this study we have demonstrated that ANA in
the relatives of children with SLE is associated with depressed serum levels of C4 and CH50. The depression
of C4 may be an inherited characteristic that precedes
the development of ANA or an acquired abnormality.
Expanded genetic studies will be necessary to determine
LEHMAN ET AL
958
if decreased C4 synthesis is inherited and in what manner. If preexistent depression of serum C4 levels, due to
abnormal rates of either synthesis or utilization, can be
demonstrated to be an inherited characteristic of the
SLE proband relatives with ANA, it may provide an explanation for the combined roles of genetic and environmental factors suspected from epidemiologic studies
of SLE. Metabolic balance studies of C4 in SLE probands and their relatives with and without ANA will be
necessary to fully assess the role of serum C4 depression
in the etiology of SLE and the serologic abnormalities
seen in first degree relatives.
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22. Teisberg P, Olaisen B, Jonassen R, et al: The genetic polymorphism of the 4th component of human complement:
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fourth component of complement and the major histocompatibility complex. N Engl J Med 296:470-475, 1977
24. Tala1 N: Immunologic and viral factors in the pathogenesis of systemic lupus erythematosus. Arthritis Rheum
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25. Daniels CA, Borsos T, Rapp HJ, et a1 Neutralization of
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