AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 28:264-268 © 1992 MUNKSGAARD
Neonatal Lupus Erythematosus in Offspring of Mothers With Experimental Systemic Lupus Erythematosus FRANCIS KALUSH, EPHRAIM RIMON, AND EDNA MOZES Department ofChemical Immunology, The Weizmann Institute of Science, Rehovot, Israel ABSTRACT: Neonatal lupus erythematosus (NLE) syndrome is a result of the transfer of autoantibodies produced by the mother, across the placenta, to the fetus. NLE is characterized by a transient dermatitis, a variety of systemic and hematological abnormalities, and isolated cases ofcongenital heart block. The latter has been reported to be due to the presence of autoantibodies specific to La (SS-B) and/or Ro (SS-A). As female mice with experimental SLE, induced by immunization with the monoclonal anti-DNA 16/6 Id, produce a variety of autoantibodies including anti-Ro and anti-La antibodies, we examined the relevance of NLE in the murine system. Offspring of SLE-afflicted BALB/c mothers possessed antibody titers to the 16/6 Id, ssDNA, and nuclear extract, which gradually declined until reduced to normal levels by day 60 after delivery. Antibody titers in the sera of the mothers remained elevated throughout this period. Electrocardiograms were recorded from groups of neonates from mothers with experimental SLE. The results indicated that a high percentage of the offspring had defects in their conduction system including first, second, and third degree heart block; significant bradycardia; and wide QRS complex. Normal patterns were observed in offspring of healthy mothers. Experiments done with mice that were exposed to SLE-related autoantibodies early in their development indicated that offspring to mothers with experimental SLE were neither protected nor more susceptible to disease induction by the 16/6 Id, (Am J Reprod Immunol. 1992; 28:264-268.) Key words: Neonatal lupus, heart block, mouse model, anti-Ro (SSA), anti-La (SSB)
inducing experimental SLE in mice by the immunization with a human monoclonal anti-DNA IgM antibody that expresses a dominant idiotype designated 16/6 Id.2-4 Following immunization with the 16/6 Id, high levels of anti-16/6 Id antibodies and anti-anti-16/6 Id antibodies (murine 16/6) were detected in the sera of all immunized mice. High titers of autoantibodies reacting with different nuclear and other antigens (ss/ds DNA, Sm, Ro, La, cardiolipin, and others) were also detected.f The serological findings were associated with increased erythrocyte sedimentation rates and significant leukopenia. All mice exhibited significant proteinuria, abundant immune complex deposition in the kidneys, expression of the murine 16/6 Id, and subsequent sclerosis of the glomeruli. Electron microscopy demonstrated electron-dense deposits in kidney sections of sick mice.f Neonatal lupus erythematosus (NLE) syndrome is a result of the transfer of autoantibodies produced by the mother across the placenta to the fetus. NLE is characterized by transient dermatitis, a variety of systemic and hematological abnormalities," and isolated congenital heart block." The latter has been reported to be due to the presence of autoantibodies specific to La (SS-B) and/or Ro (SS_A).5,8-l0 Because female mice with experimental SLE induced by immunization with the monoclonal antiDNA 16/6 Id antibody produce a variety of autoantibodies including anti-Ro and anti-La antibodies, we examined the relevance of NLE in the murine system. The results of this study demonstrate that neonates born to female mice with experimental SLE had a wide variety of defects in the conduction system. In addition, early exposure to SLE-related autoantibodies does not affect the susceptibility of mice to the induction of the disease. MATERIALS AND METHODS
INTRODUCTION
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause and cure. Despite extensive research, the information available on the etiology of SLE is very limited. A variety of different autoantibodies against nuclear components is observed in SLE. Autoantibodies to platelets and lymphocytes also occur, and they may result in thrombocytopenia and leukopenia, respectively. The principal clinical manifestations of SLE are arthritis, rash, glomerulonephritis, and involvement of the central nervous system. The idiotypic network was shown to have a role in regulating immune responses and it has been suggested that Id-anti-Id antibodies may be involved in the induction and progression of autoimmunity.' In agreement with this suggested role, our laboratory recently succeeded in Submitted for publication September 1, 1992; accepted September 9, 1992. Address reprint requests to Francis Kalush, Department of Chemical Immunology, The Weizman Institute of Science, Rehovot, Israel.
Mice BALB/c mice were obtained from alae, Blackthorn, Bicester, axon, U.K, and used for experiments at the age of 2-3 months. Offspring were obtained by breeding normal and immunized female mice with syngeneic males at our animal facilities.
The 16/6 Id The hybridoma secreting the 16/6 Id was grown continuously in culture. The 16/6 Id was obtained from the culture supernatants and the affinity purified material eluted from a goat-anti-human IgM-Sepharose column was employed in this study. Immunizations Mice were immunized with 1 fLg of the 16/6 Id, in complete Freund's adjuvant (Difco Laboratories Inc., Detroit, MI) intradermally into the hind footpads. The mice were given a booster injection 3 weeks later with the same
NEONATAL LUPUS ERYTHEMATOSUS
amount of the immunizing antibodies in aqueous solution into the hind footpads.
265
Anti 16/6 Id antibodies In offspring of mothers with Induced experimental SLE.
Radioimmunoassay Flexible plastic microtiter plates were coated with 50 1-1-1 of20 I-I-g/ml of the monoclonal 16/6 Id dissolved in phosphate buffered saline (PBS) for the detection of anti-16/6 Id antibodies. For the detection of anti-ssDNA antibodies, wells were coated with 50 1-1-1 of calf thymus ssDNA (50 I-I-g/ml) dissolved in PBS. After a 3-hour incubation the plates were washed with PBS containing 10% lowfat milk. The sera of the mice (diluted 1:10 to 1:1000) were then added and incubated for 3 hours. 125I-labeled goat anti-mouse immunoglobulin (lOs cpm/well) was added thereafter to detect bound antibodies. After extensive washing of the radioactive reagent, plates were dried and wells were cut out and counted in a gamma counter.
moth.r
I mmunohistology Immunohistology was performed by incubation of fixed cryostat sections with rhodamine conjugated rabbit antimouse immunoglobulins (forthe identification of immune complexes). Specific staining was visualized by a fluorescence microscope. RESULTS
Female BALB/c mice with experimental SLE were bred with syngeneic males and the offspring were tested for the presence of autoantibodies. As can be observed in Fig. 1, high antibody titers specific to the 16/6 Id and to ssDNA were detected in offspring of mice with induced SLE. The antibody titers declined gradually, and at day 60 after delivery they were reduced to background levels. In contrast antibody titers in the sera ofthe mothers remained elevated throughout this period. In order to determine whether congenital heart block occurs in our experimental model of SLE, electrocardiograms (ECG) were recorded in 31 offspring (16 females and 15 males) of mothers with experimental SLE, and in 20 offspring (10 females and 10 males) born to control mothers. Conduction system disorders detected in neonatal mice are summarized in Table I. As can be seen in the table, first-degree A-V block was recorded in eight offspring, second-degree A-V block in two, and complete heart block (third degree) in two other mice born to mothers with experimental SLE. Bradycardia was recorded
1:10
1:100
II
1:1000
20000
10000
Electrocardiogram Records Electrocardiogram records were registered by an electrocardiogram instrument with a paper velocity of 5 ern/sec and a standard of 2 mV. Mice were anesthetized slightly with ether during the examination. The limb leads were connected in mice in the same way as with humans. Detection ofSLE-Associated Pathological Manifestations Proteinuria was measured by a semiquantitative method using Combistix kit. Erythrocyte sedimentation rate (ESR) was determined by passing the diluted (1:1 in PBS) heparinized blood samples into microsampling pipettes. The rate of sedimentation was measured 6 hours later. Leukopenia was determined by counting the leukocytes ofthe diluted blood (1:10) in 1% acetic acid under the microscope.
•
Ll
1 •
30
45
60
DAYS
Antl.sspNA aotlbgdl.1 In offspring_ moth.rs with
of
Induced .xperlmenta' SlE
20000
m01her
•
1;10
Ecl
1:100
~
1:1000
...o
::I
10000
::
;~
=: ~:
~~ ~ :;
15304560150 DAYS
Fig. 1. Antibody responses in offspring of female BALB/c mice with induced experimental SLE. Newborn mice were periodically bled after birth. The sera of eight individual mice were tested for antibody activity. Flexible microtiter plates were coated with 16/6 Id or ssDNA. Following incubation with the diluted sera [125I]goat anti-mouse Ig was added to detect specific binding. Results are expressed as mean cpm. S.D. values did not exceed 3%.
in 10 mice and widening of the QRS complex was measured in eight offspring born to female mice with induced experimental SLE. None of the above disorders could be detected in offspring to healthy control mothers. A representative ECG of a mouse with complete heart block born to a mother with experimental SLE is shown
266
KALUSH ET AL.
TABLE I. Conductive System Disorders in Neonatal Mice No Offspring Offspring I of: tested degree Mothers withexp SLE Control Mothers
A-V Block II
III
degree
degree
Bradycardia
Wide QRS
31
8
2
2
10
8
20
0
0
0
0
0
in Fig. 2. As can be seen in the figure, the complete A-V block was diagnosed by a regular pop interval (atrial rhythm), in conjunction with an abnormal R-R interval (ventricular rhythm) and a complete A-V dissociation (B). A normal ECG with regular atrial and ventricular rhythms, with each R wave preceded by a P wave and a normal P-R interval, was observed in a control mouse (A). Because the presence of antibodies against a variety of nuclear antigens is characteristic of SLE, and a correlation has been reported between the presence of anti-Ro and anti-La antibodies and complete A-V block in human patients, sera from the experimental females that were used in these studies were tested for the presence of anti-nuclear antibodies. Immunoblotting experiments indicated that the mice with experimental SLE produced antibodies against nuclear components such as La, Ro, RNP, and Sm. In an attempt to determine whether early (in utero
and neonatal) exposure to SLE-related autoantibodies protected or sensitized mice to the induction of experimental SLE, offspring of mothers with SLE were injected with the 16/6 Id and the development of autoantibodies and clinical manifestations of SLE were monitored. No significant differences could be observed in the antibody titers specific to the 16/6 Id and to DNA between offspring of mothers with SLE and offspring of control mothers, following the immunization with the 16/6 Id. Table II summarizes the clinical manifestations observed 5 months following immunization with the 16/6 Id of offspring of mothers afflicted with experimental SLE. The clinical manifestations (i.e., high ESR, leukopenia, and proteinuria) were comparable with those observed previously in naive mice that were immunized with the 16/6 Id. 2 Figure 3 depicts immune complex deposits observed in kidney sections of offspring of mothers with SLE, following induction of experimental SLE by immunization with the 16/6 Id (A). No such deposits were observed in kidney sections of control mice (B). Thus it appears that exposure of offspring of mothers with SLE to diseaserelated autoantibodies did not affect their susceptibility to the induction of experimental SLE. DISCUSSION
The main findings of this report are that neonatal lupus erythematosus syndrome may occur in offspring born to female mice with experimental SLE and that exposure of the offspring to SLE-related autoantibodies
A
j.
B
II ,i
I
t
I
I
.... -.
~
I
1·-Fig. 2. Complete A-V block recorded in a newborn to a mother with experimental SLE. (A) Normal ECG recording shows pop and R-Rintervals at the same rate (500 beats/min), and each P wave is followed by a
R wave. (B) Complete A-V block was diagnosed by a regular pop interval (500 beats/min) in conjunction with a R-R interval at a much slower rate (120 beats/min) and complete A-V dissociation.
NEONATAL LUPUS ERYTHEMATOSUS TABLE II. Clinical Manifestations in 16/6 Id-Injected Offspring to Mothers With Experimental SLE
E.S.R. (mm/7hr) W.B.C. (cells/mm") Proteinuria (gr/lt)
Offspring to mothers with SLE, injected with 16/6Id
Control mice
12.3 ± 3.0 4433 ± 790 0.3 - 1
2.6 ± 0.9 6833 ± 654
Neonatal Lupus Erythematosus in Offspring of Mothers With Experimental Systemic Lupus Erythematosus FRANCIS KALUSH, EPHRAIM RIMON, AND EDNA MOZES Department ofChemical Immunology, The Weizmann Institute of Science, Rehovot, Israel ABSTRACT: Neonatal lupus erythematosus (NLE) syndrome is a result of the transfer of autoantibodies produced by the mother, across the placenta, to the fetus. NLE is characterized by a transient dermatitis, a variety of systemic and hematological abnormalities, and isolated cases ofcongenital heart block. The latter has been reported to be due to the presence of autoantibodies specific to La (SS-B) and/or Ro (SS-A). As female mice with experimental SLE, induced by immunization with the monoclonal anti-DNA 16/6 Id, produce a variety of autoantibodies including anti-Ro and anti-La antibodies, we examined the relevance of NLE in the murine system. Offspring of SLE-afflicted BALB/c mothers possessed antibody titers to the 16/6 Id, ssDNA, and nuclear extract, which gradually declined until reduced to normal levels by day 60 after delivery. Antibody titers in the sera of the mothers remained elevated throughout this period. Electrocardiograms were recorded from groups of neonates from mothers with experimental SLE. The results indicated that a high percentage of the offspring had defects in their conduction system including first, second, and third degree heart block; significant bradycardia; and wide QRS complex. Normal patterns were observed in offspring of healthy mothers. Experiments done with mice that were exposed to SLE-related autoantibodies early in their development indicated that offspring to mothers with experimental SLE were neither protected nor more susceptible to disease induction by the 16/6 Id, (Am J Reprod Immunol. 1992; 28:264-268.) Key words: Neonatal lupus, heart block, mouse model, anti-Ro (SSA), anti-La (SSB)
inducing experimental SLE in mice by the immunization with a human monoclonal anti-DNA IgM antibody that expresses a dominant idiotype designated 16/6 Id.2-4 Following immunization with the 16/6 Id, high levels of anti-16/6 Id antibodies and anti-anti-16/6 Id antibodies (murine 16/6) were detected in the sera of all immunized mice. High titers of autoantibodies reacting with different nuclear and other antigens (ss/ds DNA, Sm, Ro, La, cardiolipin, and others) were also detected.f The serological findings were associated with increased erythrocyte sedimentation rates and significant leukopenia. All mice exhibited significant proteinuria, abundant immune complex deposition in the kidneys, expression of the murine 16/6 Id, and subsequent sclerosis of the glomeruli. Electron microscopy demonstrated electron-dense deposits in kidney sections of sick mice.f Neonatal lupus erythematosus (NLE) syndrome is a result of the transfer of autoantibodies produced by the mother across the placenta to the fetus. NLE is characterized by transient dermatitis, a variety of systemic and hematological abnormalities," and isolated congenital heart block." The latter has been reported to be due to the presence of autoantibodies specific to La (SS-B) and/or Ro (SS_A).5,8-l0 Because female mice with experimental SLE induced by immunization with the monoclonal antiDNA 16/6 Id antibody produce a variety of autoantibodies including anti-Ro and anti-La antibodies, we examined the relevance of NLE in the murine system. The results of this study demonstrate that neonates born to female mice with experimental SLE had a wide variety of defects in the conduction system. In addition, early exposure to SLE-related autoantibodies does not affect the susceptibility of mice to the induction of the disease. MATERIALS AND METHODS
INTRODUCTION
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause and cure. Despite extensive research, the information available on the etiology of SLE is very limited. A variety of different autoantibodies against nuclear components is observed in SLE. Autoantibodies to platelets and lymphocytes also occur, and they may result in thrombocytopenia and leukopenia, respectively. The principal clinical manifestations of SLE are arthritis, rash, glomerulonephritis, and involvement of the central nervous system. The idiotypic network was shown to have a role in regulating immune responses and it has been suggested that Id-anti-Id antibodies may be involved in the induction and progression of autoimmunity.' In agreement with this suggested role, our laboratory recently succeeded in Submitted for publication September 1, 1992; accepted September 9, 1992. Address reprint requests to Francis Kalush, Department of Chemical Immunology, The Weizman Institute of Science, Rehovot, Israel.
Mice BALB/c mice were obtained from alae, Blackthorn, Bicester, axon, U.K, and used for experiments at the age of 2-3 months. Offspring were obtained by breeding normal and immunized female mice with syngeneic males at our animal facilities.
The 16/6 Id The hybridoma secreting the 16/6 Id was grown continuously in culture. The 16/6 Id was obtained from the culture supernatants and the affinity purified material eluted from a goat-anti-human IgM-Sepharose column was employed in this study. Immunizations Mice were immunized with 1 fLg of the 16/6 Id, in complete Freund's adjuvant (Difco Laboratories Inc., Detroit, MI) intradermally into the hind footpads. The mice were given a booster injection 3 weeks later with the same
NEONATAL LUPUS ERYTHEMATOSUS
amount of the immunizing antibodies in aqueous solution into the hind footpads.
265
Anti 16/6 Id antibodies In offspring of mothers with Induced experimental SLE.
Radioimmunoassay Flexible plastic microtiter plates were coated with 50 1-1-1 of20 I-I-g/ml of the monoclonal 16/6 Id dissolved in phosphate buffered saline (PBS) for the detection of anti-16/6 Id antibodies. For the detection of anti-ssDNA antibodies, wells were coated with 50 1-1-1 of calf thymus ssDNA (50 I-I-g/ml) dissolved in PBS. After a 3-hour incubation the plates were washed with PBS containing 10% lowfat milk. The sera of the mice (diluted 1:10 to 1:1000) were then added and incubated for 3 hours. 125I-labeled goat anti-mouse immunoglobulin (lOs cpm/well) was added thereafter to detect bound antibodies. After extensive washing of the radioactive reagent, plates were dried and wells were cut out and counted in a gamma counter.
moth.r
I mmunohistology Immunohistology was performed by incubation of fixed cryostat sections with rhodamine conjugated rabbit antimouse immunoglobulins (forthe identification of immune complexes). Specific staining was visualized by a fluorescence microscope. RESULTS
Female BALB/c mice with experimental SLE were bred with syngeneic males and the offspring were tested for the presence of autoantibodies. As can be observed in Fig. 1, high antibody titers specific to the 16/6 Id and to ssDNA were detected in offspring of mice with induced SLE. The antibody titers declined gradually, and at day 60 after delivery they were reduced to background levels. In contrast antibody titers in the sera ofthe mothers remained elevated throughout this period. In order to determine whether congenital heart block occurs in our experimental model of SLE, electrocardiograms (ECG) were recorded in 31 offspring (16 females and 15 males) of mothers with experimental SLE, and in 20 offspring (10 females and 10 males) born to control mothers. Conduction system disorders detected in neonatal mice are summarized in Table I. As can be seen in the table, first-degree A-V block was recorded in eight offspring, second-degree A-V block in two, and complete heart block (third degree) in two other mice born to mothers with experimental SLE. Bradycardia was recorded
1:10
1:100
II
1:1000
20000
10000
Electrocardiogram Records Electrocardiogram records were registered by an electrocardiogram instrument with a paper velocity of 5 ern/sec and a standard of 2 mV. Mice were anesthetized slightly with ether during the examination. The limb leads were connected in mice in the same way as with humans. Detection ofSLE-Associated Pathological Manifestations Proteinuria was measured by a semiquantitative method using Combistix kit. Erythrocyte sedimentation rate (ESR) was determined by passing the diluted (1:1 in PBS) heparinized blood samples into microsampling pipettes. The rate of sedimentation was measured 6 hours later. Leukopenia was determined by counting the leukocytes ofthe diluted blood (1:10) in 1% acetic acid under the microscope.
•
Ll
1 •
30
45
60
DAYS
Antl.sspNA aotlbgdl.1 In offspring_ moth.rs with
of
Induced .xperlmenta' SlE
20000
m01her
•
1;10
Ecl
1:100
~
1:1000
...o
::I
10000
::
;~
=: ~:
~~ ~ :;
15304560150 DAYS
Fig. 1. Antibody responses in offspring of female BALB/c mice with induced experimental SLE. Newborn mice were periodically bled after birth. The sera of eight individual mice were tested for antibody activity. Flexible microtiter plates were coated with 16/6 Id or ssDNA. Following incubation with the diluted sera [125I]goat anti-mouse Ig was added to detect specific binding. Results are expressed as mean cpm. S.D. values did not exceed 3%.
in 10 mice and widening of the QRS complex was measured in eight offspring born to female mice with induced experimental SLE. None of the above disorders could be detected in offspring to healthy control mothers. A representative ECG of a mouse with complete heart block born to a mother with experimental SLE is shown
266
KALUSH ET AL.
TABLE I. Conductive System Disorders in Neonatal Mice No Offspring Offspring I of: tested degree Mothers withexp SLE Control Mothers
A-V Block II
III
degree
degree
Bradycardia
Wide QRS
31
8
2
2
10
8
20
0
0
0
0
0
in Fig. 2. As can be seen in the figure, the complete A-V block was diagnosed by a regular pop interval (atrial rhythm), in conjunction with an abnormal R-R interval (ventricular rhythm) and a complete A-V dissociation (B). A normal ECG with regular atrial and ventricular rhythms, with each R wave preceded by a P wave and a normal P-R interval, was observed in a control mouse (A). Because the presence of antibodies against a variety of nuclear antigens is characteristic of SLE, and a correlation has been reported between the presence of anti-Ro and anti-La antibodies and complete A-V block in human patients, sera from the experimental females that were used in these studies were tested for the presence of anti-nuclear antibodies. Immunoblotting experiments indicated that the mice with experimental SLE produced antibodies against nuclear components such as La, Ro, RNP, and Sm. In an attempt to determine whether early (in utero
and neonatal) exposure to SLE-related autoantibodies protected or sensitized mice to the induction of experimental SLE, offspring of mothers with SLE were injected with the 16/6 Id and the development of autoantibodies and clinical manifestations of SLE were monitored. No significant differences could be observed in the antibody titers specific to the 16/6 Id and to DNA between offspring of mothers with SLE and offspring of control mothers, following the immunization with the 16/6 Id. Table II summarizes the clinical manifestations observed 5 months following immunization with the 16/6 Id of offspring of mothers afflicted with experimental SLE. The clinical manifestations (i.e., high ESR, leukopenia, and proteinuria) were comparable with those observed previously in naive mice that were immunized with the 16/6 Id. 2 Figure 3 depicts immune complex deposits observed in kidney sections of offspring of mothers with SLE, following induction of experimental SLE by immunization with the 16/6 Id (A). No such deposits were observed in kidney sections of control mice (B). Thus it appears that exposure of offspring of mothers with SLE to diseaserelated autoantibodies did not affect their susceptibility to the induction of experimental SLE. DISCUSSION
The main findings of this report are that neonatal lupus erythematosus syndrome may occur in offspring born to female mice with experimental SLE and that exposure of the offspring to SLE-related autoantibodies
A
j.
B
II ,i
I
t
I
I
.... -.
~
I
1·-Fig. 2. Complete A-V block recorded in a newborn to a mother with experimental SLE. (A) Normal ECG recording shows pop and R-Rintervals at the same rate (500 beats/min), and each P wave is followed by a
R wave. (B) Complete A-V block was diagnosed by a regular pop interval (500 beats/min) in conjunction with a R-R interval at a much slower rate (120 beats/min) and complete A-V dissociation.
NEONATAL LUPUS ERYTHEMATOSUS TABLE II. Clinical Manifestations in 16/6 Id-Injected Offspring to Mothers With Experimental SLE
E.S.R. (mm/7hr) W.B.C. (cells/mm") Proteinuria (gr/lt)
Offspring to mothers with SLE, injected with 16/6Id
Control mice
12.3 ± 3.0 4433 ± 790 0.3 - 1
2.6 ± 0.9 6833 ± 654
