Professional Documents
Culture Documents
a
This work was supported in part by NIH Grant No. NS17904.
d
Address for correspondence: National Institutes of Health, National Institute of Neurologi-
cal Disorders and Stroke, 31 Center Drive, Bethesda, Maryland 20892-2540.
433
FIGURE 1. Chemical formulas of penicillamine and other sulfhydryl compounds used as drugs in rheumatoid arthritis (after refer-
ence 18, with permission).
PENN et al.: DRUG-INDUCED MG 435
has been reported to induce MG. D-P is highly chemically reactive. It will reduce
cystine, providing therapy for cystinuria,20 and will form a mixed disulfide with the
cysteine produced. Because it is less reactive than either dithiothreitol or mercap-
toethanol, it tends to form mixed disulfides with other thiols as it does with cysteine.
We have shown previously that it will bind covalently to acetylcholine receptor
(AChR) alpha and gamma subunits and have hypothesized that it binds to one or both
alpha subunit cysteines 192 or 193 after reducing the readily reducible disulfide bond
formed from these cysteines.21 This bond is located at about 1 nm from the ligand-
binding site on the alpha subunit of AChR.22 Xu and colleagues have confirmed that
D-P acts near the ligand-binding site since they showed that D-P blocked the binding
of monoclonal antibodies to Torpedo AChR, which themselves blocked the action of
agonists.23 The D-P effect was abolished by pretreatment with N-ethylmaleimide. D-
P may similarly reduce disulfide bonds and bind to thiols on the insulin molecule. It
will also bind to various plasma proteins and this property has been used to investi-
gate its pharmacokinetics.24 Nearly all is bound irreversibly at 3 hours after iv admin-
istration to rats. Although D-P will be present long enough to be presented to im-
mune cells, immune responses to D-P and its derivatized proteins depend heavily on
species and strain differences and thus on genetic predisposition.
Animal models of immune disorders that have developed after D-P administration
vary in type and severity. Brown-Norway rats developed antinuclear antibodies, cir-
culating immune complexes, and disseminated intravascular coagulation with granu-
lomatous lesions and IgG deposits in the kidneys after about 8 weeks of oral D-P.25
Lewis and Sprague-Dawley varieties did not.26 Mice of the C57BL/KsJ (H-2d) and
C3H/HeJ (H-2k) strains, but not Balb/c (H-2d) or C57BL/6 (H-2b), developed anti-
bodies against insulin.10 The mice were not clinically ill. Similarly, A.SW (H-2s)
mice developed antinuclear antibodies specific for either DNA or histone-DNA com-
plexes after receiving D-P or quinidine.27 We have studied mice treated chronically
with D-P by daily (5 of 7) injections of 40 mg/kg (approximately 1 mg per mouse per
day). None of these developed clinical signs after 6 months. However, when mice
were treated and then exposed to a single inoculating dose of Torpedo AChR, strain A
(IAk) mice showed clinical and electrophysiological evidence of myasthenia at 9
weeks after the injection.28,38 Antibody titers in C57BL/6 (IAb) and C3H/HeJ (IAk)
were significantly higher than in matched animals who received only saline followed
by the single injection of AChR. In a similar study of guinea pigs, no antibodies to
AChR were found in D-P-treated animals, but there was evidence of proliferative re-
sponses to AChR by their spleen cells.29
Immune responses to D-P depend upon its chemical reactivity and ability to bind
to and derivatize proteins. It is also able to generate immune responses to itself by de-
rivatizing immune cells.30–33 After immunization of various murine strains with D-P
in complete Freund’s adjuvant (CFA), C3H/He, B10.AM, and A.TI mice, all IAk,
436 ANNALS NEW YORK ACADEMY OF SCIENCES
Antibodies to AChR
Antibody titers to D-P-AChR were compared to those against AChR using ELISA
and radioimmunoassay (RIA). The AChR preparations used in the RIAs were treated
with 125I-␣-bungarotoxin as described.40,42 ELISA was achieved by plating 1.5
mg/mL of purified AChR or D-P-AChR (0.3 mg/well) as described.43 Peroxidase-
conjugated antibodies, raised in goats, to IgG and IgM were used at 1:6000 and at
1:10,000, respectively. Sera from rabbits immunized with D-P-AChR reacted to a
greater degree with D-P-AChR than with AChR when examined by ELISA or RIA,
which suggests a reaction to epitopes present on D-P-AChR and not on AChR (FIG-
URES 2A and 2B, TABLE 1). Binding curves generated by varying the amounts of anti-
gen confirmed higher recognition of the D-P-modified AChR at all antigen doses
studied (FIGURE 2C). No differences were found when antisera raised to unmodified
AChR40 were tested.
We also detected antibodies to both modified and unmodified Torpedo AChR in
serum from 4 of the 5 surviving rabbits that had only been given D-P. Antibodies
were measured using RIA and ELISA. IgG titers rose steadily and were sustained to
termination at the sixth month.
Low antibody titers to D-P-modified Torpedo AChR and unmodified AChR were
also detected by RIA in a range similar to D-P-treated human patients (TABLE 1).
Titers derived from binding curves of the samples starting with those obtained during
the second month of D-P treatment (TABLE 1) were significantly elevated over con-
trols. Full binding curves against D-P-AChR and AChR showed higher binding of D-
P-AChR at higher serum concentrations, but became superimposable as sera were di-
luted out (FIGURE 3). Therefore, D-P-AChR bore additional reactive determinants as
compared to AChR, which could be detected with less dilute serum from rabbits
treated only with D-P containing relatively uncommon antibody species present in
low titers.
FIGURE 2. (A,B) ELISA examination of binding curves of sera from two groups of rabbits immunized with D-P-AChR (D-P-T) as
compared to reactions with AChR (T). Data points are means of triplicates. Error bars represent two standard deviations. (C) Binding
curve of group A rabbits against D-P-AChR and AChR generated by plating increasing doses of antigen.
PENN et al.: DRUG-INDUCED MG 439
Rabbit albumin was used in order to minimize reactions against foreign determi-
nants. It was derivatized by two methods that produced D-P attached to lysine
residues either via propionyl or via disulfide bonds.44,45 Serum samples from the 4
rabbits that had detectable antibodies to modified and unmodified Torpedo AChR af-
ter prolonged D-P administration (see above) also showed low, but measurable reac-
tions with the D-P-modified rabbit albumin (D-P-RSA) (TABLE 1). These were mea-
sured by ELISA-plating 25 g/mL (5 g/well) of rabbit albumin (RSA) or D-P-RSA
in 0.06 M carbonate buffer, pH 9.4, by incubation in a humidified atmosphere at 4 °C
overnight. Wells were washed with phosphate-buffered saline (PBS) containing
0.05% Tween 20 (PBS/T). Uncoated sites were blocked by incubation with nonfat
milk in PBS/T for 1 hour at room temperature. Following three more washes with
PBS/T, the serum to be analyzed was added to every other well in every other row in
serial fivefold dilutions starting at 1:5. After 2 hours at room temperature, wells were
washed as described above. The optimal working dilution of second antibody was de-
termined by a preliminary titration against control rabbit serum and antigen and by
assay of the peroxidase activity of the conjugate against o-phenylenediamine at 490
nm. Bound conjugate was visualized by addition of a freshly prepared solution con-
taining 2 mM o-phenylenediamine in citrate-phosphate buffer, pH 5.0, mixed with
1/Dilution
FIGURE 3. ELISA examination of binding of serum from individual rabbits (A, B, C) treated with D-P at peak response (45–60
days) to D-P-AChR (D-P-T) as compared to AChR (T). Data points are means of triplicates. Error bars represent two standard devia-
tions. Control sera, <0.1 OD at 1:5 dilution.
PENN et al.: DRUG-INDUCED MG 441
50% H2O2. The reaction was terminated after 10 minutes with H2SO4 and each well
was read at 490 nm on an ELISA reader (Biotek, Winooski, Vermont).
There was no apparent correlation between titers of anti-AChR and reactions with
D-P-RSA. The 2 rabbits with no anti-AChR also showed no reaction with D-P-RSA.
We also found antibodies to D-P, as presented on RSA, in sera from rabbits immu-
nized with D-P-modified Torpedo AChR (TABLE 1), but no reactivity to unmodified
RSA. Sera from rabbits immunized with unmodified Torpedo AChR with compara-
ble titers to AChR did not react with either D-P-RSA or RSA (data not shown). To-
gether, these findings provide evidence that rabbits treated chronically with D-P or
immunized with D-P-AChR had antibodies recognizing the D-P group.
Calf sequence peptide ␣179–191 and human sequence ␣179–191 (Trp 187/Ser
184) were synthesized using an Applied Biosystems 430A automated synthesizer and
purified by ion exchange chromatography (Pharmacia FPLC mono-Q) followed by
reverse-phase HPLC. Identity and purity of the peptides were established by both
amino acid composition and sequence analysis. We have shown that the calf se-
quence is immunogenic in IAk mice.46 When we tested the D-P-treated rabbits for
the presence of antibodies to these peptides by ELISA, significant titers were found
(FIGURE 4, TABLE 1). Indeed, titers were higher than those to AChR, D-P-derivatized
albumin, or D-P-AChR.
Lower, but detectable titers were also found in sera from rabbits immunized with
D-P-AChR (TABLE 1). No antibodies to this peptide were detected in sera from rab-
bits immunized with unmodified AChR or from control rabbits. The presence of anti-
bodies to this sequence in D-P-treated rabbits supports our hypothesis that regions
contiguous to the proposed D-P binding site could contribute to the in vivo immuno-
genicity.
We have shown that IAk mice, both strain C3H/eb (H-2k) and A (H-2a), develop
augmented antibody responses to a single inoculum of AChR after D-P treatment as
compared to antibody titers in saline-treated, AChR-primed syngeneic mice.28 Initia-
tion of an immune response to AChR should involve helper T cell activity against
epitopes on AChR, or to an AChR peptide involved in D-P binding or to a cross-re-
acting antigen targeted by D-P. We therefore examined mice treated chronically with
D-P for up to 6 months as well as mice immunized with D-P emulsified in CFA.
Mice were injected into the peritoneum (IP) with freshly prepared D-P hydrochlo-
ride (40 mg/kg) for 5 of 7 days for up to 6 months. Controls received saline. They
were then injected with saline/CFA emulsion in one hind footpad and IP. Other
groups were hyperimmunized with D-P in Freund’s adjuvant using a priming dose in
442 ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 4. Binding of serum from individual rabbits (Rbt A, B, C) and from D-P-AChR
(DPT)–immunized rabbits to human alpha subunit sequence peptide 179–191 (Trp 187/Ser
184) examined by ELISA. Data points are means of triplicates.
CFA and two boosts in incomplete Freund’s adjuvant. Popliteal lymph nodes (PLN)
and spleens were removed 9 days after the adjuvant injections.47 Single cell suspen-
sions were prepared in Click’s medium supplemented with Hepes buffer, 15 mM, pH
7.0, and 10% horse serum.48 Cells (5 × 106/mL, 0.1 mL) were plated into wells of mi-
crotiter plates in triplicate. Peptide, in 0.1 mL, was added in varying doses; AChR
was added at 0.5 to 1 mg/mL as previously determined. Cultures were allowed to in-
cubate for a total of 96 hours in an atmosphere of 95% air/5% CO2. Tritiated (3H)
thymidine, 1 Ci, was added for the final 16 hours. Cells were then harvested onto
glass fiber paper using a cell harvester (Mini-mash automated sample harvester,
Whitaker M.A. Bioproducts, Walkersville, Maryland). 3H-Thymidine, incorporated
into DNA, was counted, after the paper strips dried, in a liquid scintillation counter.
We found significant responses (stimulation index > 2.0) of PLN cells from mice
treated with D-P for 4 to 6 months to the alpha subunit peptide 179–196, whereas
controls did not react (FIGURE 5). Responses to AChR were not significantly different
from controls. In contrast, PLN cells from mice immunized and boosted with D-P in
PENN et al.: DRUG-INDUCED MG 443
FIGURE 5. Responses of popliteal lymph node cells from C3Heb/FeJ mice against AChR and
peptide calf sequence alpha 179–196-Acm. Terms—D-P/CFA: mice were primed with D-
P/CFA; D-P: mice were treated with D-P and then injected with CFA in saline; D-P + peptide:
mice were primed with D-P/CFA and boosted with peptide/CFA; control: mice were primed
with saline and boosted with CFA in saline. Final injections were given 9 days before cells were
obtained. Stimulation index (SI): cpm experimental/cpm control. Media cpm: D-P/CFA 320;
D-P 376; D-P + peptide 520. An asterisk indicates a significant SI over normal control at ⱕ2.0.
adjuvant showed significant responses to AChR and low, but significant, responses to
peptide. Spleen cell responses failed to reach significance in either treatment para-
digm.
We found very low antibody titers in 4 of 60 mice treated for at least 4 months
(data not shown); they did not show any clinical signs.
It has been shown that D-P derivatizes spleen cells that are likely to be dendritic
cells, B cells, or both.32–34 Helper T cells from mice of the H-2k strain will respond to
D-P-derivatized spleen cells.32,33 It is therefore possible that the immune system will
also respond to D-P derivatives of other cells or proteins. The presence of D-P-AChR
in treated mice of specific H-2 strains, especially those bearing IAk, would then gen-
erate immune reactions against AChR. Our data suggest that indeed reactions devel-
op against an alpha subunit sequence in the immediate vicinity of the most likely
binding site for D-P. It is intriguing that actual priming with D-P in CFA into foot-
pads produced responses to AChR, while chronic exposure to D-P followed by intra-
dermal CFA generated response to the peptide sequence. Neither protocol resulted in
444 ANNALS NEW YORK ACADEMY OF SCIENCES
antibody responses to AChR. The reactive T cells may be of the TH1 type since D-P
in CFA has been shown to generate delayed hypersensitivity associated with LY1,
CD4 T cell phenotypes.
We have studied 6 patients with D-P-MG (TABLE 2) and serum samples from an-
other 29 patients treated with D-P, but without clinical symptoms that warranted fur-
ther evaluation. All, but one, had been treated for rheumatoid arthritis; the other for
scleroderma. The anti-AChR titers in serial samples from 3 of the 6 D-P-MG patients
fell toward the normal range over 6 to 12 months. Serial samples were not available
in the other 29 patients. Although information on histocompatibility testing was not
available on all 35 patients, 4 of the 6 with D-P-MG showed HLA haplotypes report-
ed to occur with increased frequency in D-P-MG (2 Bw35, 2 DR7, 2 DR1).17 Control
samples came from patients with spontaneous MG, who were matched roughly for
age, sex, and anti-AChR titer; from patients with polymyositis, rheumatoid arthritis
with rheumatoid factor not receiving D-P [3 with MG, 6 without], Lambert-Eaton
syndrome,2 and neuropathies; and from normal individuals (control bank) reported
previously.42 All samples were obtained by venipuncture and were stored, in aliquots,
at –70 °C until use. Informed consent was obtained from all patients.
Antibodies to AChR
Antibody titers against both human and Torpedo AChR were assessed by RIA as
described previously40,42 and by ELISA as described above for rabbit serum. Patients
with D-P-MG all showed significantly elevated titers of anti-AChR, measured by the
standard RIA against AChR-enriched human muscle extract (TABLE 2). Therefore,
AChR also contributed epitopes that generate antibodies when AChR alone is the
antigen, a conclusion also reached by a study of D-P-MG antibodies using competing
monoclonal antibodies of defined specificity.15
Serum from all 6 patients also reacted with Torpedo AChR when tested by RIA or
ELISA (TABLE 2). This allowed comparisons with Torpedo AChR modified by D-P.
Reactions with D-P-AChR were significantly greater than those against unmodified
AChR at the more concentrated regions of the ELISA binding curves in 3/4 patients
(data not shown). Slightly higher titers against D-P-AChR, measured by RIA, were
significant in only 2 patients of these 4. Both IgM and IgG anti-AChR were detected.
Three had higher IgG titers, but anti-AChR were exclusively IgM in 1 patient.
ELISAs on D-P-modified human albumin were performed as for rabbit sera with
the following modifications. The coating concentration for modified and unmodified
human albumin (HSA) was 25 g/mL (5 g/well) in borate buffer, pH 8.4. Uncoated
TABLE 2. D-P Myasthenia Gravis
Duration of Antibodies to HLA
D-P Rx Severity ____________________________ ______________________
Patient (Months) of MGa AChR-Hb DPT/Tb DPT/Tc D-P-HSAd A B DR Peptide
G 5 II 6.0 1.0/0.8 160/160 1560 2, w30 17, 18 1, 7 25
K 6 II 14.5 7.8/11 320/160 625 w24, 11 27, w35 n.d. 125
M 12 II 7.5 3/4 80/80 1560 28, 36 5, w35 2, 7 625
Mr 10 III 46.0 11.7/9.3 80/40 10,240 3, w24 7, 27 1, 6 n.d.e
P 24 III 250.0 3.7/3.3 320/320 250 n.d. n.d. n.d. 125
T 4 III 100.0 7.0/4.0 160/160 1560 n.d. n.d. n.d. 125
a
II: Mild generalized disease sparing oropharyngeal muscles. III: Moderate generalized disease with mild or moderate
oropharyngeal muscles or wheelchair-bound.
b
H: Human AChR preparations; control values < 0.5 pmoles/mL. Affinity-purified Torpedo AChR (T) or D-P-modified Tor-
pedo AChR (DPT); controls subtracted.
c
Titers from ELISA binding curves of serum versus D-P-AChR (DPT) and Torpedo AChR (T) expressed as the reciprocal of
dilution at which the experimental-matched mean control value is at 1:10 dilution (OD: 0.100 ± 0.009).
d
Titers from ELISA binding curves of serum versus D-P-derivatized human albumin (D-P-HSA) expressed as the reciprocal
of dilution at which the experimental-matched mean control is at 1:12.5 dilution (OD: 0.075 ± 0.012).
e
n.d. = not determined.
446 ANNALS NEW YORK ACADEMY OF SCIENCES
sites were blocked with 2.5% chicken serum. Horseradish peroxidase–conjugated an-
tibodies raised in goats was used. Anti-IgG was at a dilution of 1:2500; anti-IgM at
1:8000.
Sera from patients with generalized D-P-MG recognized D-P presented on human
albumin (D-P-HSA), but did not react with HSA alone above controls (TABLE 2).
Background reactivity with unmodified HSA, quenched by blocking with 2.5%
chicken serum in borate buffer, was similar to that found with control sera. Control
samples including the 12 from patients matched for age, sex, and titer of anti-AChR
and 6 with rheumatoid arthritis did not. These experiments establish that, in subjects
with reactions to D-P-AChR, the D-P group is recognized as presented on another
carrier, to which it has been linked by disulfide bonds.
Antibodies to ␣179–191
Among D-P-MG patients, there was an IgM response of modest titer in patient M
and a primarily IgG response in patients T and K (TABLE 2).
We conclude that treatment with D-P and the development of D-P-MG are associ-
ated with the development of immune responses to D-P itself. D-P triggers immune
responses that can be measured using D-P-AChR, unmodified AChR, or D-P-conju-
gated albumins. In addition, D-P triggers responses to AChR. These occur in rela-
tionship to specific genetic backgrounds as shown by response of humans and mice.
Antibodies to a peptide segment that constitutes most of the prime alpha toxin-bind-
ing site and is contiguous to the D-P reducible disulfide bond within 1 nm of the lig-
and-binding site can be measured. These were found in those treated with D-P on a
chronic basis, especially rabbits. Therefore, B cells are involved in the immune re-
sponses to both peptide and drug. Although D-P treatment alters the equilibrium
binding of ACh and thus may induce a conformational change, the absence of weak-
ness in rabbits suggests that this alteration does not produce the conformational epi-
tope responsible for experimental myasthenia in rabbits; however, it may in humans.
Nevertheless, we hypothesize that the immunogenicity of this region of the alpha
subunit is potentiated by the presence of D-P covalently bound by a disulfide. This
could alter MHC binding to this epitope or influence T cell responses. There are
precedents for both. Substitution of critical residues in a subdominant peptide from
rat interphotoreceptor retinoid protein with corresponding residues from the defined
bovine immunogenic peptide resulted in increased binding of the substituted peptide
to MHC and increased induction of uveoretinitis.49 In addition, derivatization of
lysozyme with a hapten, phosphorylcholine, converted low antibody responder mice
into high responders by generating more efficient T cell stimulation.50 This may ex-
plain the development of antibodies to AChR as well as experimental myasthenia in
strain A mice.28 A third possibility involves epitope spreading, with the derivatized
peptide serving to initiate additional T helper cell responses to other critical epitopes
that trigger antibody production.51,52 Another possibility would simply involve direct
triggering of B cells by the presence of the hapten, D-P, resulting in binding to B cells
of critical conformational determinants.
In this example of drug-induced autoimmunity, the alteration of a self protein by
PENN et al.: DRUG-INDUCED MG 447
an external influence plays a critical role. We have shown that this sulfhydryl reduc-
ing agent can act at multiple sites in the sequence of initiation of immune responses
and we suspect that different sites are important in different species. Clearly, in hu-
mans of specific genetic backgrounds, D-P also acts to trigger production of patho-
genic antibodies that cause MG, but that will disappear along with clinical MG after
the drug is discontinued. We suspect that very similar immune responses are involved
in triggering MG by captopril and thiopronine. It is probable that derivatization of
other proteins by D-P during therapy results in a similar sequence of immune re-
sponse, although different histocompatibility antigens will bind to different D-P-
modified peptides.
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