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Etiology and mechanisms of drug-induced lupus

Article  in  Current Opinion in Rheumatology · October 1999

DOI: 10.1097/00002281-199909000-00006 · Source: PubMed

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 Toxicology 209 (2005) 135–147

Drug-induced lupus
Robert L. Rubin ∗
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA

Abstract

Autoantibodies and, less commonly, systemic rheumatic symptoms are associated with treatment with numerous medications
and other types of ingested compounds. Distinct syndromes can be distinguished, based on clinical and laboratory features, as
well as exposure history. Drug-induced lupus has been reported as a side-effect of long-term therapy with over 40 medications. Its
clinical and laboratory features are similar to systemic lupus erythematosus, except that patients fully recover after the offending
medication is discontinued. This syndrome differs from typical drug hypersensitivity reactions in that drug-specific T-cells or
antibodies are not involved in induction of autoimmunity, it usually requires many months to years of drug exposure, is drug
dose-dependent and generally does not result in immune sensitization to the drug. Circumstantial evidence strongly suggests
that oxidative metabolites of the parent compound trigger autoimmunity. Several mechanisms for induction of autoimmunity
will be discussed, including bystander activation of autoreactive lymphocytes due to drug-specific immunity or to non-specific
activation of lymphocytes, direct cytotoxicity with release of autoantigens and disruption of central T-cell tolerance. The latter
hypothesis will be supported by a mouse model in which a reactive metabolite of procainamide introduced into the thymus results
in lupus-like autoantibody induction. These findings, as well as evidence for thymic function in drug-induced lupus patients,
support the concept that abnormalities during T-cell selection in the thymus initiate autoimmunity.
© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Autoimmunity; Systemic lupus erythematosus; T cells; Thymus function; Oxidative drug metabolism

1. Overview munity. The vast majority of implicated agents are de-

Exposure to a wide variety of synthetic compounds
has been causally related to the appearance of autoim-
Abbreviations: ANA, anti-nuclear antibody; GVHD, graft-
versus-host disease; IL, interleukin; MHC, major histocompatibil-
ity complex; MPO, myeloperoxidase; NSAID, non-steroidal anti-
inflammatory drugs; PAHA, procainamide-hydroxylamine; RBC,
red blood cells; SLE, systemic lupus erythematosus; TCR, T cell
receptor; TREC, T cell receptor excision circle
∗ Tel.: +1 505 272 4640; fax: +1 505 272 6029.
E-mail address: rlrubin@salud.unm.edu.
liberately ingested medications, and these are associ-
ated most often with development of lupus-like signs
and/or symptoms. However, it is useful to distinguish
bonafide drug-induced lupus from other categories of
xenobiotic-related autoimmune diseases based on the
features of the syndromes and their natural history as
summarized in Table 1 because the underlying disease
mechanisms are probably distinct.
From a clinical perspective drug exposure that is
temporally related to symptoms of systemic lupus ery-
thematosus (SLE) encompasses both drug-induced lu-

0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2004.12.025
136 R.L. Rubin / Toxicology 209 (2005) 135–147
Table 1
Autoimmune diseases due to ingested or injected xenobiotics
Syndrome Main features
Drug-induced lupus Lupus-like (Table 3)
Drug-induced lupus SLE (Table 3)
flares/exacerbation
Autoimmune Anemia and/or
hemolytic anemia (positive Coombs)
Eosinophilia-myalgia Myalgia, etc.
Toxic oil syndrome Myalgia, pleuropulmonary
Cytokine toxicity Musculoskeletal
pus and drug-associated exacerbation of SLE or ini-
tiation of SLE flares. The latter group involves cases
of pre-existing SLE which remain or recur after with-
drawal of the implicated medication, while bonafide
drug-induced lupus usually occurs in the setting of a
previously normal immune system and disappears af-
ter discontinuation of the medication. Careful patient
history is required to distinguish these phenomena, but
their mechanistic significance is probably profoundly
different as discussed below.
Drugs or environmental agents may also be asso-
ciated with other distinct autoimmune diseases such
as hemolytic anemia induced by certain medications,
eosinophilia-myalgia syndrome associated with l-
tryptophan ingestion, toxic oil syndrome associated
with ingestion of aniline-adulterated cooking oil, sil-
icosis associated with inhaled silica or asbestos dust
and autoimmunity associated with vinyl chloride expo-
sure. These syndromes have unique features (reviewed
in Kaufman and Varga, 1999), and their mechanistic
bases should not be confused with that of drug-induced
lupus.
There are also some environmental agents suspected
of causing lupus-like disease based largely on stud-
ies in experimental animals. A lupus-like syndrome
was induced in monkeys by alfalfa sprouts and l-
canavanine (Malinow et al., 1982), and several case
reports of lupus-like disease associated with the inges-
tion of abnormally large amounts of alfalfa seeds or
tablets (Malinow et al., 1981; Roberts and Hayashi,
1983) implicate l-canavanine as capable of inducing
or exacerbating lupus if excessive quantities are in-
gested. The other principal example in this category is a
scleroderma-like autoimmunity associated with heavy
metals in rats and mice (Robinson et al., 1986; Hult-
Causative agents Exposure duration
requirement
Many medications (Table 2) Months to years
Numerous medications (see text) Hours to days
Penicillin, stibophen, quinidine Days to months
(␣-methyldopa and others)
l-Tryptophan contaminants Months
Oleic acid esters of aniline Months
and/or phenylaminopropanol
IL-1, IL-2, interferons, anti-TNF␣ Weeks
man et al., 1989). The major target of mercury-induced
autoantibodies is the nucleolar protein fibrillarin, a spe-
cific serologic marker for a subset of patients with scle-
roderma (Hultman et al., 1989). While these animal
models are of considerable mechanistic interest, these
agents have not been definitely implicated in autoim-
mune disease in humans.
Various immune modulating agents appear to pro-
duce autoimmune features in a minority of treated
patients. Cyclosporine A has been reported to pre-
cipitate graft-versus-host disease (GVHD) after with-
drawal from patients receiving autologous bone mar-
row transplants (Dale et al., 1989; Jones et al., 1989).
Gold therapeutics have been implicated in immune-
mediated disease (Romagnoli et al., 1992), but this is
usually limited to skin reactions or buccal irritation
(Lockie and Smith, 1985) and falls outside the usual
presenting symptoms and clinical progression of SLE
(Wallace, 1993) or drug-induced lupus.
Therapeutic biologics such as interleukin (IL)-2,
interferon-␣, -␤, and -␥, and tumor necrosis factor
(TNF)-␣ inhibitors have been occasionally associated
with a variety of musculoskeletal manifestations and
serological features of lupus-like autoimmunity. Up
to 14% of rheumatoid arthritis patients treated with
anti-TNF␣ developed anti-DNA antibodies, although
these were predominately of the IgM isotype; only one
patient in this prospective study of 156 patients de-
veloped lupus-like symptoms (Charles et al., 2000).
SLE is associated with dysregulation of cellular im-
munity, and TNF␣ protects against lupus nephritis in
a mouse model of SLE (Jacob et al., 1991). It is pos-
sible that therapeutic manipulation of cytokine levels
in individuals predisposed to autoimmunity may dis-
rupt cytokine-mediated immune homeostasis, leading
 R.L. Rubin / Toxicology 209 (2005) 135–147 137

to autoantibody induction and disease. However, drug- Table 2

induced lupus related to small molecule pharmaceuti- Drugs currently in use that can induce lupus-like disease
cals occurs by de novo induction of autoimmunity in Agenta Riskb
the setting of a formerly normal immune system, and Anti-arrhythmics
appears to be related to disruption of the central im- Procainamide (Pronestyl) High
Quinidine (Quinaglute) Moderate
mune tolerance machinery as discussed below. Disopyramide (Norpace) Very low
Propafenone (Rythmol) Very low
Anti-hypertensives
2. Drug-induced lupus Hydralazine (Apresoline) High
Methyldopa (Aldomet) Low
Lupus-like symptoms of muscle and joint pain, fever Captopril (Capoten) Low
Acebutolol (Sectral) Low
and occasionally pleuritis and pericarditis occasionally Enalapril (Vasotec) Very low
develop as a side effect of long-term medication with Clonidine (Catapres) Very low
40 drugs currently in use (Rubin, 1999). Table 2 di- Atenolol (Tenormin) Very low
vides these drugs into therapeutic classes and indicates Labetalol (Normodyne, Trandate) Very low
Pindolol (Visken) Very low
their approximate risk levels based on the number of Minoxidil (Loniten) Very low
reports. In all cases drugs that induce lupus also in- Prazosin (Minipress) Very low
duce autoantibodies in a much higher frequency. By Anti-psychotics
far the highest risk drugs are procainamide and hy- Chlorpromazine (Thorazine) Low
dralazine, with approximately 20% incidence for pro- Phenelzine (Nardil) Very low
cainamide and 5–8% for hydralazine during 1 year of Chlorprothixene (Taractan) Very low
Lithium carbonate (Eskalith) Very low
therapy at currently used doses. The risk for develop-
ing lupus-like disease for the remainder of the drugs is Antibiotics
Isoniazid (INH) Low
much lower, considerably less than 1% of treated pa- Minocycline (Minocin) Low
tients. Quinidine can be considered moderate risk while Nitrofurantoin (Macrodantin) Very low
sulfasalazine, chlorpromazine, penicillamine, methyl- Anti-convulsants
dopa, carbamazepine, acebutalol, isoniazid, captopril, Carbamazepine (Tegretol) Low
propylthiouracil and minocycline are relatively low Phenytoin (Dilantin) Very Low
risk. The remaining 27 drugs should be considered very Trimethadione (Tridone) Very low
Primidone (Mysoline) Very low
low risk based on the paucity of case reports in the lit- Ethosuximide (Zarontin) Very low
erature. Some drugs of very low risk may be falsely
Anti-thyroidals
implicated or are currently of negligible risk because Propylthiouracil (propyl-thyracil) Low
customary treatment doses have been decreased, but
Anti-inflammatories
most reports on drug-induced lupus are convincing be- d-Penicillamine (Cuprimine) Low
cause cessation of therapy usually results in prompt Sulfasalazine (Azulfidine) Low
resolution of symptoms and eventually autoantibodies. Phenylbutazone (Butazolidin) Very low
It should be appreciated that criteria for reaching a di- Diuretics
agnosis of drug-induced lupus-like disease are not as Chlorthalidone (Hygroton) Very low
rigorous as those for diagnosis of SLE. While drug- Hydrochlorothiazide (Diuchlor H) Very low
induced lupus is not a major clinical problem because Miscellaneous
it can be fully cured by discontinuing use of the medi- Anti-tumor necrosis-␣ (Remicade) Very low
Lovastatin (Mevacor) and other statins Very low
cation, it is of mechanistic interest that a low molecular
Levodopa (Dopar) Very low
weight xenobiotic has capacity to initiate and sustain a Aminoglutethimide (Cytadren) Very low
well-defined systemic autoimmune disease. Interferon-␣ (Wellferon) and other cytokines Very low
The clinical and laboratory features of Timolol eye drops (Timoptic) Very low
procainamide- and hydralazine-induced lupus are aCommonly used brand names are enclosed in parentheses.
shown in Table 3 and compared with SLE. The bRisk refers to likelihood for lupus-like disease, not autoantibody
onset of symptoms can be slow or acute, although induction, which is usually much more common.
138 R.L. Rubin / Toxicology 209 (2005) 135–147

Table 3
Prevalence of clinical and laboratory abnormalities in drug-induced and idiopathic lupus
Feature Hydralazine-induced Procainamide-induced Systemic lupus
lupus (%) lupus (%) erythematosus (%)
Symptoms
Arthralgia/arthritis/myalgia 80 85 80
Pleuritis/pleural effusion <5 50 44
Fever/weight loss/fatigue 40–50 45 >80
Hepatosplenomegaly 15 25 5–10
Pericarditis <5 15 20
Rash 25 <5 71
Glomerulonephritis 5–10 <5 42
Neuropsychiatric <5 <5 32
Signs
Anti-nuclear antibodies >95 >95 >95
Lupus erythematosus cells >50 80 71
Anti-histone Abs >95 >95 54
Anti-[(H2A–H2B)–DNA] Ab 43 96 70
Anti-denatured DNA Ab 50–90 50 82
Anti-native DNA Ab <5 <5 28–67
Anti-cardiolipin Ab 5–15 5–20 35
Rheumatoid factor Ab 20 30 25–30
Anemia 35 20 65
Elevated erythrocyte sed. rate 60 60–80 >50
Leukopenia 5–25 15 50
Thrombocytopenia <5 <5 30–50
Elevated gammaglobulins 10–50 25 32
Hypocomplementemia <5 <5 51

For sources see Rubin (2002); Ab = antibody.

an interval of 1–2 months typically passes before the
diagnosis is made. Approximately 50% of patients
have constitutional symptoms of fever, weight loss
and fatigue. Musculoskeletal complaints are also
commonly observed, with arthralgia heading the list
for both drugs. Arthritis is a less common feature
with lupus induced by procainamide (20%) than by
hydralazine (50–100%), whereas serositis (pleuritis
and/or pericarditis) and/or myalgia are more common
presenting features of procainamide-induced lupus.
By contrast, hydralazine-induced lupus is associated
with a higher frequency of skin rashes although these
are not usually malar rashes typical of SLE. Lupus-like
disease associated with quinidine or minocycline is
often atypical in that the former can manifest only as
polyarthalgias (Cohen et al., 1988) and the latter with
symmetrical polyarthritis and evidence of hepatitis
and pneumonitis (Christodoulou et al., 1999). How-
ever, the clinical features of an isolated, individual
case of drug-induced lupus is not characteristic of
a particular drug. The symptoms of drug-induced
lupus usually resolve within days to weeks after
discontinuing the offending drug without introduction
of anti-inflammatory medications; this observation
provides a key (although retrospective) diagnostic tool.
The frequency of serologic abnormalities in lupus
induced by procainamide and hydralazine are essen-
tially identical (Table 3). As with idiopathic SLE the
most commonly observed abnormality is the presence
of anti-nuclear antibodies (ANA), and these autoanti-
bodies usually react with chromatin, the histone–DNA
macromolecular complex in the cell nucleus. Within
this structure the (H2A–H2B)–DNA subnucleosome
particle shows predominant antigenicity (Burlingame
and Rubin, 1996). However, unlike in patients with
SLE, anti-chromatin antibodies rarely react with na-
tive (double-stranded) DNA. Other laboratory features
noted in a minority of patients include a mild anemia,
leukopenia, and hypergammaglobulinemia. Although
the reactivity of drug-induced antibodies is highly re-
stricted, it is neither characteristic of nor dependent on
the particular drug and is similar to the anti-chromatin
 R.L. Rubin / Toxicology 209 (2005) 135–147
autoantibodies that arise spontaneously in the absence
of any drugs (i.e. in SLE). This apparent paradox may
be resolved by the finding that a metabolite of a lupus-
inducing drug has capacity to non-specifically disrupt
central immune tolerance to chromatin (see below),
possibly related to the origin of these autoantibodies
in SLE as well.
3. Drugs that may exacerbate SLE
A report in 1945 is often cited as the first descrip-
tion of drug-induced lupus (Hoffman, 1945). In actual-
ity this patient probably had a hypersensitivity-like re-
action to sulfadiazine associated with exacerbation of
preclinical SLE or with the onset SLE. This and sub-
sequent, similar reports helped to entrench the view
that many cases of idiopathic SLE are “unmasked”
during drug therapy in patients with a lupus diathesis
(Alarcon-Segovia, 1967). This idea is difficult to dis-
count or prove but now appears unlikely. Various drugs
have been noted to have a temporal relationship with
the exacerbation of SLE or with the onset of chronic
SLE prior to diagnosis (Wallace and Dubois, 1987). In
these cases SLE remains after withdrawal of the im-
plicated agent. In case-controlled studies of this phe-
nomenon 12% of SLE patients with drug allergies were
considered to display disease exacerbation, predomi-
nately lupus rash (Petri and Allbritton, 1992; Wang et
al., 1993). Since SLE patients are significantly more
prone to develop drug allergies especially to antibi-
otics such as sulfonamides, penicillin/cephalosporin
and erythromycin (Petri and Allbritton, 1992), these
agents should be avoided in patients with SLE.
A wide variety of drugs have been thought to ex-
acerbate SLE including antibiotics, anti-convulsants,
hormones, non-steroidal anti-inflammatory drugs
(NSAIDs), and dermatologic; particularly impli-
cated are hydralazine, sulfonamides, penicillin, para-
aminosalacylic acid, hydrochlorothiazide, cimetidine,
phenylbutazone and mesantoin. Sulfonamides, tetracy-
clines, griseofulvin, piroxicam, and benoxaprofen are
reported to be photosensitizers of varying frequency;
the rash or dermatitis related to these drugs typically
has a history of rapid onset and behaves as a drug
hypersensitivity-type reaction that may be triggered
by exposure to ultraviolet light. The majority of ad-
verse drug reactions in previously diagnosed SLE pa-
139
tients are of this category (Petri and Allbritton, 1992;
Wang et al., 1993). Another, possibly related category
of patients is those with acute or subacute cutaneous lu-
pus erythematosus related to photoactive medications;
these patients may have systemic disease and may even
fulfill criteria for a diagnosis of SLE (Sontheimer and
Provost, 1997). Some of these drugs are also associ-
ated with typical drug-induced lupus and are included
in Table 2. Drug-induced aseptic meningitis in SLE
patients is occasionally associated with ibuprofen and
other NSAIDs (e.g., sulindac, tolmetin, diclofenac).
Unknown or suspected environmental chemicals such
as hair dyes and permanent wave preparations are also
occasionally implicated as aggravating agents in SLE
and related diseases.
Whether or not an environmental or pharmaceutical
agent might aggravate or unmask incipient SLE should
be considered as a clinical problem distinct from drug-
induced lupus because, by definition, symptoms of the
latter resolve after discontinuation of therapy, although
in severe cases full recovery may require up to 1 year.
If drugs or environmental agents are truly causative in
initiating or aggravating SLE, the mechanistic basis is
probably different from that of drug-induced lupus be-
cause the steady-state blood levels of lupus-inducing
drugs must generally be sustained for many months to
years for development of drug-induced lupus. In con-
trast for most cases believed to be exacerbated SLE,
exposure is of very low level or infrequent when the
suspected agent is environmental or of relatively short
duration when a drug is implicated. The association
between drugs and the onset of SLE resembles the lu-
pus flares following exposure to sunlight, exercise or
pregnancy.
4. Drug-induced immune hemolytic anemia
Long-term therapy with some drugs is associated
with development of hemolytic anemia due to anti-
bodies bound to red blood cells (RBC) in vivo (direct
Coombs test positivity). In the penicillin-type, antibody
to the drug binds to RBC as a result of adsorption of
the drug or its metabolite to the RBC membrane. In the
methyldopa-type, the drug is not required for (and does
not affect) antibody binding, and anti-RBC antibodies
typically have specificity for rhesus locus or other in-
trinsic RBC antigens. These antibodies rarely produce
140 R.L. Rubin / Toxicology 209 (2005) 135–147
frank hemolytic anemia, possibly because their isotype
or low avidity does not support complement fixation.
Hemolytic anemia is commonly associated with the
stibophen-type of drug-induced antibodies (as is quini-
dine and quinine) in which presumably immune com-
plexes consisting of the drug or drug metabolite bind
to RBC (see Aster R, in these proceedings).
The mechanism underlying the penicillin-type of
anti-RBC response is frequently used as the basis for
models for autoantibody elicitation in drug-induced lu-
pus. Interestingly, the autoantibodies associated with
drug-induced lupus behave more like the methyldopa-
type of autoimmune response in that the likelihood
for autoantibody appearance is dose-dependent (rather
than a hypersensitivity-type reaction to even low dose
drug exposure), but the drug is not required for anti-
body binding to its target antigen. In fact many of the
drugs associated with Coombs positivity of this drug-
independent type (methyldopa, l-dopa, mefanamic
acid, procainamide, chlorpromazine and streptomycin)
are also known to cause drug-induced lupus (Table 2),
although there is generally no correlation between pos-
itive Coombs test and other autoantibodies or symp-
toms. However, patients with methyldopa-induced
hemolytic anemia have been reported to have positive
lupus erythematosus cells and ANA. The mechanism
for induction of this type of anti-RBC autoantibody is
unknown. However, despite the drug-independence of
anti-RBC binding, a drug-altered RBC model is com-
monly (inappropriately) invoked, and this model is in-
correctly applied to the origin of autoantibodies asso-
ciated with drug-induced lupus as well.
5. Summary of features of drug-induced lupus
Table 4 compares the overall features of drug-
induced lupus with classical drug hypersensitivity such
Table 4
Comparison between overall features of classical drug hypersensitivity and drug-induced lupus
Drug hypersensitivity reactions
Drug-altered B or T cell epitope, MHC or TCR mediate disease
Rapid recurrence of symptoms upon challenge
Symptoms triggered by low or transient drug exposure
as drug-induced autoimmune hemolytic anemia. Be-
cause the clinical expression, time course and natural
history of drugs that exacerbate SLE are so heteroge-
neous it is not readily possible to place them into either
of these categories, and their pathogenic mechanisms
are probably varied as previously discussed. Also,
cytokine-active biologics that induce asymptomatic au-
toimmunity in patients with an autoimmune diathe-
sis are not included because their underlying disease-
promoting mechanism is presumably a reflection of
their known capacity to disrupt cytokine networks.
Drug-induced lupus does not behave like a classi-
cal drug hypersensitivity reaction in that it lacks drug-
specific T cells or antibodies and the target autoanti-
gens are not directly affected by the inducing drug.
Also, the time course for development of drug-induced
lupus tends to be much slower than that of classical
drug allergies (Table 1), and reintroduction of a lupus-
inducing drug is not associated with memory of prior
exposure if systemic autoimmunity had been allowed
to normalize. However, there are predisposing factors
in drug-induced lupus including slow drug acetylator
phenotype (Perry et al., 1970; Mongey et al., 1999),
HLA-DR4 (Batchelor et al., 1980) or -B47 (Gastineau
et al., 1985), complement protein C4 null allele (Speirs
et al., 1989), female gender (Totoritis et al., 1988) and
Caucasian race (Mongey et al., 1999). Finally, while
drug hypersensitivity can be triggered by relatively low
or transient doses of the inciting agent at least after sen-
sitization, the probability of expressing drug-induced
autoantibodies and symptomatic lupus increases as the
dose and duration of exposure increases (Woosley et
al., 1978). This feature suggests a phenomenon that de-
pends on at least one event of low probability, so that
drug-induced lupus is more likely as time and dose in-
crease. Circumstantial evidence strongly suggests that
this event is metabolic transformation of the drug to a
reactive product.
Drug-induced lupus
Autoantibody binding unaffected by drug; drug-specific T
cells unrelated to autoimmunity
Once systemic autoimmunity has faded, drug reintroduction
restarts the induction period
Strong correlation between development of autoimmunity
and accumulative dose
 R.L. Rubin / Toxicology 209 (2005) 135–147 141

6. Oxidative drug metabolism
The following features of drug-induced autoimmu-
nity are difficult to explain by a direct action of the
ingested, parent compound on some component of the
immune system:
1. Lupus-inducing drugs are highly diverse in chemi-
cal structure and pharmacological action (Table 2),
yet the laboratory and clinical features of lupus in-
duced by all the drugs are essentially the same.
2. Except for their pharmacological action, lupus-
inducing drugs are largely inert at normal doses.
Drug-induced lupus is an idiosyncratic drug reac-
tion not predicted by any known property of the
implicated drugs.
3. Drugs reach a steady-state concentration within a
few hours, but drug-induced autoimmunity and lu-
pus require many months or years of continuous
treatment for manifestation.
The requirement for metabolic transformation of the
ingested drug to a reactive compound would account
for many of the features of drug-induced lupus. In vivo
metabolism of dissimilar drugs to a product with a com-
mon, reactive property could explain how compounds
with widely different pharmacological and chemical
characteristics could produce the same adverse reac-
tion. The low probability for a productive metabolic
event could account for the long lag time for autoim-
munity to unfold.
Phagocytic leukocytes including neutrophils, mono-
cytes, macrophages and skin Langerhans cells have
drug metabolizing capacity due to the presence within
these cells of various enzymes with promiscuous sub-
strate properties such as myeloperoxidase (MPO),
prostaglandin H synthase or, less commonly, the cy-
tochrome P450s. Neutrophils are clearly the greatest
drug-metabolizing engine outside the liver because of
their preponderance in the circulation, apparently un-
limited hematopoietic regenerative capacity, ability to
freely circulate and populate essentially any organ or
tissue including lymphoid tissue where autoimmunity
presumably develops and capacity to generate in re-
sponse to stimulants a robust extracellular oxidizing
machinery. It is this latter feature that is particularly
important because generation of reactive drug metabo-
lites outside a cell sets up a condition for delivering
these agents some distance from their immediate site
of formation.
Essentially all pharmacological classes of lupus-
inducing drugs (Table 2) but not their non-lupus-
inducing analogues have been demonstrated to un-
dergo oxidative metabolism by activated neutrophils
including procainamide, hydralazine, phenytoin, quini-
dine, dapsone, propylthiouracil, penicillamine, chlor-
promazine, isoniazid and carbamazepine. The gen-

Fig. 1. Extracellular transformation of procainamide to procainamide-hydroxylamine by neutrophils. Activation of neutrophils by opsonized

particles or certain soluble factors triggers the ectoenzyme NADPH oxidase to produce superoxide anion (O2 − ) in the extracellular environment.
O2 − spontaneously dismutates to hydrogen peroxide (H2 O2 ). Degranulation often follows, releasing myeloperoxidase. If a drug with an
appropriate functional group is present, it will participate in electron transfer with the H2 O2 –MPO intermediate. Consequently, the functional
group accepts an oxygen atom from H2 O2 , resulting in a new compound.
142 R.L. Rubin / Toxicology 209 (2005) 135–147
eral mechanism responsible for drug transformation is
shown in Fig. 1 using the metabolism of procainamide
to procainamide-hydroxylamine (PAHA) as the proto-
type (Rubin and Curnutte, 1989). Neutrophil-mediated
drug metabolism by this mechanism requires the en-
zymatic action of MPO, as evidenced by the compet-
itive inhibition of MPO activity by all lupus-inducing
drugs tested and the correlation of this property with
neutrophil-dependent drug cytotoxicity (Jiang et al.,
1994). N-acetylation of hydralazine and procainamide
competes with N-oxidation of these drugs, accounting
for the lower probability for development of autoimmu-
nity in people with the rapid acetylation phenotype. Re-
active intermediates of lupus-inducing drugs are strong
candidates for triggering autoimmunity (Rubin, 1994).
7. Mechanisms
7.1. Drugs act as haptens or agonists for
drug-specific T cells
This concept as well as much of the older litera-
ture on drug-induced lupus is based on the presumed
capacity of lupus-inducing drugs or their metabolites
to form stable complexes with self-macromolecules
or to directly stimulate lymphocytes. Autoantibodies
might develop if an immune response to the drug
in the form of a hapten or to a self-antigen altered
by the drug induces antibodies that cross-react with
or cause spreading of the immune response to na-
tive self-macromolecules. A number of lupus-inducing
drugs (hydralazine, chlorpromazine, carbamazepine,
phenylbutazone, and nitrofurantion) or of their oxida-
tive metabolites (procainamide, isoniazid and propy-
lthiouracil) have capacity to cause significant enlarge-
ment of the draining popliteal lymph node when in-
jected subcutaneously into the hind foot pad of mice
(Kammüller et al., 1989; Kubicka-Muranyi et al., 1993;
Goebel et al., 1999). In this popliteal lymph node as-
say (PLNA) T cells apparently respond to drug-altered
self proteins although it is possible that a direct, non-
covalent binding of the drug to the T cell receptor
(TCR) (Engler et al., 2004) underlies responses in some
cases. Since monocytes, macrophages and Langerhans
cells can present antigens to T cells, they have received
special attention as a potential source of both—drug
biotransformation producing drug conjugates as well
as immune presentation of the conjugate to initiate an
immune response (Griem et al., 1998). Autoreactive B
cells within the microenvironment of a drug-specific
T cell response might become activated by cytokine-
mediated bystander mechanisms. However, mice that
developed enlarged lymph nodes in response to lupus-
inducing drugs or their oxidative metabolites failed to
develop autoreactive T cells or autoantibodies, indicat-
ing that drug-specific T cells do not typically lead to
autoimmunity. In addition the autoantibodies that arise
in people with drug-induced lupus or drug-induced cy-
topenias are limited in specificity, a feature not consis-
tent with a bystander activation scenario. However, it
is possible that drug-specific T cells could accelerate
development of autoimmunity if an incipient autoim-
mune response were independently underway.
7.2. Cytotoxic drug metabolites cause pathology
Cytopenias associated with certain drugs may
be related to the capacity of various reactive drug
metabolites to directly cause cell death in a non-
immune-mediated process. Demonstration that oxida-
tive metabolites of many lupus-inducing drugs can kill
under certain in vitro conditions at pharmacologically
relevant concentrations (Rubin et al., 1987; Wheeler et
al., 1988; Adams et al., 1989; Jiang et al., 1994) or en-
hance reactive oxygen species generation by murine
macrophage (Adams et al., 1990) and human neu-
trophils (Adams et al., 1989) is consistent with this
view. Reports implicate apoptosis rather than necrotic
cell death in cytotoxicity mediated by sulfamethoxa-
zole hydroxylamine (Hess et al., 1999) or by the nitre-
nium ion of clozapine (Williams et al., 2000). Extra-
cellular cytotoxic drug metabolites generated by neu-
trophils in vitro (see Fig. 1) have been demonstrated for
amodiaquine, carbamazapine, chlorpromazine, cloza-
pine, hydralazine, isoniazid, procainamide, propylth-
iouracil, quinidine and sulfonamides at therapeutically
feasible concentrations. In addition to direct toxicity,
cells sensitized by drug binding to the cell surface
at sub-toxic concentrations could be destroyed by an
immune-mediated mechanism. Lymphocytes from pa-
tients with a history of agranulocytosis secondary to
clozapine therapy were more sensitive to the cytotoxic
effects of oxidative metabolites of clozapine than nor-
mals or patients who did not develop agranulocytosis
(Gardner et al., 1998; Tschen et al., 1999), suggest-
 R.L. Rubin / Toxicology 209 (2005) 135–147
ing killing was mediated by drug-specific antibody or
cytotoxic T cells.
Cytotoxicity of drug metabolites could be an in-
dependent pathogenic mechanism especially in cer-
tain susceptible populations, but such a process can-
not explain the autoimmune abnormalities in drug-
induced lupus. It is formally possible that drug toxi-
city alters degradation, clearance or processing of self-
materials by antigen presenting cells, producing ab-
normal macromolecular forms or unusual peptides.
These “cryptic” T cell autoepitopes may induce classi-
cal adaptive immune responses because immune tol-
erance to such unusual forms of self-materials was
never established. Autoreactive B cells pre-existing
in the immune repertoire could present such cryp-
tic epitopes to T cells, resulting in B cell activa-
tion and autoantibody secretion. Alternatively, repet-
itive macromolecular structures released from dying
cells could theoretically elicit T-independent autoim-
mune responses. This type of scenario has been pro-
posed to account for autoantibodies associated with di-
verse medications, environmental agents and viruses,
but there remains little in the way of experimental
support.
7.3. Drugs non-specifically activate lymphocytes
Mouse splenocytes exposed to procainamide or hy-
dralazine while activated in vitro displayed an in-
creased proliferative response to autologous antigen
presenting cells without the need for cognate antigen,
killed autologous macrophage and promoted B cell dif-
ferentiation into antibody-secreting cells (Cornacchia
et al., 1988). Autoantibodies and glomerulonephritis
were produced after adoptive transfer of such drug-
treated cells into mice (Yung et al., 1995). The au-
toreactive nature of drug-treated cells was due to the
inhibition by procainamide or hydralazine of DNA
methyltransferase in CD4+ T cells (Yung et al., 1995).
Hypomethylation of promoter sequences is associated
with enhanced gene transcription, and drug-treated
T cells showed increased expression of lymphocyte
function antigen-1 (LFA-1), an important adhesion
molecule that helps stabilize the interaction between
T cells and antigen presenting cells. Longer contact
between the TCR on T cells and the major histocom-
patibility complex (MHC) on antigen presenting cells
may promote T cell activation upon contact with low
143
affinity self-antigens. While the immunopathological
features of this mouse model resemble the more global
autoimmune characteristics of a GVHD (Gleichmann
et al., 1984) or of idiopathic SLE (Table 3) rather than
drug-induced lupus, it is possible that autoreactive T
cells developing through an independent mechanism
become more aggressive due to such a drug-induced
LFA-1 over-expression process.
7.4. Drug metabolites disrupt central immune
tolerance
Because no autoimmune or even hyperimmune ef-
fect had been elicited by treatment of any component of
the peripheral immune system with drug metabolites,
we injected PAHA into the thymus of young adult mice
to tested whether disruption of central T cell tolerance
by a lupus-inducing drug could result in autoimmunity.
Two intrathymic injections with PAHA resulted in a de-
layed but sustained production of IgG anti-chromatin
antibodies (Kretz-Rommel et al., 1997). The autoan-
tibodies in PAHA-treated mice had a specificity re-
markably similar to that of patients with procainamide-
induced lupus in reacting with the (H2A–H2B)–DNA
complex (Table 3), suggesting that the immune system
had undergone similar perturbations in both species. In
the peripheral immune organs of PAHA-treated mice,
chromatin-reactive T cells were detected at a time-point
when anti-chromatin antibodies started to rise, suggest-
ing that PAHA action in the thymus resulted in the ex-
port of autoreactive T cells to the periphery where they
provided T-helper cell function to B cells with poten-
tial to produce autoantibodies. Transfer into naı̈ve mice
of autoreactive peripheral T cells derived from PAHA-
injected animals elicited a similar autoantibody profile,
indicating that autoreactive T cells that emigrated from
the thymus to the periphery accounted for autoantibody
production in this system (Kretz-Rommel and Rubin,
1999) by acting on constitutive B cells with capacity to
secrete anti-chromatin antibodies (Kretz-Rommel and
Rubin, 2001).
PAHA did not reverse self-tolerance of the mature
thymocyte and did not prevent deletion of high affin-
ity autoreactive T cells in the thymus. Instead, PAHA
apparently interfered with the establishment of toler-
ance to endogenous self-antigens that are normally pre-
sented by the MHC on thymic epithelial cells during
the positive selection of thymocytes (Kretz-Rommel
144 R.L. Rubin / Toxicology 209 (2005) 135–147

Fig. 2. Proposed mechanism for PAHA-induced autoimmunity. At the top are depicted three possible developmental fates of CD4+ CD8+
(double positive, immature) T cells in the thymic cortex bearing TCRs differing in affinity for self-antigens. T cells with negligible affinity for
self-peptides + the MHC die by neglect. Engagement of the TCR by self-antigens presented on thymic epithelial cells (TEC) activates signal
transduction pathways, initiating a program of cellular differentiation that includes enhanced expression of the TCR and CD69. Normally,
selected thymocytes acquire non-responsiveness (anergy) to the selecting self-antigens. The presence of a xenobiotic such as PAHA appears to
act at this stage, preventing establishment of anergy. Because some of these cells have low affinity TCRs, they are ignored by the negative selection
machinery that causes the active deletion in the thymic medulla of thymocytes with high affinity for self-antigens. After differentiating into
CD4+ mature T cells and emigrating to the periphery, these non-self-tolerant T cells will encounter chromatin-derived peptides on professional
antigen presenting cells (APC), similar to those that were involved in their positive selection. Because they have a reduce activation threshold
as a consequence of exposure to PAHA during their development, they can respond, resulting in IL-2 production and clonal expansion. Upon
encountering chromatin-specific B cells which concentrate the T cell antigen on their MHC, a T–B immune unit is created, initiating systemic
autoimmunity.

and Rubin, 2000). As a result mature T cells are pro-
duced that are capable of undergoing spontaneous acti-
vation when they encounter similar self-antigens in the
periphery. The implication of this scenario is that the re-
stricted autoantibody production associated with drug-
induced lupus reflects the putative predominance of
pre-T cells undergoing positive selection on chromatin-
derived antigens and that the subsequent establish-
ment of tolerance to these self-antigens is compro-
mised by non-specific toxicity of the lupus-inducing
drug (Fig. 2).
8. Thymus function in drug-induced lupus
It is widely assumed that the thymus is non-
functional in the adult, raising doubts about the rel-
evance of the mouse model of drug-altered central T
cell tolerance. Semi-quantitative measurement of thy-
mus function can be made by determining the content
of TCR rearrangement excision circles (TRECs) in pe-
ripheral blood lymphocytes. TRECs are by-products
of the DNA rearrangement process accompanying
nascent T cell production in the thymus and can be
 R.L. Rubin / Toxicology 209 (2005) 135–147
readily detected in recent thymic emigrants (Douek et
al., 1998). Eventually the TRECs disappear from pe-
ripheral T cells due to cell division or death, so TREC
levels in the periphery correlate with relatively recent
T cell production in the thymus. Studies with normal
donors demonstrated TRECs in patients in their seventh
decade (Jamieson et al., 1999) and along with other
evidence supports the notion that the thymus remains
functional throughout life.
Individuals from 53 to 78 years old and who devel-
oped drug-induced lupus during treatment with pro-
cainamide had in their circulating lymphocyte pool
recent thymic emigrants marked by TRECs, and the
level of these cells was significantly correlated with
anti-chromatin antibody activity (Rubin et al., 2001).
These observations suggest that thymus function is im-
portant in drug-induced lupus and that the intensity of
the anti-chromatin antibody response depends on the
amount of overall thymus function. These results link
the mouse model and human drug-induced lupus, sup-
porting the view that production of autoreactive T cells
in this syndrome is initiated in the thymus by the ca-
pacity of reactive drug metabolites to disrupt central T
cell tolerance.
Acknowledgment
Supported by National Institutes of Health Grant
ES06334.
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