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Serum activity that confers acid lability to ╨Ю┬▒-interferon in systemic lupus erythematosusits association with disease activity and its independence from circulating ╨Ю┬▒-interferon.

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We conducted a longitudinal evaluation of a
patient with systemic lupus erythematosus who constitutively exhibited elevated levels of circulating ainterferon ( a - I F N ) . This study demonstrated that the
serum levels of an activity that renders the endogenous
a-IFN acid labile are positively correlated with disease
activity. This IFN acid lability-inducing activity can also
be found in the sera of systemic lupus erythematosus
patients who have active disease but who do not have
circulating a-IFN.
Previous reports have documented that up to
50% of patients with systemic lupus erythematosus
(SLE) have abnormally elevated serum levels of the
antiviral cytokine, a-interferon (a-IFN), and that there
is a positive correlation between the clinical activity of
SLE and serum titers of a-IFN (1,2). Because a-IFN
possesses many potent and diverse immunoregulatory
From the Departments of Microbiology and Medicine, New
York University School of Medicine; the Public Health Research
Institute of the City of New York; and the Department of Rheumatic
Diseases, Hospital for Joint Diseases, New York University Medical Center, New York, New York.
Dr. Yee’s work was supported by Medical Scientist Training Program grant 5-T32-GM-07308 from the NIGMS, NIH. Dr.
Yip’s work was supported in part by grant CA-37385 from the NCI,
Arthur M. F. Yee, MS: Department of Microbiology, New
York University School of Medicine; Yum K. Yip, PhD: Department of Microbiology, New York University School of Medicine
and the Public Health Research Institute of the City of New York;
Harry D. Fischer, MD: Hospital for Joint Diseases; Jill P. Buyon,
MD: Department of Medicine, New York University School of
Medicine and Hospital for Joint Diseases.
Address reprint requests to Jill P. Buyon, MD, Department
of Rheumatic Diseases, Hospital for Joint Diseases, 301 East 17th
Street, New York, NY 10003.
Submitted for publication September 11, 1989; accepted in
revised form December 8, 1989.
Arthritis and Rheumatism, Vol. 33, No. 4 (April 1990)
functions, its role in the etiopathogenesis of SLE has
been the subject of great speculation; much attention
has been focused on the observation that serum a-IFN
from SLE patients is apparently acid labile (3). When
this serum a-IFN is dialyzed at pH 2 and then reequilibrated to a neutral pH, its antiviral activity is abolished.
The term “acid labile a-IFN” has been used to
specifically distinguish it from the many purified and
well-characterized subspecies of a-IFN, all of which
are acid stable (4). Accordingly, it has been hypothesized that the physicochemical properties that confer
acid lability to this a-IFN are responsible for biologic
activities that cause or potentiate the autoimmune
state in SLE and other disorders (3). Stimulation of
polyclonal B cell proliferation, inhibition of T suppressor cell functions, and modulation of HLA class I
antigen expression are among some of the many
known biologic activities of conventional a-IFN; aberrations in some of these functions have been proposed for the induction or promotion of autoimmunity
in these diseases by acid labile a-IFN (3). Alternatively, it is also possible that serum acid labile a-IFN
reflects later processes in the evolution of the disease,
for example, as an immunopathologic manifestation or
as a physiologic corrective mechanism.
In a recent report, we established that partially
purified a-IFN from the sera of SLE patients is not
intrinsically acid labile and is, in fact, physicochemically indistinguishable from conventional a-IFN (5).
These dqta indicated that another activity present in
SLE sera is responsible for the apparent acid lability of
endogenous a-IFN. This conclusion is supported by
the observation that leukocytes isolated from SLE
patients whose sera contain acid labile a-IFN are able
Figure 1. Changes over time in the titer and acid lability of serum a-interferon (a-IFN) in a patient with
= residual
systemic lupus erythematosus. U= changes in a-IFN titer in the patient's serum; 0----0
a-IFN activity of,the corresponding samples after dialysis in 100 mM glycine-HCI (pH 2.0) and reequilibration
in phosphate buffered saline (PBS) (pH 7.4); 0 = points at which the serum a-IFN titer was the same after
dialysis in PBS or acid buffer. a-IFN titers are presented on a logarithmic scale. Exacerbations of the
patient's illness are marked by arrows and are numbered accordingly. * = dosages of glucocorticoids
administered to the patient at the time of serum sampling. All dosages signify mg of prednisone per day,
unless indicated by the letter "M," which denotes methylprednisolone.
to secrete only acid stable a-IFN when grown in
culture (4).
The present study was initiated to address the
question of whether any correlation exists between the
serum levels of IFN acid lability-inducing activity and
the clinical course of SLE. Although a change in the
acid lability of serum a-IFN in SLE over time has been
noted (6), to date, there have been no studies investigating the relationship between the acid lability of
serum a-IFN and the severity of disease. During a
I-year period, we measured the titers and acid lability
of a-IFN in serial serum samples from a patient with
SLE and membranoproliferative glomerulonephritis
,who constitutively exhibited elevated levels of serum
a-IFN. In addition, in order to determine whether the
presence of the IFN acid lability-inducing activity is
independent from the presence of circulating a-1FN,
serial serum samples from 7 other SLE patients who
did not exhibit serum a-IFN were evaluated for the
ability ,to confer acid lability to exogenously added
recombinant a-IFN.
Patient population. Sera were obtained from
individuals who fulfilled at least 4 criteria for the
diagnosis of SLE as established by the American
Rheumatism Association (7). One patient, previollsly
reported to have elevated levels of circulating a-IFN
was studied serially during the course of 1 year and
is identified as the index case (Figure 1). Seven additional patients who did not have detectable circulating
IFN were serially evaluated for the presence of IFN
acid lability-inducing activity. Six of these 7 patients
were considered to have had severe disease activity at
some time in the study period, as defined by major
organ system involvement (either renal, hematologic,
and/or neurologic), requiring acute hospitalization and
Table 1. Interferon (IFN) acid lability inducing-activity in 16
serum samples from 7 patients with systemic lupus erythematosus*
IFN titer, IU/ml
With added a,-1FNt
disease activity
Glycine (pH
Serum PBS (pH 7.4)
alone dialysis buffer dialysis buffer
Active (FPGN, anemia)
Active (skin rash)
Active (arthritis)
Active (MPLN)
Active (thrornbocytopenia)
Active (nephrosis)S
Active (cerebral infarcts)
Active (MPLN, skin rash)
* Serial samples are presented chronologically. FPGN = focal
proliferative glomerulonephritis; MPLN = membranoproliferative
lupus nephritis.
1. Exogenous a2-IFN was diluted in the serum samples to a final titer
of 64 IUlml (see Patients and Methods). Samples dialyzed in glycine
buffer were reequilibrated in phosphate buffered saline (PBS) before
being assayed for residual IFN activity. A 4-fold or greater loss of
IFN activity is regarded as significant (4).
$' Renal biopsies were not available.
>60 mg of prednisone per day for control of disease.
Stable clinical status was defined as the absence of
SLE symptoms and resolution of acute organ involvement; in some cases, however, >5 mg of prednisone
per day was required for control of underlying disease.
One patient (patient 2, Table 1) had a flare of disease,
which consisted of severe cutaneous lesions without
major organ system involvement. In all instances, the
primary investigator (AMFY) evaluated samples without knowledge of the disease activity of the patients.
Normal sera were obtained from volunteer donors at
New York University Medical Center.
IFN assays and acid lability tests. Antiviral activity was determined in triplicate in 96-well microtiter
plates by previously described methods, using only the
middle 6 rows of each plate (43). Human foreskin
fibroblasts (strain FS-4) were cultured in the presence
of 2-fold serial dilutions of test samples in minimal
essential medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum. After 24 hours,
the cells were infected with encephalomyocarditis
virus (EMCV) without prior washing of the cells. IFN
titers are defined as the dilutions at which 50% of the
cells are protected from the cytopathic effects of
EMCV 24 hours after infection and are expressed as
IU/ml based on human leukocyte a-IFN #G023-901-527 (National Institutes of Health, Bethesda,
MD). This assay is capable of detecting as little as 4
IUlml of IFN. Inhibition of antiviral activity by antibodies specific for human a-IFN has been previously
described (43).
Samples were tested for acid lability by dialysis
in 100 mM glycine-HC1 (pH 2.0) for 24 hours at 4°C
followed by dialysis in phosphate buffered saline
(PBS; pH 7.4) for another 24 hours (4). Control preparations were dialyzed in PBS throughout the 48-hour
period. A 4-fold or greater loss of IFN activity is
regarded as a significant difference (4). In cases where
exogenous a,-IFN was added to SLE sera, stocks of
a,-IFN were diluted 10-fold in the appropriate serum
samples prior to all dialysis steps, thus ensuring that
the a-IFN was exposed to serum components throughout the entire procedure. Recombinant human a,-IFN
was kindly provided by Dr. Paul Trotta (Schering
Corp., Bloomfield, NJ).
Association between titers of serum a-IFN and
disease activity in SLE. The index patient, a 27year-old black woman, was diagnosed with SLE in
July 1986 at the age of 24. At the time of diagnosis, she
had arthritis, myalgias, fevers, rash, anemia, antibodies to Smith antigen and double-stranded DNA (dsDNA), and depressed values of complement C3, C4,
and CH50; a 24-hour urinary protein excretion level
was 145 mg, with a creatinine clearance of 118 ml/
minute. The patient was started on a regimen of
prednisone, 60 mg/day. During the first year of disease, the patient developed proteinuria, reaching a
maximum level of 1.5 gm/24 hours; treatment with
azathioprine was started at this time. The patient's
condition remained stable, and there was a decrease in
her level of proteinuria. A gradual tapering of the
patient's prednisone dosage to 8 mg/day during the
winter and spring of 1988 was tolerated.
The levels of serum a-IFN were first measured
in this patient in June 1988 and were serially evaluated
through May 1989, as shown in Figure 1 . At all time
points, the patient’s serum IFN activity could be
completely inhibited by polyclonal antibodies specific
for a-IFN (data not shown). The initial evaluation
revealed an elevated serum a-IFN level of 16 IU/ml
(normal C4). In August 1988, the patient became
increasingly leukopenic and anemic and exhibited rising serum titers of antibodies to dsDNA and falling
complement levels. Her serum titer of a-IFN rose to
256 IU/ml. An increase in the prednisone dosage from
8 mg to 40 mg per day was necessary to control the
patient’s disease. Her condition stabilized shortly
thereafter, and her serum a-IFN titer subsequently
declined to 32 IU/ml. The patient’s renal status again
deteriorated as her 24-hour excretion of protein increased to 2 gm. An open renal biopsy performed in
October 1988 revealed membranoproliferative glomerulonephritis. The serum a-IFN titer remained at 32
In February 1989, the patient was hospitalized
with arthritis, fever, dehydration, bilateral pleural
effusions, anemia, increasing titers of anti-dsDNA
antibodies, decreasing levels of C3, C4, and CH50,
and a 24-hour urinary excretion of 3 gm of protein.
White cell casts were observed in the sediment. The
serum a-IFN level rose sharply to a peak value of
1,024 IU/ml. This second period of disease exacerbation necessitated an increase of glucocorticoid dosage
from 9 mg of prednisone per day to 48 mg of methylprednisolone per day. As her condition stabilized
briefly, the serum a-IFN titer decreased to 64 IU/ml,
and the glucocorticoid regimen was changed to 50 mg
of prednisone per day.
The patient was rehospitalized in March 1989
with rapidly progressive glomerulonephritis, cardiomegaly, serositis, and severe anemia. As observed on
the 2 previous occasions of acute disease exacerbation, there was a concomitant rise in her serum a-IFN
level, reaching 512 IU/ml. Intravenous cyclophosphamide was added to her regimen of prednisone, 50 mg/
day. However, despite the treatment with cyclophosphamide, the patient’s condition progressed to endstage renal disease. By May 1989, the patient’s condition had stabilized, with a decline of serum a-IFN
titers to levels comparable with those obtained at the
initiation of the study (16 IU/ml). The prednisone
dosage was tapered to 30 mg/day by the end of the
study period.
Association between the acid lability of serum
a-WN and disease activity in SLE. The acid lability of
the index patient’s serum a-IFN was measured concurrently with the titer. As demonstrated in Figure 1 ,
serum a-IFN, in samples obtained at different time
points during the patient’s disease course, exhibited
varied degrees of acid lability, and more importantly,
there was a positive correlation between disease severity and the acid lability of the a-IFN. Notably, at
the start and at the end of the study period, when the
patient was in relatively stable condition, her serum
a-IFN was not acid labile or was only slightly acid
labile. In contrast, during the 3 periods of severe
disease exacerbation, the acid lability of serum a-IFN
was extreme. For example, during the second flare of
lupus, 1,024 IU/ml of cu-IFN was completely inactivated after acid treatment.
It should also be noted from Figure I that
neither serum a-IFN nor the appearance of the IFN
acid lability-inducing activity appeared to be affected
by increases in the glucocorticoid dosage.
Independence of IFN acid lability-inducing activity from serum a-IFN. In order to determine
whether the presence of IFN acid lability-inducing
activity in SLE serum is independent of the presence
of circulating a-IFN, 16 serum samples from 7 additional patients who did not exhibit any endogenous
a-IFN were studied serially. Sera were collected during both stable and active phases of disease. Recombinant human a,-IFN was diluted 10-fold to a final
concentration of 64 IUlml in these serum samples and
then tested for acid lability. The results are presented
in Table 1. When dialyzed in PBS, the exogenously
added a2-IFN retained complete activity in all test
samples. However, when acid treated, 6 of the 16 SLE
sera, all of which were obtained at a time of disease
exacerbation, had a significant loss of a2-IFNactivity.
None of the samples drawn during a period of stable
disease had any significant reduction of a,-IFN activity after acid treatment. Serum from 1 patient (patient
2) showed no capacity to inactivate a,-IFN at any
time, which is of particular interest because this patient was the only one without major organ involvement during the course of this study. Sera drawn from
7 normal individuals contained neither endogenous
a-IFN nor any IFN acid lability-inducing activity
(data not shown).
Acid labile a-IFN has remained an enigma
because of its peculiarly close association with im-
munoregulatory and rheumatic diseases (4,&10) and
because all of the many well-characterized subspecies
of a-IFN are acid stable. Recently, we elucidated the
nature of acid labile a-IFN in SLE by demonstrating
that acid lability is not an intrinsic physicochemical
property of this a-IFN, but rather the result of another
activity present in the sera of SLE patients (5). In the
present study, we extended these earlier observations
by serial evaluation of the IFN system in a patient with
SLE who constitutively exhibited elevated circulating
levels of a-IFN. In addition, we examined sera from
SLE patients who do not exhibit circulating a-IFN, to
investigate their capacity to render exogenously added
recombinant a,-IFN acid labile.
Consistent with previous reports (1,2), there
was a positive correlation between disease activity and
serum levels of a-IFN in the index patient presented
here. Moreover, the a-IFN never completely disappeared from circulation, even when the patient’s disease was clinically stable. This is intriguing since the
patient’s disease was never in complete remission, and
glucocorticoids were necessary to keep her condition
stabilized at all times. Thus, persistent elevation of
serum a-IFN may accompany insidious disease activity and reflect a poor prognosis.
The severe exacerbations that occurred during
the patient’s clinical course were associated not only
with peaking titers, but also with extreme acid lability
of serum a-IFN. During disease flares, there was as
much as a 1,000-fold loss of a-IFN activity after acid
treatment. In contrast, when the patient’s condition
was stable, her serum a-IFN was generally unaffected
by acid treatment. These findings suggest that there is
an increase in IFN acid lability-inducing activity during periods of lupus flares, and may explain why serum
a-IFN is not acid labile in some reported cases of SLE
(4,6), presumably in those patients who were clinically
stable. We have also shown here that sera from SLE
patients who do not exhibit circulating a-IFN can
confer acid lability to exogenously added recombinant
a,-IFN, demonstrating that the presence of the IFN
acid lability-inducing activity in SLE sera is independent from the presence of circulating a-IFN. The
presence of this activity is also associated with disease
severity in these individuals. The IFN acid labilityinducing activity was clearly associated with periods
of disease exacerbation in the index patient and ‘in 6 of
the 7 additional ‘SLE patients who were studkd. In
contrast, serum a-IFN has been reported to be present
in only 40-50%, of SLE patients (1,4,5). These data
suggest that the IFN acid lability-inducing activity
may be more closely correlated with clinical status
than is the absolute titer of serum a-IFN. Further
studies will be required to determine whether the IFN
acid lability-inducing activity is of useful prognostic
In our attempts to elucidate the mechanism by
which serum a-IFN in SLE is rendered acid labile,
preliminary findings indicate that the inactivation of
a-IFN after acid treatment is effected by the entrapment of IFN molecules within high molecular weight
complexes (manuscript in preparation). Since the experimental conditions used to test acid lability can also
cause the dissociation (i.e., at acid pH) and reassociation (i.e., at neutral pH) of non-covalently linked
protein complexes, it is tempting to conjecture that
acid treatment enables the release of complexed and
sequestered IFN inhibitors, which, upon pH neutralization, can trap free IFN molecules. Although IFN
inhibitors have been reported in the serum of SLE
patients, these were not acid dependent (1 1). Alternatively, acidification and neutralization may facilitate
the entrapment and inhibition of a-IFN by large scavenging proteins in a manner analogous to the inhibition
of various serum cytokines by a,-macroglobulin (12).
The ability of SLE sera, but inability of normal sera, to
confer acid lability to a-IFN may reflect the inappropriate expression or presence of abnormal quantities
of such serum proteins. These possibilities are currently being evaluated.
To our knowledge, this is the first report of an
association between disease severity and acid lability
of serum a-IFN in SLE. However, an analogous
situation does exist in the acquired immunodeficiency
syndrome (AIDS), where an abrupt increase in the
acid lability of serum a-IFN frequently precedes a
rapid deterioration of clinical status (13). Since it is
known that elevated serum a-IFN titers are also
associated with a poor prognosis in AIDS (10,13), it is
an intriguing conclusion that both serum levels of
a-IFN and of the IFN acid lability-inducing activity
are correlated with disease severity. This is consistent
with the observation that AIDS patients with the
highest levels of circulating a-IFN are also those with
the most acid labile a-IFN (10). It will be of interest to
determine if the IFN acid lability-inducing activity in
SLE, AIDS, and other immunoregulatory diseases is
due to the presence of a similar factor (or factors) in
the circulation of these patients.
The apparent parallels in the IFN system in
SLE and AIDS underscore the increasingly recognized similarities in the clinical presentations of the 2
diseases (14,15). Malar rashes, anti-DNA autoantibodies, renal disease, and arthritis are among the many
SLE-associated findings that have also been found in
AIDS patients. It is clear that AIDS can mimic SLE
and other autoimmune diseases, and that a better
understanding of the IFN system and the IFN acid
lability inducing activity in these disorders may provide insight into any common pathogenic pathways.
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