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Impaired delayed hypersensitivity in systemic lupus erythematosus.

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Impaired Delayed Hypersensitivity in Systemic Lupus
David A. Horwitz
When identical twin sisters discordant for systemic lupus erythematosus
(SLE) were immunized with keyhole limpet hemocyanin (KLH) the affected
twin failed to develop delayed hypersensitivity and her lymphocytes failed
to proliferate in response to KLH in vitro, unlike her normal sister. Subsequently, recently diagnosed SLE patients were skin tested with common
antigens and 12 of 14 did not develop a positive skin reaction, in contrast
to most patients with tuberculosis, who showed at least one positive response. There was no difference, however, in the leukocyte response to
phytohemagglutinin between the two groups. These findings raised the
question of a selective defect in cellular immunity in patients with SLE.
Although immune complexes are believed to
trigger vascular inflammatory lesions in systemic lupus erythematosus (SLE) (l), the primary events which lead to the formation of
these pathogenetic complexes are unknown.
Patients with this disease show increased
immunoglobulin metabolism. They characteristically demonstrate numerous autoantibodies
(2), show increased titers of antibodies to a
number of viruses (3) and immunoglobulin
turnover studies indicate increased synthesis of
IgG (4). In contrast, cellular immunity in SLE
patients is poorly understood. We have had the
unique opportunity to study the immune response of identical twin sisters discordant for
SLE. After immunization with keyhole limpet
From the Division of Rheumatic Diseases, Department
of Internal Medicine, University of Virginia School of
Medicine, Charlottesville. Virginia 22901.
DAVID A. HORWITZ, MD. Assistant Professor of Internal Medicine, university of Virginia School of Medicine,
Charlottesville, Virginia. T h e author is a recipient of a
postdoctoral fellowship from the Arthritis Foundation.
This work was supported in part by United States Public
Health Service institutional grants RR05431-10, RR30405 and NSF institutional grant GU 3784.
Reprint requests should be addressed to: Dr. David A.
Submitted for publication Sept 23, 1971; accepted Dec
29, 1971.
hemocyanin (KLH) the affected twin failed to
demonstrate delayed and in vitro hypersensitivity to this antigen when compared with her
normal sister, although both twins demonstrated a normal antibody response. Subsequently, other patients with S L E of recent
onset were also studied. T h e purpose of this
communication is to report impaired hypersensitivity in S L E and the possible implications
of this finding.
Antigens. In these studies the following antigens were
used: candida (Hollister Stier, Yeadon, Pennsylvania), trichophyton (Hollister Stier), streptokinase-streptodornase
(SK-SD) (Varidase, Lederle, Pearl River, New York),
mumps (Eli Lilly, Indianapolis, Indiana) and tuberculin
[purified protein derivative (PPD)] (Park Davis, Detroit,
Michigan). Preservative-free P P D (Lot 974775) for tissue
culture studies was generously supplied by D r H B Devlin
(Parke Davis).
Keyhole limpet hernocyanin was obtained as an ammonium sulfate precipitate from the Pacific Biomarine Supply
Company, Venice, California. This material was prepared
essentially in the associated form by a method modified
from Campbell and co-workers (5).
Patients. At age 51 one of monozygotic twin sisters
developed arthralgias, nephrotic syndrome, positive LE
preps and decreased complement. At age 57, 1%years after
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
her disease had stabilized and previous steroid therapy had
been discontinued, both twins were immunized subcutaneously with 2 mg of KLH. T o date, the other twin sister remains healthy and without antinuclear antibodies. The
white blood count and differential of each were normal at
the time of these studies. Detailed case reports of the twins
will appear elsewhere (6).
Other patients with SLE included 13 females and 1
male, age range 14-69 (median 33). T h e diagnosis was established by the presence of signs and symptoms consistent
with that disease, combined with the presence of antinuclear
antibodies demonstrated by the immunofluorescence technic
using mouse liver as nuclear substrate. Twelve manifested
hypergammaglobulinemia ( > 2 g% gammaglobulin), and
none were azotemic. Nine of these patients were studied
within 3 months after the onset of their presenting complaint. Age- and sex-matched patients with active tuberculosis, hospitalized at the Blue Ridge Sanitarium, served as
Skin Tests. Eleven of 14 SLE patients were skin-tested
prior to prednisone therapy and 3 were receiving 10 mg or
less at the time they were studied. All patients rereived .1
ml intradermal injections of the following antigens: candida
1 :30, trichophyton 1:30, SK-SD 1:40. T h e twins received
KLH 10 pg. Millimeters of erythema and induration were
recorded at 5, 24 and 48 hours. Early (5 hour) reactions
were clearly distinguished from late (48 hour) reactions.
were added and incubated for 6 days. Tritiated thymidine
1 pCi/ml, specific activity 67 Ci/mM, was added 2 hours
prior to termination and the incorporation of isotope
measured by scintillation spectrometry (7). Serum antibody titers to KLH were determined by Dr. John S.
Davis IV using the tanned red cell hernagglutination
technic (8).
Statistical Analysis. Lymphocyte counts were determined from a hemocytometer leukocyte count, 200 cell
differential count and the results from patients with SLE
and tuberculosis compared with the Fisher exact prohabilit y test. T h e Wilcoxon Rank-Sum Test (9) was used to
analyze the radioisotopic data obtained.
Prior to immunization neither twin demonstrated hemagglutinating antibody to K L H and
their leukocytes showed no response to K L H as
measured by the incorporation of 3Hthymidine. Twenty-eight days after immunization, the healthy sister demonstrated a typical
delayed cutaneous response as well as a rise in
antibody titer to KLH. Although antibody to
In Vitro Studies. Control and stimulated leukocyte
cultures containing 5 x lo6 washed blood leukocytes were
prepared in triplicate using previously described technics (7). Fifteen percent heat-inactivated type AB human
serum obtained from a healthy donor was used in all cultures rather than autologous plasma. Phytohemagglutinin
M (Difco, Detroit, Michigan) . I ml was added where indicated and the cultures incubated for 3 and 4 days to determine the peak response of each patient. T o other cultures,
preservative-free P P D 10 pmoles/ml and KLH 15 pg/ml
Table 1. Comparison of Delayed Hypersensitivity
and Serum Antibody Response to KLH 28 Days
After Immunization
Monozygotic twins
Induration 48 hours
after 10 pg KLH
anti body
10 x 1 0 m m
Day of Incubation
Fig 1. Kinetics of 3H-thymidine incorporation by
the twins’ mononuclear leukocytes in resDonse to
KLH 28 days postimmunization.
indicate counts per minute of stimulated and control cultures of normal sister. -- and ... indicate
counts per minute of twin sister. Error bars indicate
the range of triplicate cultures.
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
K L H was also found in the serum of the sister
with SLE, this individual failed to develop a
cutaneous response to this antigen (Table 1).
At this time, the healthy sister’s leukocytes
showed a strong proliferative response to K L H
in contrast to her sister. Figure 1 indicates the
incorporation of 3H-thymidine by each sister’s
leukocytes on successive days in culture. T h e
peak response observed occurred on Day 6. In
these studies, in vitro hypersensitivity correlated with delayed hypersensitivity rather than
the humoral antibody response to KLH.
T h e twins were skin tested with the three test
antigens, as well as mumps and tuberculin. T h e
affected twin failed to show a delayed response
to any antigen while her sister responded to
SK-SD and trichophyton. These studies demonstrate impaired delayed hypersensitivity in
the twin with S L E when compared with her
healthy sister.
Figure 2 illustrates the leukocyte response to
phytohemagglutinin (PHA) on successive days
in culture. Both twins’ leukocytes showed a
proliferative response to PHA. However, the
peak response of the affected twin was approximately 20% less than her normal sister
and appeared 1 day later.
Delayed cutaneous reactivity to antigens
in SLE and tuberculosis. T h e immune defect described in the twin with S L E may have
been present at the onset of her disease or may
be a late manifestation of a chronic disease. In
an attempt to clarify this issue, a group of patients with S L E of recent onset were skin-tested
and their reactivity to common antigens compared with age and sex-matched controls with
tuberculosis. Figure 3 indicates that all normal
individuals manifested s t r o n g delayed skin
reactions to SK-SD. Each normal usually responded to either candida or trichophyton.
Skin reactivity of the group with tuberculosis
was only slightly less than the normal individuals. After intradermal SK-SD, 88% of
the patients with tuberculosis showed 48-hour
skin reactions similar to the controls. In sharp
contrast to these results, 12 of the 14 patients
with S L E did not show a typical delayed response to any of the antigens tested. T h e 2 patients with S L E showing more than 10 mm of
induration to SK-SD were asymptomatic at
the time they were tested. One of these also
reacted to candida.
Figure 4 shows that 7 of 8 patients with S L E
with less than 1200 lymphocytes per cubic millimeter failed to show a positive skin test.
However, 5 of 6 nonlymphopenic patients were
also anergic, which suggests that other factors
besides reduced numbers of lymphocytes contribute to impaired cutaneous reactivity in
Day of lncu bation
Fig 2. Kinetics of 3H-thymidine incorporation by
the twins’ mononuclear leukocytes in response to
PHA. Control cultures yielded less than 100 counts/
m i n per million mononuclear leukocytes.
Mononuclear leukocyte response to
PHA in SLE and tuberculosis. Since P H A
stimulates predominantly thymus-derived
cells (10) and these cells are required in delayed
hypersensitivity (1 l ) , blood leukocytes from
patients with S L E were cultured with P H A and
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
Candido SK-SD
Candida SK-Sd
Candida SK-SD
Day of Incubation
Fig 3. Delayed hypersensitivity t o candida. streptokinase-streptodornase and trichophyton in SLE and matched controls with. tuberculosis. Each point indicates the mean of
two diameters measured perpendicular to each other.
Arthritis and Rheumatism,Vol. 15,No. 4 (July-August 1972)
.c?;: 2.0 0
Fig 4. Peripheral blood lymphocyte counts of
patients with SLE an.d tuberculosis. Bar indicates
median values (P = 0.05). Closed circles indicate
patients with at least one positive skin test. Open
circles indicate nonresponders.
their peak response compared with matched
patients with tuberculosis. All cultures contained serum obtained from a single source,
since inhibitors to PHA may occur in the serum
of patients with many diseases (12). Figure 5
indicates that, unlike the skin test response,
there was no difference in the uptake of 3Hthymidine between the two groups (P<O.1).
T h e present studies suggest a dissociation
between humoral and cellular immunity in
SLE. T h e finding of defective delayed hypersensitivity in one monozygotic twin with
SLE was confirmed by further studies involving
a group of recently diagnosed patients with
SLE. T h e present results parallel the very recent observations of Bitter (13) and Abe and
Homma (1 4) who reported depressed delayed
hypersensitivity to all antigens they tested.
Previously, Block and co-workers skin tested
patients with SLE with five ubiquitous antigens
and noted significant hyporeactivity only to tu-
Fig 5. 3H-thymidine incorporation by mononuclear leukocytes in response t o PHA. Leukocytes
were incubated with PHA 3 and 4 days and the peak
response of patients with SLE and matched controls compared. The horizontal bars indicate
median values ( P > 0.1).
berculin (15). Many of their reported skin
reactions were not typical of delayed responses
peaking at 24 rather than 48 hours. Five-hour
responses were not reported.
T h e mononuclear infiltrate of delayed hypersensitivity results from 1) antigen-stimulated,
thymus-derived lymphocytes that produce
migration inhibitory factors and other lymphokines which 2) results in the accumulation
activation of marrow-derived cells which comprise the majority of cells found in the intradermal lesion (11, 16). In addition, macrophages appear to be necessary for lymphocytes
to respond to antigen (17). Since mononuclear
leukocytes are required for the expression of
this reaction, can the observed cutaneous hyporeactivity be a manifestation of reduced blood
leukocytes? Although lymphopenia was frequently observed in these studies, nonlymphopenic patients also manifested anergy.
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
Other studies from this laboratory indicate that
patients with SLE have normal numbers of
circulating mononuclear phagocytes (18). Apparently other factors besides decreased leukocyte counts must be implicated.
Defective delayed hypersensitivity in diseases
such as Sjogren’s syndrome, sarcoidosis, leprosy
or Hodgkin’s disease is correlated with poor
lymphocyte responsiveness to P H A (1 9-22).
T h e failure to find decreased incorporation of
3H-thymidine by PHA-stimulated lymphocytes
in patients with SLE and anergy was surprising.
Unlike the twin studies, the kinetics of the
P H A response in these patients was not determined. A mild abnormality might not have been
detected. T h e present findings, however, corroborate the results of Patrucco, Rothfield and
Hirschhorn who reported normal leukocyte
transformation following P H A stimulation in
SLE (23). Delbarre and colleagues reported
poor PHA-induced lymphocyte transformation
in patients with active SLE. These results are
difficult to interpret since autologous plasma
was used in many cell cultures (24). Unpublished studies from this laboratory indicate that
SLE plasma will inhibit the proliferative response of normal lymphocytes to PHA.
T h e observation that the SLE twin’s lymphocytes responded to PHA, not antigen, is of
interest. Since P H A stimulates predominantly
thymus-derived cells (10), these findings suggest that these cells are present but unresponsive. T w o instances of lymphocyte malfunction
in the presence of normal P H A responsiveness
have been reported. Greaves, Torrigiani and
Roitt observed that antibodies to immunoglobulin light chain determinants inhibited the
mixed lymphocyte reaction probably by blocking the lymphocyte surface receptor site for antigen (25). Smithwick and Berkovich reported
that measles virus suppressed the lymphocyte to
tuberculin (26). In each of these studies these
factors, either external or internal to the lymphocyte, did not interfere with the stimulatory
effect of PHA.
Finally, a dissociation between humoral and
cellular immunity has been reported in the
genetically transmitted “mouse lupus,” the
New Zealand mouse disease. These mice demonstrate a hyperactive antibody response to a
number of antigens, but their lymphocytes show
an impaired ability to mount a cell-mediated
response (27). It is possible that a defect in cellular immunity may in some way be related to
autoantibody formation and increased immunoglobulin metabolism. In some strains of mice
neonatal thymectomy induced the appearance
of autoantibodies (28), and in one strain thymectomy was followed by the appearance of an
immune complex glomerulonephritis similar
pathologically to the nephritis of SLE (29). It
is possible that the cellular defect in delayed
hypersensitivity described in these experiments
reflects a more fundamental loss of cellular control of the antibody response which eventually
leads to the autoimmune manifestations of SLE.
Further studies are in order to pinpoint the
cellular defect in SLE.
T h e author wishes to thank Dr. ,John S. Davis IV
for the antibody determinations included in this study, Mrs.
Martha Ann Garrett for expert technical assistance and Dr.
Forrest Pitts, Director of the Blue Ridge Sanitarium,
Charlottesville, for making patients available for study. The
clinical studies in this report have been approved by the
Human Experimentation Committee of the University of
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