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Immunoglobulin deposits in skin in systemic lupus erythematosus.

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77
IMMUNOGLOBULIN DEPOSITS IN
SKIN IN SYSTEMIC LUPUS
ERYTHEMATOSUS
M. 0. WIERZCHOWIECKI, F. P. QUISMORIO, and G. J. FRIOU
Immunoglobulin deposits in the dermal-epiderma1 junction of clinically normal skin from patients
with SLE were eluted by acid buffer. T h e eluates contained antinuclear and antibasement membrane antibody activities. T h e anti-BM antibody reacted with
skin and esophageal but not glomerular basement
membrane. Enzymatic studies indicated that the antibody reacted with carbohydrate moeities in the basement membrane. The anti-BM antibody was not
present in corresponding sera of SLE patients.
Cutaneous lesions are one of the most common
clinical manifestations of SLE. Studies by light microFrom the Clinical Immunology and Rheumatic Disease
Section, Department of Medicine, University of Southern California School of Medicine.
Supported partly by grants from The Arthritis Foundation and NIH Grant AM 05483-08.
Presented in part in the Annual Meeting of American
Rheumatism Association, Los Angeles, California, June 1973.
Michal Wienchowiecki, MD, was on a Fellowship from
the Senior Fulbright-Hays Program. Present Address: Department of Internal Medicine, Poznan Medical Academy, Poznan,
Poland; Francisco P. Quismorio, MD, Assistant Professor in
Medicine, University of Southern California School of Medicine;
George J. Friou. MD, Professor of Medicine, Head, Clinical Immunology and Rheumatic Disease Section, Department of Medicine, University of Southern California School of Medicine, Los
Angeles, California 90033.
Address reprint requests to F. P. Quismorio, MD, USC
School of Medicine, 2025 Zonal Avenue, Los Angeles, California
90033.
Submitted for publication April 2, 1974; accepted June
20. 1974.
Arthritis and Rheumatism, VoL 18, No. 1 (January-February 1975)
scopy have revealed inflammation, necrosis, fibrin deposition, and eosinophilic bodies (1,2). Immunofluorescent studies have established the presence of
immunoglobulin deposits in the dermal-epidermal
junction, blood vessel walls, and dermal structures
(3-5). Electron-dense deposits have also been identified by electron microscopy at the basement membrane
zone, in walls of blood vessels, or among collagen
fibers in locations similar to immunofluorescent findings (6). The present studies were undertaken to
elaborate further on the nature of immunoglobulin
deposits, employing elution and fluorescent antibody
techniques.
MATERIALS AND METHODS
Fifty-four skin specimens were obtained by punch
biopsy of lesions from 12 patients with SLE, 2 patients
with discoid lupus erythematosus (DLE), 2 drug-induced
SLE, 22 with other diseases including 8 patients with
bullous pemphigoid, 6 with pemphigus vulgaris, a n d 6 patients with rheumatoid arthritis (RA). Clinical diagnosis
was confirmed by established histologic a n d serologic features. All but 1 patient with SLE had acute exacerbation of disease a t the time of study. I n 4 patients with
SLE, clinically normal skin was obtained during autopsy.
Immunofluorescent Studies (IF)
Direct immunofluorescent tests were performed as follows: GP-cryostat sections of skin were air-dried for 60
minutes, washed for 30 minutes with phosphate buffered
saline (PBS),O.OlM, pH 7.0, a n d then stained with mono-
WIERZCHOWIECKI E T AL
78
valent fluorescein isothiocyanate (FITC) conjugated goat
antisera specific for human IgG, IgM, IgA, and C3 (Hyland Laboratories). For blocking experiments, to confirm
specificity of these conjugates corresponding unconjugated
goat antihuman antisera were used.
Indirect immunofluorescent tests using normal human skin, rabbit esophagus, and rat kidney as substrates
were done to detect circulating antibasement membrane
zone (anti-BMZ) antibodies, intercellular antibodies (antiICS) (7), and antinuclear antibodies in human sera and
eluates obtained from skin.
I n studies to determine the relationship between
basement membrane antigens binding immunoglobulins
to skin in SLE and those involved in bullous pemphigoid,
serum from the latter type of patient was used. For this
purpose, sera from 5 patients with bullous pemphigoid,
known to contain antibasement membrane antibody, were
pooled. The gamma globulin fraction was obtained by
ammonium sulfate fractionation and conjugated with FITC
(molar fluorescein/protein ration == 3.6) (8).
Elution from homogenized tissue material. Clinically
normal skin was obtained from the anterior chest and abdomen of 4 patients with SLE and 2 control subjects 12-24
hours after death. T h e specimens were kept frozen at
-70°C until used. They were subjected to elution according
to the method of Koffler et a1 (12) with certain modifications. All procedures .were carried out at 4°C. After the
adipose tissue was removed, 3.5 g of skin were homogenized
in an ice-jacketed VirtisB homogenizer for 2 minutes at
medium speed. The homogenate was washed six times in
cold normal saline and then suspended in 25 ml of 0.02M
citrate buffer, p H 3.2, and stirred for 1 hour at room temperature and an additional 12 hours at 4°C. After centrifugation, the supernatant was dialyzed against multiple
changes of PBS for 72 hours, and finally concentrated to
one-tenth its volume by ultrafiltration with positive pressure through a Diaflow@membrane.
Biochemical Studies
1 . Immunodiffusion studies were carried out in 0.7% agarose in 0.01M PBS, p H 7.0.
2. Indirect IF with substrates of normal human skin, rabbit
esophagus, and rat kidneys with monovalent goat antihuman FITC antisera (Hyland Labs) was performed. To
confirm the specificity of staining, blocking with nonconjugated goat antihuman antisera was used.
3. Indirect IF with rabbit esophagus substrate after treatment with biochemical reagents was carried out as described
above (2).
4. An attempt was made to compare antigenic determinants involved in reaction with lupus skin eluates and
anti-BMZ sera in bullous pemphigoid. Double-layer indirect IF was done with rabbit esophagus, using eluates in
the first incubation step and FITC conjugated antibasement membrane antiserum prepared from bullous pemphigoid sera in the second step.
Biochemical treatment of sections of skin was carried out to characterize further antigens that might be responsible for binding immunoglobulins. Such studies were
carried out on specimens of clinically normal skin obtained
at autopsy from patients who died of SLE. These skin
sections were stained by direct immunofluorescence after
biochemical treatment, to determine whether the procedure removed immunoglobulins from skin sections. In
studies designed to investigate the reaction of immunoglobulins eluted from SLE skin, normal human skin and
rabbit esophagus were treated identically. Thc eluates from
skin (see below) were incubated on treated normal skin and
esophagus, followed by appropriate conjugated antiserum.
In various biochemical treatments, cryostat sections
of skin on slides were incubated with the following different solutions (9,lO):
1. Sodium metaperiodate, 0.01M in PBS, p H 7.0, for 2
hours, and at room temperature for 1 hour.
2. Deoxyribonuclease I, 4 mg% in saline, p H 7.0, containing 0.4mM MgC12, for 1 hour at 37°C.
3. Collagenase, 0.2 mg/ml in TRIS-HCL buffer, p H 7.4,
with 0.01M calcium chloride, at 37’C, for 10 hours.
4. Neuraminidasc Type VI, 0.2 mg/ml in 0.2M acetate
buffer, pH 5, at 37°C for 20 hours.
5. TRIS-HCL buffer, p H 7.4 with 0.01M CaCl, at 37’C,
for 10 hours.
6. Acetate buffer, 0.2M p H 5. at 37”C, for 20 hours.
7. PBS, p H 7.0. 0.01M at 37°C for 20 hours.
Elution Studies
Microelution was attempted with 4p-cryostat sections of skin lesions from 10 patients with SLE and from
3 control subjects, using the procedure described by Feltkamp and Boode (11). Cryostat sections (25 per slide) were
covered with 0.2 ml of 0.02M citrate buffer, pH 3.2, and
incubated in a moist chamber for 12 hours at 4°C. Eluates
were then reconstituted to p H 7.2 by adding 0.4M NaOH.
Studies of Eluates
(from Macroelution Method)
Microscopy
A Leitz Wetzlar fluorescence microscope was equipped for transmitted light with “Osram” HbOW Hg super
pressure lamp and excitation filters, 50mm UGI +50mm
BG38, and for incident light with a xenon lamp and excitation filter 50mm TAL 479 and Barrier filter 17mm
K580. Photographs were taken on Kodak High-speed Ektachrome film 160 ASA.
RESULTS
T h e direct IF studies of skin lesions are summarized in T a b l e 1. In all 12 patients with SLE characteristic immunoglobulin deposits were found i n the
basement membrane zone (BMZ). I n 8 patients, the
pattern of deposits was stippled, while i n 2 a homogeneous a n d i n 1 a mixed pattern was present. Large
immunoglobulin deposits were found along the base-
IMMUNOGLOBULIN DEPOSITS IN SLE SKIN
79
Table 1. Localization of Immunoglobulin Deposits in Skin Lesions by
Direct lmmunofluorescence in Patients with Various Diseases
Fluorescence of Dermis
(Vessels, Elastic Fibers)
Basement Membrane Zone Staining (BMZ)
No.
Tested
SLE
DLE
Drug-induced SLE
Pemphigoid
Pemphigus vulgaris
RA
Other diseases
Normal controls
No.
Positive
IgM
IgG
IgA
C8
IgG
IgM
IgA
C.
12
2
2
8
6
6
12
6
ment membrane in 1 patient. Deposits of the IgG class
were seen in 11 patients, IgM in 9, IgA in 1, and C3
in 3. I n 5 patients, various dermal constituents, including blood vessel walls and elastic fibers, were
stained. In I patient with DLE, BMZ deposits were
seen only in skin lesions. I n 7 of 8 patients with bullous pemphigoid, there was linear staining of BMZ.
I n addition, 3 showed patchy immunoglobulin deposits within the dermis. I n 5 of 6 patients with pemphigus vulgaris the typical intercellular staining (ICS)
pattern was observed. Studies were negative in other
patients, including 2 with drug-induced SLE and normal controls.
Table 2 shows the results of biochemical treatment of cryostat sections of SLE skin lesions on BMZ
staining of deposits by anti-Ig conjugate. Sodium metaperiodate and neuraminidase almost completely abolished staining. There was no significant difference in
the intensity of staining after treatment with other
enzymes.
Eluates obtained from the microelution procedure did not show antibody activity in double diffusion or indirect immunofluorescent studies. Of four
eluates obtained from homogenized skin from patients
Table 2. Eflect of Prior Biochemical Treatment of
Clinically Normal SLE Skin on Im~nunoglobulinDeposits
Seen on Direct Imniunofluorescence
with SLE, three gave precipitin lines with antihuman
IgG and one with anti-IgM. Results of indirect IF
with these eluates are shown in Table 3. I n three
eluates, antinuclear antibodies (ANA) were found and
in two anti-BM antibodies reactive with normal skin,
especially rabbit esophagus, were present. T h e type
of basement membrane staining was linear (Figures
1, 2). There was no BM staining with rat kidney substrate. T h e anti-BM antibodies belonged to the IgG
class in two eluates and to IgM in one. T h e ANA
of eluates were of the IgG class in three, and of the
IGM in two eluates. T w o eluates demonstrated homogeneous and one the shaggy pattern of nuclear staining.
I n the two-step indirect IF, with eluate as the first
step and FITC-conjugated anti-BM antiserum from
bullous pemphigoid serum as second, fluorescence of
rabbit esophagus BM was partially decreased. This
blocking of the reaction of bullous pemphigoid antibody by eluates from SLE skin suggests that some
antigenic determinants are similar for the two. Biuchemical treatment of rabbit esophagus was carried
out before incubation with eluates in indirect I F tests
(Table 4). T h e results showed no staining after sodium
metaperiodate treatment.
N o antibody activity was found in two eluates
from normal controls.
Degree of Fluorescent Staining in Patients
Treatment
Buffered saline
Na metaperiodate
Neuraminidase
Acetate buffer, pH5
Collagenase
Tris-HC1 buffer, pH7
Deoxyribonuclease
1
2
2+
2+
3
4
-
4 f
1+
2f
2 f
2+
2 f
*
+
3+
3+
4+
-
2 f
2f
4+
-
*
-
*
2+
*Destroyed tissue. Fluorescence graded from I+ to 4 f / .
-
*
k
DISCUSSION
We found that immunoglobulin deposits in
clinically normal skin from patients with SLE have
both antinuclear and antibasement membrane antibody activity. Beyvin and Thivolet (13) reported antibasement membrane antibodies in eluates from the
skin of 2 patients with SLE but they did not find
antinuclear antibodies. Recently, Landry and Sams
WIERZCHOWIECKI ET AL
80
Table 3. Antibody Specificity of SLE Skin Eluates Tested by Indirect Immunofluoretcence"
Rabbit Esophagus
(A) SLE Eluate 1
(B) SLE Eluate 2
(C) SLE Eluate 3
(D) SLE Eluate 4
(E) Normal Skin Eluate 1
(F) Normal Skin Eluate 2
BMZt
Nuclei
2+
3+
2+
3+
I+
*
-
Rat Kidney
Human Skin
BMZ
Nuclei
BMZ
Nuclei
-
*Degree of fluorescent staining graded from 1+ to 4+.
tBMZ = basement membrane zone.
(14,15) reported investigations with results similar to
ours. W e have not observed anti-BM antibodies in the
circulation, even in patients with SLE whose skin
eluates gave positive results. I t is possible that these
antibodies are high-affinity, low-titered antibodies,
which are rapidly recovered from the circulation by
binding rapidly to basement membrane.
T h e enzymatic studies indicated that some antigenic determinants to which the SLE anti-BM antibody is reacting appear to be associated with the carbohydrate moiety of the basement membrane. T h e
action of sodium metaperiodate and neurainidase indicated that certain of the antigenic determinants consist of sialic acid-rich glycoprotein. T a n and Kunkel
(9) observed similar results with sodium metaperiodate
treatment of SLE skin lesions. Partial blockade of the
fluorescein-labeled bullous pemphigoid anti-BM antiserum by SLE anti-BM antibody indicated similarities
i n certain antigenic specifities of the two antibodies.
Landry and Sams (14,15) reported similar observations. Furthermore, the SLE anti-BM antibody activity
of eluates, like the pemphigoid anti-BM antibody,
reacts with BM of rabbit esophagus but not with that
from rat kidney. Cross-reactivity with human glomerular basement membrane however was not tested.
Immunochemical studies of isolated basement membrane from various organs of different species have
demonstrated two chemically distinct antigenic components. One is a collagen-like glycoprotein and the
other is a noncollagen glycoprotein (16). Recent studies have shown that the basement membrane reactive
antibody in Goodpasture's syndrome is associated with
a collagen-like glycoprotein (10). We found that collagenase treatment of SLE skin did not release the
bound immunoglobulins. This suggested that the
SLE anti-BM antibody is not reactive with the collagen moeity. However, to establish definitely this
nonreactivity to collagen the effectiveness of the colla-
genase digestion should be confirmed using fluorescein-labeled antihuman collagen antiserum. Experiments o n the treatment of rabbit esophagus with
collagenase prior to reaction with SLE anti-BM antibody were not conclusive because the tissue was
limited by the small amount of antibody available
for study.
T h e significance of the SLE anti-BM antibody
in the pathogenesis of tissue damage is unclear. T h e
passive transfer of bullous pemphigoid plasma containing high titer of anti-BM activity to experimental
animals resulted in in vivo deposition of the antibody
but failed to produce an inflammatory reaction or a
cutaneous lesion (17). On the other hand, Lerner and
associates (18) demonstrated that passive transfer of
IgC with antiglomerular basement membrane activity
resulted in the production of glomerulonephritis in
recipient animals. These observations may reflect
variations i n biologic properties between these different basement membrane reactive antibodies, or the
experimental animals used in the transfusion experiments were not appropriate. I t is also conceivable that
the anti-BM antibody in SLE may appear as an immunologic consequence to cutaneous damage brought
about by exposure to sun or other noxious agent(s).
I n thermal burns, antiepithelial antibodies appear in
the circulation after the tissue injury (7).
Table 4. Eflect of Prior Biochemical Treatment of Rabbit
Esophagus Sections on the Reactiaity of SLE Skin Eluates
with the Basement Membrane Zone (BMZ)
Degree of BMZ Staining*
Phosphate-buffered saline
Na metaperiodate
DNase
Eluate 1
Eluate 2
I f
2 f
I f
2+
-
-
*Staining graded from I + to 4+ with anti-Ig conjugate.
I M M U N O G L O B U L I N DEPOSITS IN SLE SKIN
Fig 1. Reaction of SLE skin eluate with rabbit esophagus
showing antibasenlent membrane antibody activity. Indirect
immunofluorescent test with antihuman gamma globulin conjugate. T h e mucosal layer is in the upper part of the picture
( X 560).
Fig 2. Reaction of SLE skin eluate with rabbit esophagus
showing antinuclear antibody. T h e mucosa is in the upper
portion of the photomicrograph and the basement membrane
in the lower portion ( X 560).
REFERENCES
1. Tuffanelli DL, Dubois EL: Cutaneous manifestations
of systemic lupus erythematosus. Arch Dermatol 90:377,
1964
2. Tuffanelli DL, Kay D, Fukuyama K: Dermal-epidermal
junction in lupus erythematosus. Arch Dermatol 99:
652, 1969
3. Cormane R H : “Bound” globulin in the skin of patients
with chronic discoid lupus erythematosus and systemic
lupus erythematosus. Dermatologica 129:304, 1964
4. Burnham T K , Neblett T R , Fine G: T h e application
of the fluorescent antibody technique to the investigation of lupus erythematosus and various dermatoses.
J Invest Dermatol 43:451, 1963
5. Cormane RH, Szabo E, Hauge LS: Immunofluorescence
of the skin: the interpretation of the staining of blood
vessels and connective tissue aided by new techniques.
Br J Dermatol 82:26, 1970
6. Grishman E, Churg J: Ultrastructure of dermal lesions
81
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in systemic lupus erythematosus. Lab Invest 22: 189,
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Quismorio FP, Bland S, Friou GJ: Autoimmunity in
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Exp Immunol 8:701, 1971
Spendlove RS: Optimal labelling of antibody with
fluorescein isothiocyanate. Proc SOCExp Med 122:380,
1966
T a n EM, Kunkel HG: An immunofluorescent study of
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McIntosh RM, Griswold W: Antigen identification in
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Feltkamp T E W , Boode JH: Elution of antibodies from
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82
tion dermoepidermigne dans le lupus erythemateux
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14. Landry M, Sams WM Jr: Basement-membrane antibodies in two patients with systemic lupus erythematosus. Lancet 1:821, 1972
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Invest 52: 1871, 1973
WIERZCHOWIECKI E T AL
16. Kefalides NA: The chemistry and structure of basement membrane. Arthritis Rheum 12427, 1969
17. Sams WM Jr, Gleich GJ: Failure to transfer bullous
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18. Lerner RA, Glassock RJ, Dixon FJ: The role of antiglomerulobasement membrane antibody in the pathogenesis of human glomerulonephritis. J Exp Med 126:
989, 1967
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