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Skin basement membrane immunofluorescence in rheumatoid arthritis.

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653
SKIN BASEMENT MEMBRANE
IMMUNOFLUORESCENCE IN
RHEUMATOID ARTHRITIS
LACK OF DIAGNOSTIC CORRELATION
DANIEL C. DeBOER. ROLAND W. MOSKOWITZ, and B E N 0 MICHEL
Thirty-nine patients with rheumatoid arthritis were
studied for the presence of skin basement membrane immunofluorescence. Punch biopsies from normal sun-exposed skin of the forearm were negative for basement
membrane immunofluorescence in all cases, except one
which was read as questionable. No correlation with serum
antinuclear antibody or lupus erythematosus cells was observed. Skin immunofluorescence studies are helpful in
differential diagnosis when patients with a clinical picture
of rheumatoid arthritis present with serum antinuclear
antibodies and lupus erythematosus cells. Positive basement membrane immunofluorescence is strong evidence of
systemic lupus erythematosus.
Serum antinuclear antibody (ANA) and lupus
erythematosus (LE) cell formation, which are typically
found in systemic lupus erythematosus (SLE), are seen
in other connective tissue diseases such as rheumatoid
arthritis (RA). The diagnostic and prognostic significance of these tests when positive in patients with RA is
unclear.
Demonstration of immunoglobulins at the dermal-epidermal junction of the skin by immunofluores~
From the Department of Medicine, Case Western Reserve
University, Cleveland, Ohio.
Daniel C. DeBoer, M.D.: Resident, Division of Dermatology; Roland W. Moskowitz, M.D.: Professor and Director. Division
of Rheumatic Diseases: Beno Michel, M.D.: Associate Professor,
Division of Dermatology.
Address reprint requests to Roland W. Moskowitz, M.D.,
2073 Abington Road, Cleveland, Ohio 44106.
Submitted for publication January 21, 1976; accepted October 15. 1976.
Arthritis and Rheumatism, Vol. 20, No. 2 (March 1977)
cence (IF) has become a well accepted and valuable test
since described by Burnham and coworkers (1) and
Cormane (2). Subsequent studies in limited numbers of
patients have shown basement membrane IF to occur
only rarely in patients with RA (3-7). Two of these
studies suggested a concurrence between basement
membrane IF and serum antinuclear antibody ( 5 7 ) .
The present study was undertaken t o correlate
basement membrane IF with serum antinuclear antibody and LE cell formation in patients with RA, and
to determine if the IF study would be helpful in differential diagnosis of RA and SLE.
MATERIALS A N D METHODS
Patients
Thirty-nine patients were studied. RA patients with
positive studies for serum antinuclear antibody, L E cells, or
both were primarily selected. Thirty patients (group I ) had
definite or classic R A by American Rheumatism Association
criteria: 9 patients (group 2) had definite or classic RA with LE
cell formation. Population characteristics included 21 black
and 18 white persons, 33 females and 6 males, an age range
from 13 to 79 years, and disease duration of 5 months to 31
years (mean: 13 years). Seventeen of 30 patients in group I ,
and 7 of 9 in group 2 were taking oral prednisone. In most
patients the dosage was 10 mg or less daily. Eleven patients in
group 1 and 3 patients in group 2 were receiving oral hydroxychloroquine. One patient in group 1 was taking azathioprine,
I 0 0 rng/day, and 1 patient cyclophosphamide.
Methods
Diagnostic studies included serum ANA, anti-DNA,
serum rheumatoid factor (RF), erythrocyte sedimentation
DeBOER ET AL
654
rate, WBC, hematocrit, and VDRL. Three-millimeter punch
biopsies were taken from the sun-exposed dorsum of the wrist
of all patients; an additional biopsy was taken from the sunprotected medial upper arm of 1 1 patients.
Skin biopsies were quick-frozen in liquid nitrogen and
sectioned in a cryostat at 5 microns. The dried sections were
washed in phosphate-buffered saline (PBS) at pH 7.2 for 30
minutes, then incubated with fluorescein-conjugated goat antiwhole human immunoglobulin at room temperature in a moisture chamber for 30 minutes. The conjugate’s fluorescein/protein weight ratio was 9.5 pg/mg, and its fluorescein/protein molar ratio was 3.9. Protein-bound FlTC
(fluorescein isocyanate) measured 242 pg/ml, and the total
protein concentration was 25.6 pg/mI. The dilution factor was
1 :8, and the final concentration 1 U/ml.
The sections were washed in PBS for 30 minutes, dried,
and examined under a Leitz microscope with an HB 200 high
pressure mercury lamp as the source of ultraviolet light and
BG12 filter. Previous and ongoing studies in this laboratory
utilizing this technique have demonstrated positive basement
membrane immunofluorescence in skin biopsy specimens from
sun-protected areas in 53% percent of patients with SLE as
defined by the American Rheumatism Association criteria (8).
These results parallel those of other investigators of this disease (3,9).
The antinuclear antibodies were determined in one of
two ways. The first method involved spleen imprints, and the
second, rat liver sections. Fluorescein-conjugated goat antihuman immunoglobulin was the conjugate in both methods.
Studies utilizing spleen imprints were performed with undiluted sera, and the reactions were graded on an intensity scale
of 1 to 4. Rat liver sections were studied in serum dilutions of
1 : 10, I : 100, and 1 : 1000. and the results are reported as the
highest dilution to give a positive reading.
ANA determinations of grade + I or with a titer of
I : 10 were interpreted as weakly positive reactions. ANA of
grade +2 or +3 or a titer of I : 100 was described as moderately
positive. ANA of grade + 4 or a titer of I : 1000 was read as
strongly positive.
Anti-DN A determinations were performed by a hemagglutination technique utilizing DNA (Worthington Biochemical Corp, Freehold, N J ) coupled to formalinized human
0-positive red blood cells. Appropriate positive and negative
serum controls were utilized in all studies. Sera revealing hemagglutination in a titer of 1 :5 or greater were considered
positive.
Table 1. Results oj Serum Serologic Studies and Basement Membrane
Iinmunofluorescence Testing in Two Patient Groups
RA (30)
(Group 1 )
24/30 (80%)
7
15
2
4/12 (33%)
R F*
24/30 (80%)
IFt
0
* RF: serum rheumatoid factor.
t IF: dermal-epidermal immunofluorescence.
ANA
Weak
Moderate
Strong
Anti-DNA
R A with L E cells (9)
(Group 2)
7/9 (78%)
3
3
I
2/4 (50%)
7/9 ( 7 8 7 ~ )
1 (9
The rheumatoid factor (latex fixation) studies were
carried out by Rhein’s modification of the Singer-Plotz test
( 10). Agglutination in a dilution of 1 : 20 or greater was considered positive. Appropriate controls were run simultaneously.
RESULTS
The serum A N A was positive in 24 of 30 patients
with R A a n d n o LE cell formation (group 1 ), a n d in 7 of
9 patients with R A and positive LE cell formation
(group 2) (Table I ) .
Serum anti-DNA studies were positive in 4 of 12
patients with RA, a n d in 2 of 4 patients with R A and LE
-cell formation (Table I ) . T h e titer exceeded 1 : 20 in 1
case.
Rheumatoid factor was present in serum in most
of the patients with R A , whether or not LE cells were
present. There was no correlation between the quantitative titers of serum rheumatoid factor and antinuclear antibody.
Twenty-four patients received prednisone. N o
statistically significant difference in A N A reactivity was
seen when these patients were compared to the nonsteroid-treated group.
Basement membrane immunofluorescence was
questionably present in 1 patient with R A in whom
grade 1 antinuclear antibody activity a n d LE cells were
also seen. I n all other patients it was absent.
DISCUSSION
The inital reports by Burnham, Nebleet, and
Fine in 1963 ( I ) and Cormane in 1964 (2) of basement
membrane immunofluorescence in skin biopsies from
patients with discoid lupus erythematosus and SLE have
been confirmed by other investigators (3,5,6,11-14).
Studies of basement membrane immunofluorescence in patients with R A have shown IF t o be negative,
questionable, weak, or only infrequently positive (3-7).
Limited studies suggested a relationship between serum
antinuclear antibody a n d basement membrane IF (5,7),
a correlation not well studied in most prior reports. The
present study demonstrated absent skin basement membrane IF in a well-defined population of patients with
RA, with or without serum immunologic abnormalities.
Although variable effects of corticosteroid therapy on
basement membrane IF have been reported (3,5,6,15,16),
several studies suggest that these agents, especially at
lower dosage levels, d o not appear to significantly influence the results (3,6,15.16). Fourteen patients were
receiving oral hydroxychloroquine. To the authors’
knowledge the effect of this agent on basement mem-
BASEMENT MEMBRANE IMMUNOFLUORESCENCE
brane I F has not been evaluated. T h e finding of negative basement membrane IF in t h e 25 remaining patients with RA, however, supports a negative correlation
between this diagnostic study and rheumatoid arthritis
per se.
Baldassare and coworkers (17) reported basement membrane immune complex deposits in only 2 of
16 patients with R A when punch biopsies of skin were
tested with fluorescein-conjugated anti-whole human ser u m antibody. Eight of these 16 patients were shown to
have deposits in the dermal-epidermal junction when
studies used fluorescein-conjugated antiserum against
human IgM alone. T h e increased prevalence of basement membrane globulin deposits in studies using antihuman IgM may reflect the effects of higher concentrations of test antibody as compared to studies using antiwhole human immunoglobulin.
It is interesting t o note in this regard that granular deposits of IgM were found in the epidermal basement zone of punch biopsies of the skin taken from the
extensor surface of the forearm of 23 healthy volunteers
in studies reported by de la Faille-Kuyper and colleagues (18). Although their studies suggest that IgM
deposits may be found a s a nonspecific observation in
normal persons, other workers have not noted conspicuous fluorescence of the dermo-epidermal junction
in non-light-exposed skin of healthy individuals (4,17).
Follow-up evaluations of so-called normal persons with
IgM deposits would be of value to exclude eventual
development of immune complex disease.
Gilliam has recently demonstrated D N A binding
to skin connective tissue (19). This phenomenon would
explain localization of anti-DNA antibody o r D N A anti-DNA complexes t o the dermal-epidermal junction
in SLE and the resultant basement membrane immunofluorescence. T h e absence of basement membrane IF in
rheumatoid arthritis, as seen in the present study, would
not be unexpected because circulating anti-DNA o r
DNA-anti-DNA complexes in high titer are not characteristic of this disorder.
T h e present study demonstrates that t h e skin of
patients with rheumatoid arthritis is negative for immunoglobulin deposits at the epidermal-dermal junction in
spite of positive serum antinuclear antibody a n d LE
cells. This result contrasts sharply with t h e high incidence of immunoglobulin deposits in the sun-exposed
skin of patients with systemic lupus erythematosus. T h e
absence of a positive IF band does not exclude SLE.
However, in patients with a clinical diagnosis of rheumatoid arthritis, a positive band test would be strong
evidence in favor of SLE.
655
REFERENCES
I . Burnham TK, Nebleet TR, Fine G: The application of
the fluorescent antibody technique to the investigation of
lupus erythematosus and various dermatoses. J Invest
Dermatol 41:451-456, 1963
2 . Cormane RH: “Bound” globulin in the skin of patients
with chronic discoid lupus erythematosus and systemic
lupus erythematosus. Lancet 1:534-535, 1964
3. Burnham TK, Fine G: The immunofluorescent band test
for lupus erythematosus. 111. Employing clinically normal
skin. Arch Dermatol 103:24-32, 1971
4. Gilliam JN: The significance of cutaneous immunoglobulin deposits in lupus erythematosus and NZB/NZW F,
hybrid mice. J Invest Dermatol 65:154-161, 1975
5. Kalsbeek GL, Cormane RH: The occurrence of immunoglobulins in the dermo-epidermal junction of the skin in
lupus erythematosus and related syndromes. Dermatologica 135:205-215, 1967
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skin in patients with rheumatoid arthritis. Dermatologica
134:351-355, 1967
8. Cohen AS, Reynolds WE, Franklin EC, et al: Preliminary
criteria for the classification of systemic lupus erythematosus. Bull Rheum Dis 21:643-648, 1971
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an tin uclear-antibody-positive diseases. Ann Intern M ed
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1 I. Tan EM, Kunkel HG: An immunofluorescent study of the
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13. Pohle EL, Tuffanelli DL: Study of cutaneous lupus by
immunohistochemical methods. Arch Dermatol 97:520526, 1969
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Venerol (Stockh) 48578-588, 1968
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Dermatol 52:333-338, 1969
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656
17. Baldassare A, Weiss T, Tsai C, et al: Immune complexes in
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Rheum 19:788, 1976 (abstr)
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HB: An immunochemical study of the skin of healthy
DeBOER ET AL
individuals. Acta Derm Venerol (Stockh) 54:271-274,
1974
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tissue as a localizing factor for DNA:anti-DNA complexes. Arthritis Rheum 20:117-118, 1977 (abstr)
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