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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

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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

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7.

8.

9.

10. 11.

12.

13.

in systemic lupus erythematosus. Lab Invest 22: 189, 1970 Quismorio FP, Bland S, Friou GJ: Autoimmunity in thermal injury: occurrence of rheumatoid factors, antinuclear antibodies and antiepithelial antibodies. Clin 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 the skin lesions in systemic lupus erythematosus. Arthritis Rheum 9:37, 1966 McIntosh RM, Griswold W: Antigen identification in Goodpasture’s syndrome. Arch Pathol 92392, 197 1 Feltkamp T E W , Boode JH: Elution of antibodies from biopsy tissue. J Clin Pathol 23:629, 1970 Koffler D, Schur, PH, Kunkel HG: Immunological studies concerning the nephritis of systemic lupus erythematosus. J Exp Med 126:606, 1968 Beyvin AJ, lhivolet J: Les globulines fixes a la jonc-

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tion dermoepidermigne dans le lupus erythemateux sont-elles des anticorps anti-membrane basale? Presse Med 79:1070, 1971 14. Landry M, Sams WM Jr: Basement-membrane antibodies in two patients with systemic lupus erythematosus. Lancet 1:821, 1972 15. Landry M, Sams WM Jr: Systemic lupus erythematows: studies of the antibodies bound to skin. J Clin Invest 52: 1871, 1973

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16. Kefalides NA: The chemistry and structure of basement membrane. Arthritis Rheum 12427, 1969 17. Sams WM Jr, Gleich GJ: Failure to transfer bullous pemphigoid with serum from patients. Proc Soc Exp Biol Med 136:1027, 1971 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