Archives of
Arch. Derm. Res. 261, 123-134 (1978)
Deermatological searcn 9 Springcr-Verlag 1978
Autoimmunity in Psoriasis* A Compl~mentlmmunofluorescenceStudy* Ernst H. Bdtttner l, Maria Jarzabek-Chorzetska 2, Stefania Jablonska 2, Tadeusz P. Chorzelski ~ and Genowefa Rz~sa 2 Department of Microbiology, Suny at Buffalo, Schools of Medicine and Dentistry, Buffalo, New York 14214, U,g.A. 2 Department of ~etmatology, Warsaw Academy of Medicine, 02-008 Warsaw, Poland
Summary. The stratum corneum (SC) antibodies are present in all human sera as seen by indirect immunofluorescent (IF) staining. They appear to bind in vivo to the stratum corneum of psoriatic lesions. They fix complement in vitro in a two step complement iF test system using either anti C4 or anti C3 conjugates as indicators. IF tests with proper controls showed that the SC antigen in psoriatic scales is coated not only with IgG but in a majority of the lesions also with complement. In the present studies in fully developed lesions complement was detectable m 88% of the specimens studied and m about 50% of very fresh hnear lesions of unintentional K6bner type. These as well as some previously published observations afford indirect evidence for the participation of SC antibodies and the ensuing fixation of complement in the development of psoriatic lesions.
Zusammenfassung. Antik6rper gegen Stratum corneum (SC) befinden sich in Seren aller Menschen und k6nnen mittels der indirekten Immunofluorescenzmethode festgestellt werden. In PSoriasis-Herden scheinen sie in vivo im Stratum corneum fixiert zu sein. Sie fixieren Komplementin vitro, was man durch Komplement-Fixierungs-Tests unter Benutzung von anti C4 oder anti C3 Konjugaten beweisen kann. IF-Teste mit unterschiedliehen Kontrollen haben aufgezeigt, dab Stratum corneum als Antigen in Psoriasis-L~isionen nicht nut von IgG, sondern in der Mehrheit der Ver~inderungen auch von Komplement umgeben ist, In v611ig entwickelten L~isionen wurde das Komplement in 88 % der Biopsien festgestellt, weniger hgufig - in ungef~ihr 50 % - in sehr frfihen linearen Hautver/inderungen vorrl ~l'yp des zufiilligen K6bnerschen Ph~inomens. Diese und friihere Beobachtungell erbrlngen einen indirekten Beweis, dab Antik6tper SC an der Entstehung der Psoriaslsherde teilnehmen, und die Komplementfixierung hier von Bedeutung sein kfnnte. This study was supported by grants from the Summerhill Foundation and from the Polish Academy of Sciences No. 10.5 Offprint requests to: Dr. S. Jablonska (address see above) 0340-3696/78/0261/0123/$ 2.40
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A g r o u p of r e p o r t s o n i m m u n o l o g i c studies of psoriasis using r e d cell tagging [ 1 - 1 0 ] a n d I F [11, 12] m e t h o d s i n d i c a t e that the f o r m a t i o n of psoriatic p l a q u e s involves a n a u t o i m m u n e m e c h a n i s m . E s s e n t i a l l y all n o r m a l adult sera have s t r a t u m c o r n e u m (SC) a n t i b o d i e s a n d these do a p p e a r to fix to the SC a n t i g e n in psoriatic scales in vivo. H o w e v e r , they d o n o t a p p e a r to b i n d in vivo to the h o r n y layer of the n o r m a l skin [ 1 1 - 1 3 ] . Since the SC a u t o a n t i b o d i e s occur in virtually all h u m a n sera they can h a r d l y b e c o n s i d e r e d a b n o r m a l i m m u n e r e s p o n s e s n o r can they b e ascribed to a n u n u s u a l a n t i g e n i c stimulus. W h i l e the n o r m a l bacterial flora has b e e n i m p l i c a t e d as the a n t i g e n i c s t i m u l o u s [4] this r e m a i n s to b e p r o v e n . T h e n o r m a l SC a n t i g e n fails to react with the u n i v e r s a l SC a u t o a n t i b o d i e s a p p a r e n t l y b e c a u s e it is s e q u e s t e r e d b o t h b y its a n a t o m i c l o c a t i o n in a n d a b o v e the s t r a t u m g r a n u l o s u m a n d b y the n o n reactive b i o c h e m i c a l f o r m in which it n o r m a l l y occurs. T h e findings set forth in this p a p e r deal with a n indirect c o m p l e m e n t I F m e t h o d as well as the c o n v e n t i o n a l indirect I F m e t h o d for d e m o n s t r a t i n g SC a n t i b o d i e s in in vitro test systems o n n o r m a l skin, with direct I F tests for in vivo fixation o f I g G a n d c o m p l e m e n t in psoriatic scales a n d with c o m p a r i s o n s of the two for e s t i m a t e s of free SC a n t i g e n . T h e findings suggest a possible p a t h o g e n i c effect of the o b s e r v e d i m m u n o l o g i c events.
Material and Methods IF complement studies were performed in 60 active, fully developed psoriasis cases and in 30 linear fresh lesions of unintentionalK6bner phenomenon type. Controls have been performed on 15 normal human biopsies. Some experiments have been carried out on the skin of 5 monkeys. Skin Biopsy Specimens. Specimens of psoriatic lesions were taken during the active stage of the disease. Normal skin biopsy specimens served a s controls; specimens were screened to select those which gave a suitably strong indirect IF staining reaction. Almost all normal skin specimens tested contained some SC antigen; the few which appeared to be negative had to be rejected as controls. Specimens were either fresh frozen or frozen after being held in Michel's transport medium and washed in the indicated buffer [ 14]. Comparisons between IF reactions with direct frozen skin specimens and specimens held in Michel's transport medium indicated that the SC antigen, the fixed IgG and the fixed complement remained unchanged in biopsy specimens in the meditma [15]. Sera and Conjugates. Fresh normal human sera afforded a source of both SC antibodies and of human complement. Normal human sera were screened to select those which had above average (40-160) titers of SC autoantibodies as detected in a standard indirect IF test system or titrated for complement in a complement IF test for SC antibodies. Labeled antibodies to human IgG and to human complement component C4 and C3 were prepared and characterized as described previously [16,17]. The characteristics of the conjugates were as follows: C-427 Goat antihuman IgG: - Total protein 12 mg/ml, total fluorescein 85.4 ~tg/ml, molar F/P 2.9, antibody 8 units/ml, final dilution for IF staining - I : 32, final antibody concentration 1/4 unit/ml. C-452 Goat antihuman C4 : - Total protein 9.9 mg/ml, total fluorescein 45 ~tg/ml, molar F/P 1.9, antibody 4 units/ml, final dilution for IF staining - 1 : 16, final antibody concentration 1/4 units/ml. C-413 Goat antihuman C3 : - Total protein 11.2 mg/ml, total fluorescein 68.5 ~tg/ml, molar F/P 2.4, antibody 8 units/ml, final dilution for IF staining - 1 : 32, final antibody concentration 1/4 unit/ml. Conjugates absorbed with human sera for nonspecific staining controls were used at the following dilutions: C-427 - 1 : 16, C-452 - 1 : 8, 1 : 8, C-413 - 1 : 16. Serum Studies by Indirect IF Tests. The complement indirect IF staining method for SC antibodies was carried out by allowing complement to fix at the same time that the SC antigen-antibody corn-
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plexes and by treating with labeled anticomplement reagent. Both anti C3 and anti C4 conjugates served to reveal the SC antibody reactions. Sera with SC antibodies were diluted in tris buffer with 000665M CaCI2 and MgC12for complement indirect IF tests. They were selected for studies of biopsies on the basis of their reactivity in chessboard type titrations with the two step complement IF tests. Sera heated to 56~ for 30 min or diluted in a tris buffer with EDTA served as negative controls. Standard indirect IF tests [ 16] for IgG class antibodies were performed in parallel with these complement IF tests using heated sera as positive controls.
Biopsy Studies. The methods used included direct IF tests for in vivo fixation of IgG, C3 and C4, and the comparison of direct and indirect IF staining for IgG and complement. Standard incubation was for 30 rain and washing for 10 rain after serum treatment and for 30 min after conjugate treatment. Comparisons of indirect IF and complement indirect IF staining with fresh and heat inactivated normal sera on normal skin sections serve as a check on the reactivity of the SC antibodies, of the complement and of the conjugates. In IF tests in which all controls give the expected reactions the following determinations were made: a) whether IgG and C4 or C3 have bound to the SC in vivo at the site of the lesions in a pattern comparable to that obtained by indirect IF staining (presumed to be in vivo bound autoantibodies and complement) and b) whether only some or essentially all of the SC antigen sites which are reactive with SC antibodies were saturated with the in vivo bound IgG and C4 by comparisons of indirect and direct IF test reaction patterns. Microscopy and Reading. An American Optical Company model 10 microscope equipped with a darkfield condenser with a toric lens and an HBO 200 mercury vapor light source was used for most of these studies. In some experiments readings were made with both the UV and BV types of illumination as specified in the methods of Kawamura et al. [18], but most experiments were read with only the UV type of illumination using a Schott UG-1 primary of exciter filter and no secondary filters.
Results Table 1 summarizes the experimental format used and Figures 1 and 2 depict the I F reactions obtained respectively with normal skin and psoriasis lesion biopsy specimens. Absorbed anti-IgG conjugate (free of antib'odies) failed t o give positive staining reaction either in normal or psoriatic skin as indicated in the first column of Table 1 and portrayed in Figures l a and 2 a (no serum) and in Figure 2 b (with serum). The same held true of the absorbed anti C4 conjugate (Fig. l e , 2 e and 2f) thus indicating that nonspecific staining (NSS) due to Fc binding did not play a role in the SC staining in these cases. Direct I F staining for IgG and C4 consistently gave negative I F reactions on normal skin as shown in Figures 1 b and 1 f. As had b e e n found in previous studies [12] psoriatic lesions consistently give positive direct IF staining for IgG (Fig. 2c) and usually also for C4 though somethimes only weak or negative reactions occur as in Figure 2 g. In most psoriatic biopsy specimens the direct I F staining for IgG give an irregular linear staining pattern with dots, dashes and commas such as the one shown in Figure 1 c. This psoriatic pattern is easily recognized morphologically. In addition to this pattern some fresh lesions contain homogenously stained cell-like structures such as those shown in Figure 2. On the other hand more advanced psoriatic lesions give more diffuse staining reactions in direct IF tests for IgG, C4, C3 (and other Ig), particularly over dermal papillae. Figure 2c illustrates strongly positive I F staining of diffuse structures and of amorphous masses in the cornified layers. A d j a c e n t to the densely stained areas some psoriatic pattern can also be seen. Indirect IF staining with SC antibodies using both heat inactivated serum (Fig. 1 c) and fresh serum (Fig. 1 d) gave positive staining of the cornified layer. Importantly, complement indirect IF staining gave negative reactions with heated serum (Fig. 1 g) but positive IF staining was obtained
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Fig. 1 a - h . Frozen sections of a normal human skin biopsy specimen. These were treated in the first step either with phosphate buffered saline (PBS) or with SC antibodies in the form of a 1 : 10 dilution of fresh or heated inactivated (56 ~ for 30 rain) normal human sera with a SC antibody titer of 1:40 and in the second step with conjugated anti IgG (a to d) or anti complement component C4 (e to h). These conjugates were either absorbed free of their specific antibodies or were diluted to 1/4 unit/ml. • 250. a Treated with fresh serum with SC antibodies and secondly with absorbed anit IgG conjugate. Note negative IF in horny layer, b Treated with PBS and anti-IgG conjugate with 1/4 unit/ml. Note that this direct IF staining reaction also yields a negative SC reaction on normal skin sections. r Treated with heat inactivated normal serum with SC antibodies and anti-IgG conjugate. Note positive indirect IF staining of the stratum granulosum and corneum, d Treated with fresh normal serum with SC antibodies and anti-lgG conjugate. Note again the positive indirect IF reaction comparable to Figure 1c in the horny layers, e Treated with fresh serum with SC antibodies and absorbed anti C4 conjugate. Note negative IF staining in the SC. f Treated with PBS and anti C4 conjugate diluted to i/4 unit/ml. Note that this direct IF staining procedure also gives a negative SC reaction, g Treated with heat inactivated serum with SC antibodies and anti C4 conjugate as for Figure 1 f. Note that the heated serum gives a negative complement indirect IF reaction in the horny layers, h Treated with fresh normal serum with SC antibodies and anti C4 conjugate as for Figure I f. Note positive IF staining both in the strata granulosum and corneum; this serum had complement fixing antibodies to epidermal nuclei which fail to react in the standard indirect IF test. (See Fig. I d)
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Fig. I e - h
with fresh serum containing SC antibodies (Fig. 1 h). The histologic distribution of SC antigen varies with the type of skin biopsy specimen examined. In most normal skin specimens the "SC antigen" occurs in the granular layer and at least in the lower parts of the cornified layer. This is particularly evident in Figures 1 c and 1 d. The serum used for these studies also contained a complement fixing A N A as may be seen in Figure 1 h. Both conventiox/al indirect IF with an anti-IgG conjugate and complement indirect IF staining with an anti-C4 conjugate gave the expected positive reactions on sections of a psoriatic lesion as depicted respectively in Figures 2d and 2h and indicated in Table 1. The intensity of the psoriatic pattern staining in the direct and indirect IF tests with IgG or complement afford a measure of the amount of free SC antigen. The amount of free SC antigen in biopsies in which the direct and indirect tests yielded a similar intensity and distribution of a psoriatic pattern as "none". This suggests that the SC antibodies have saturated the antigen in vivo or that in vivo complement fixation has gone to completion. Each of the 60 fully developed psoriatic lesions examined had in vivo deposits of IgG in the SC in a typical psoriatic staining pattern. Most (53 of 60) also had
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Fig. 2 a-h. Frozen section of a skin biopsy specimen of a fully developed psoriatic lesion. The cornified layer tends to desquimate both as seen on these sections and in vivo. Section from which the horny layer is lost are not suitable for IF studies of this type. The reagents used for the treatment of these sections are the same as those used for the normal skin sections depicted in Figure 1. That is, either PBS or heat inactivated or fresh normal serum with SC antibodies were used in the first step and either absorbed or unabsorbed anti IgG (Fig. 2 a to d) or anti C4 (Fig. 2 e to h) conjugates served as second step reagents. • 600. a Treated first with PBS and secondly with absorbed anti lgG conjugate. Note negative reaction in the SC. This as welt as the reaction depicted in Figure 2b serve as nonspecific staining (NSS) controls, b Treated first with fresh normal serum and then with absorbed anti IgG conjugate. Note again the negative reaction in horny layer. This as well as the NSS test shown in Figure 2 a rule out Fc binding of conjugates, c Treated first with PBS and then with anti-IgG conjugate diluted to 1/4 unit/ml. Note strongly positive direct IF staining reaction for IgG (DIF-IgG) in two adjacent focal areas, one in the upper and one in the mid stratum corneum region.d Treated with fresh normal serum and secondly with the anti-IgG conjugate for an indirect IF (IIF) test with SC antibodies. Note
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Fig. 2e-h positive IF reaction in two adjacent loci in the SC comparable to the ones shown in Figure 2c. This indicates that the SC antigen sites are either completely or almost completely saturated with IgG presumably due to in vivo bound SC antibodies, e Treated with PBS and absorbed anti C4 conjugate. Note negative NSS reaction in the SC except for a trace staining of scattered particles. These resemble nuclear staining in some NSS tests of this type. f Treated with fresh normal serum and secondly with absorbed anti C4 conjugate. Note again negative reaction except for scatter particle staining. This is a form NSS which can easily be distinguihed from the more linear, "'psoriatic" pattern seen on direct and indirect IF staining, g Treated with PBS first and then with anti C4 conjugate diluted to 1/4 unit/ml for DIF-C4 staining. Note single focus of weakly positive staining on the left side of the SC. About half of the psoriasis lesions examined gave such reactions also upon direct IF staining for C3 or C4. C3 tends to give more positives than C4. h Treated with fresh normal and secondly with anti C4 conjugate for a complement indirect IF (CIIF) staining with SC antibodies. Note positive staining in the horny layer. Since this is significantly stronger than the reaction shown in Figure 2g this specimen was rated as being partly satured with respect to C4 on the SC antigen sites. DIF for complement and CIIF staining of psoriatic scales tend to reveal more cytoid structures which may be inflammatory cells
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Table 1. Direct, indirect and complement IF plus nonspecific staining tests of human stratum corneum First step diluent and human sera
Second step conjugates used and tissues examined
Buffered saline (PBS) + 4% bovine albumin (BSA) Normal serum (NHS) heated with stratum corneum antibodies (SCAb) NHS fresh with SCAb and complement (C)
No antibodies
With antibodies
Absorbed anti-IgG*
Anti IgG 1/4 Unit/m!
Anti C4 1/4 Unit/ml
normal
psoriasis
normal
psoriasis
Absorbed anti C4
- *
-
-
+, + +, + + +
-
+, + +, + + +
(not needed)
(not needed)
+ + +
+ + +
-
+, + +, + + +
- *
-
+ + +
+ + +
+ + +
+ + +
* - = no fluorescence; if IF is positive = nonspecific staining Interpretations: If the indirect tests with IgG and complement (C) are significantly greater than the direct tests with IgG or C (+, + +), then only partial in vivo saturation of SC is reported. If IF in indirect tests with IgG and C appear the same as the direct tests with IgG and C (+ + +), then apparent in vivo saturation of the SC antigen is reported. If IF in indirect tests with IgG or C are positive and in the direct tests is negative, then SC antigen is reported as all free
Table 2. Direct IF findings in psoriatic and normal skin biopsy specimens Character of skin lesions
Fully developed psoriatic lesions Fresh linear psoriatic lesions of K6bner type Normal skin specimens
No. of cases
Direct IF staining reactions
Indirect IF staining
In vivo fixed Complement IgG C3 or C4
with IgG
with complement
+
-
+
-
+
-
+
60
60
0
53
7
60
0
54
6
30
25
5
15
15
30
0
30
0
15
0
15
0
15
14
1
14
1
in v i v o d e p o s i t s of c o m p l e m e n t in a similar p a t t e r n . T h e s e p r e s e n t e d in the m o r e a d v a n c e d lesions w i t h p r o m i n e n t i n f l a m m a t o r y cell staining (Fig. 2 h) and s o m e t i m e s with b a n d s of staining at t h e d e r m a l e p i d e r m a l j u n c t i o n . M o s t , b u t n o t all of K 6 b n e r t y p e lesions y i e l d e d p o s i t i v e direct I F staining ( 2 5 / 3 0 ) I g G ; o n l y half of t h e m had d e m o n s t r a b l e c o m p l e m e n t deposits. Since t h e p o s i t i v e direct I F staining p a t t e r n s f r e q u e n t l y p r e s e n t e d as i s o l a t e d l o c i it m a y well be that t h e n e g a t i v e findings w e r e d u e to s a m p l i n g e r r o r s t h o u g h this r e m a i n s to be d e t e r m i n e d . I n d i r e c t and c o m p l e m e n t i n d i r e c t I F staining tests y i e l d e d p o s i t i v e r e a c t i o n s with all of t h e p s o r i a t i c lesions e x a m i n e d and w i t h all b u t o n e of the n o r m a l skin b i o p s y s p e c i m e n s .
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Discussion The findings of SC antibodies set forth in this report as well as those reported previously [1-13] are of two types, notably those which show that they are true complement fixing autoantibodies [ 1-5,7,10,11] and the observations which indicate that these SC antibodies react in vivo in virtually all typical lesions of psoriasis, in a significant proportion of cases of eczema, and occasionally in other dermatoses [4,6, 8,9, 11,12]. It may be presumed from these findings that the primary events which make it possible for the SC antibodies to react in the SC include not only some break in the dermal-epidermal barrier but also a biochemical change from its normal form which is non-reactive in vivo to an abnormal reactive form. It remains to be determined just what the relative import of these two factors is and what the nature of the biochemical changes may be.
Autoantibody Nature of SC Antibodies and Their Antigen. Since all sera contain SC reactive factor(s) one may question whether they are antibodies. If we define antibodies as immunoglobulins which give specific and sensitive reactions with their homologous antigen(s) in serologic tests, then reported observations on SC antibodies relevant to each of the components of this definition may be summarized as follows: a) Immunoglobulin nature of the SC antibodies is shown by mixed agglutination on tissue sections [8] and by indirect IF tests [ 11] since both methods employ anti-IgG antibodies as indicators. b) The specificity of SC antibodies for the stratum eorneum can be demonstrated morphologically by immune adherence [1], mixed agglutination [8] and by immunofluorescence [11]. The specificity of the SC antibodies for purified soluble SC antigens has been demonstrated by indirect hemggglutination methods [7]. c) The sensitivity of the SC antibodies is demonstrated by the high titers obtained with selected sera. Titers as high as 16,384 have been reported by indirect hemagglutination [7]. Indirect IF titers usually do not exceed 160 [11]. d) At least one SC antigen has been isolated and purified. It is a carbohydrate with an e~ linked glucose determinant [5,10,12]. e) The serologic tests with which these SC antibodies can be demonstrated include not only the above mentioned immune adherence, mixed agglutination, indirect hemagglutination and indirect IF tests but also complement fixation by immune adherence [1] and now also the complement IF system described in this report. These observations serve to identify the SC antibodies as true antibodies. Their in vitro reactivity with horny layers of the skin of antibody producers [3,11] affords documentation of their autoantibody nature.
In vivo Reactivity of SC Antibodies. Mixed agglutination revealed the presence of IgG and rheumatoid factor in psoriatic lesions [8]. Extracts of psoriatic scales contained IgG, IgM, IgA and C3. Acid elution of well washed psoriatic scales yielded SC antibodies, rheumatoid factor and C3 [9]. The initial and present studies on complement IF reveal in psoriatic scales and in certain other dermatoses IgG [ 11,12], IgA, IgM (unpublished) and at least in some cases C3 and C4 deposits in the cornified layers. These appear to be bound in vivo to the site of SC antigen. These various observations point to in vivo reactions of SC antibodies in the carrified layer in psoriasis. Such reactions occur in most if not all cases of psoriasis
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and occasionally in other dermatoses that have been studied thus far [12]. The association of psoriatic staining patterns seen occasionally in other dermatoses with psoriasiform changes in the histologic picture remains to be determined. The passive agglutination studies and other studies of Krogh and his associates [4,6-10] indicate that in vivo reactions of SC antibodies may entail unexpected mechanisms. That is, if the SC antibodies do play a role in psoriasis it may be way of first changes in SC antigens and then increasing permeability of the epidermis to the serum protein. If this be true then the increased passage of SC antibodies and complement, coming from the capillaries and fixing in vivo in a biochemically altered stratum corneum may contribute to the self perpetuating nature of the disease.
Passage of SC Antibodies to the SC. The available evidence indicates that in the normal state amino acids can pass through the epidermis to the granular layer but large protein molecules cannot under normal conditions. The tritium labeling studies of Fukuyama and Epstein show dearly that amino acids not only permeate but are incorporated into proteins in the granular layer [19-21]. However, minor trauma, as well as a variety of dermatoses permit serum proteins to pass through to the cornified layer though they usually fail to fix to the SC antigen sites as is seen in most cases of psoriasis [8,9,11,13]. It can be presumed that in psoriasis the stratum corneum must be changed by some unknown event which makes it an antigenic substrate for the reaction with SC antibodies, and the fixation of complement may induce chemotaxis of leukocytes. It is conceivable that the attraction of leukocytes to the SC, notably the squirting papillae suggested by Pinkus [22,23] may be responsible for the observed in vivo "saturation" of the SC antigen sites with IgG (presumably SC antibodies) and not infrequently also with complement. IF findings tend to support the interpretation of Pinkus and to afford one possible explanation.
Comparisons of SC Antibody and Pemphigus Antibody Reactions The appearance of SC antibodies in all normal sera and the inability of the hidden SC antigen in the normal skin to react in vivo serve to distinguish these antibodies from the rare disease specific antibodies of pemphigus which can react in vivo with accessible antigens in normal skin as indicated in Table 3. That is, studies on the sera and normal skin biopsy specimens of pemphigus patients with intercellular antibodies serve to demonstrate that insofar as IgG molecules can pass beyond the basement lamina in vivo binding can take place in the apparently normal skin [24]. This stands in contrast to the SC antibodies which fail to give demonstrable IF reactions beyond the strict confines of frank psoriatic lesions. Yet, as indicated in Table 3, both are circulating antibodies predominantly of the IgG class and both bind to their respective layers of the skin in vivo in their respective lesions. On the one hand the observed participation of leukocytes in the evolution of psoriatic lesions may be due to the in vivo fixation of complement in psoriatic scales and to the leukotactic factors released as a result of C3 and of C 5 - C 6 reactions in the complement cascade. Since some primary mechanism such as trauma must be responsible for the passage of SC antibodies, complement and other sermn proteins into the horny layer and some biochemical changes must convert the SC antigens
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Autoimmunity in Psoriasis Table
3. Comparisons of pemphigus antibodies with stratum corneum antibodies
Antibodies
Occurrence in sera
Complement fixation
In vivo fixation of IgG
in vitro
in vivo
in lesion
adjacent to lesion
Pemphigus antibodies
pemphigus sera only
not fixed (25)
variable (25-27)
yes (24)
yes (24)
SC antibodies
all human sera
fixed (2 and this report)
variable - positive (this report) - in most active cases
yes (11)
no (I 1)
to a reactive form, it seems probable that the role of the SC antibodies is that of a participating or contributing factor rather than the single causative factor, whereas pemphigus antibodies appear to play a primary role in the pathogenesis of that disease [28,29]. The concepts of a u t o i m m u n i t y of Witebsky [30] and Burnet [31] appear to encompass the relation of pemphigus antibodies to the bullous eruptions they appear to produce. O n the other hand, the relationships observed between SC antibodies and psoriatic lesions can best be explained by concepts of the type set forth by Boyden [32] and more recently by G r a b a r [33] in the form of the "transporteur" hypothesis.
References
1. Krogh, H. K., T6nder, O.: Adherence of erythrocytes to stratum corneum of skin tissue sections. Int. Arch. Allergy 34, 170-180 (1968) 2. Krogh, H. K.: Role of complement in the adherence of erythrocytes to stratum corneum. Int. Arch. Allergy 34, 397-408 (1968) 3. Krogh, H. K.: Antibodies in human sera to stratum corneum. Int. Arch. Allergy 36, 415-426 (1969) 4. Krogb, H. K.: The occurrence of antibodies to stratum corneum in man. Int. Arch. Allergy 37, 649-659 (1970) 5. Krogh, H. K.: The antigen involved in immune adherence with stratum corneum and human serum. Int. Arch. Allergy 38, 78-92 (1970) 6. Krogh, H. K., T6nder, O.: Subcorneal pustular dermatosis. Pathogenetic aspects. Brit. J. Derm. 83, 429-434 (1970) 7. Krogh, H. K., Maeland, J. A., T6nder, O.: Indirect haemaggtutination for demonstration of antibodies to stratum eorneum of skin. Int. Arch. Allergy 42, 493-502 (1972) 8. Krogh, H. K., T6nder, O.: Immunoglobulins and antiimmunoglobulin factors in psoriatic lesions. Clin. Exp. Immunol. 10, 623-634 (1972) 9. Krogh, H. K., T6nder, O.: Antibodies in psoriatic scales. Scand. J. Immunol. 2, 45-51 (1973) 10. Maeland, J. A., Krogh, H. K., T6nder, O.: Heterogeneity of antigen from callus of man. Int. Arch. Allergy 46, 619-628 (1974) 11. Beutner, E. H., Jablonska, S., Jarzabek-Chorzelska, M., Maciejowska, E., Rzesa, G., Chorzelski, T. P.: Studies in immunodermatology. VI. IF studies of autoantibodies to the stratum corneum and of in vivo fixed IgG in stratum corneum of psoriatic scales. Int. Arch. Allergy 48, 301-323 (1975)
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12. Jablonska, S., Chorzelski, T. P., Jarzabek-Chorzelska, M., Beutner, E. H.: Studies in immunodermatology. VII. Four compartment system studies of IgG in stratum corneum and of stratum corneum antigen in biopsies of psoriasis and control dermatoses. Int. Arch. Allergy 48, 324- 340 (1975) 13. Krogh, H. K.: Antibodies to straum corneum in man. In: Immunopathology of the skin: Labeled antibody studies, p. 402-414, E. H. Beutner, T, P. Chorzelski, S. F. Bean, R. E. Jordon (Eds.). Stroudsburg, Pa: Dowden, Hutchinson and Ross 1973 14. Michel, B., Milner, Y., David, K.: Preservation of tissue fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and buIlous diseases. Preliminary report. J. invest. Derm. 59, 449-452 (1973) 15. Hale, W. L., Beutner, E. H.: Comparison of two buffer solutions as transport media for immunofluorescent (IF) studies of epithelial tissue with in vivo bound gamma globulins. ASM Meetings, April 1975 (Abstr.) 16. Beumer, E. H,, Nisengrad, R. J.: Defined immunofluorescence in clinical immunopathology. In: Immunopathology of the skin: Labeled antibody studies, p. 197-247, E. H. Beutner, T. P. Chorzelski, S. F. Bean, R. E. Jordon (Eds.). Stroudsburg, Pa: Dowden, Hutchinson and Ross 1973 17. Diaz Gill, G. A., Beutner, E. H., Eisenberg, R.: Quantitative studies of immunofluorescent staining. VI. Defined complement immunofluorescent staining for antinuclear antibodies in systemic lupus and other collagen diseases. Int. Arch. Allergy 45, 868-879 (1973) 18. Kawamura, A., Mizuoka, K., Matuhasi, T., Fukuoka, Y.: Methods used in Japan for FTA-ABS tests (fluorescent treponemal antibody test). In: Immunopathology of the skin: Labeled antibody studies, p. 424-445, E. H. Beutner, T. P. Chorzelski, S. F. Bean, R. E. Jordon (Eds.). Stroudsburg, Pa: Dowdon, Hutchinson and Ross 1973 19. Fukuyama, K., Epstein, W. L.: Synthesis of RNA and protein during epidermal cell differentiation in man. Arch. Derm. 98, 75-79 (1968) 20. Fukuyama, K., Epstein, W. L.: Comparative autoradiographic study of keratohyalin granules containing cystine and histidine. J. Ultrastr. Res. 51, 314-325 (1975) 21. Fukuyama, K.,Epstein, W.L.: Heterogenous proteins in keratohyalin granules studied by quantitative autoradiography. J. invest. Derm. (In press) 22. Pinkus, H.: Psoriasiform tissue reactions. Aust. J. Derm. 8, 31-35 (1965) 23. Pinkus, H.: Lichenoid tissue reactions. Arch. Derm. 107, 840-846 (1973) 24. Beutner, E. H., Chorzelski, T. P., Jordon, R. E.: Autoimmunity in pemphigus and bullous pemphigoid. Springfield, Ill. : Charles Thomas 1970 25. Jordon, R. E., Sams, W. M., Diaz, G. A., Beutner, E. H.: Negative complement immunofluorescence in pemphigus. J. invest. Derm. 57, 407-410 (1971) 26. Cormane, R. H., Chorzelski, T. P.: "Bound" complement in the epidermis of patients with pemphigus vulgaris. Dermatologica (Basel) 134, 463-466 (1967) 27. van Joost, Th., Cormane, R. H., Pondman, K. W.: Direct immunofluorescent study of the skin on occurrence of complement in pemphigus. Brit. J. Derm. 87, 466-474 (1972) 28. Jarzabek, M.: Passive transfer of pemphigus with human sera. Doctoral Thesis (in Polish), Warsaw, Poland 1970 29. Beutner, E. H., Chorzelski, T. P. : Experimental studies on autosensitization in bullous diseases and on transfer of pemphigus. In: Immunopathology of the skin: Labeled antibody studies, p. 330-353, E. H. Beutner, T. P. Chorzelski, S. F. Bean and R. E. Jordon (Eds.). Stroudsburg, Pa: Dowden, Hutchinson and Ross 1973 30. Witebsky, E.: Ehrlich's side-chain theory in the light of present immunology. Ann. N. Y. Acad. Sci. 59, 168-181 (1954) 31. Burnet, F. M.: The clonal selection theory of aquired immunity. Cambridge U~aiv.Press, London (1959) 32. Boyden, S.: Pathology. Autoimmunity and inflammation. Nature 201, 200-201 (1964) 33. Grabar, P.: Hypothesis. Autoantibodies and immunological theories: An analytical review, Clin, Immun. Immunopath. 4, 453-466 (1975) Received September 27, 1977
Arch. Derm. Res. 261, 123-134 (1978)
Deermatological searcn 9 Springcr-Verlag 1978
Autoimmunity in Psoriasis* A Compl~mentlmmunofluorescenceStudy* Ernst H. Bdtttner l, Maria Jarzabek-Chorzetska 2, Stefania Jablonska 2, Tadeusz P. Chorzelski ~ and Genowefa Rz~sa 2 Department of Microbiology, Suny at Buffalo, Schools of Medicine and Dentistry, Buffalo, New York 14214, U,g.A. 2 Department of ~etmatology, Warsaw Academy of Medicine, 02-008 Warsaw, Poland
Summary. The stratum corneum (SC) antibodies are present in all human sera as seen by indirect immunofluorescent (IF) staining. They appear to bind in vivo to the stratum corneum of psoriatic lesions. They fix complement in vitro in a two step complement iF test system using either anti C4 or anti C3 conjugates as indicators. IF tests with proper controls showed that the SC antigen in psoriatic scales is coated not only with IgG but in a majority of the lesions also with complement. In the present studies in fully developed lesions complement was detectable m 88% of the specimens studied and m about 50% of very fresh hnear lesions of unintentional K6bner type. These as well as some previously published observations afford indirect evidence for the participation of SC antibodies and the ensuing fixation of complement in the development of psoriatic lesions.
Zusammenfassung. Antik6rper gegen Stratum corneum (SC) befinden sich in Seren aller Menschen und k6nnen mittels der indirekten Immunofluorescenzmethode festgestellt werden. In PSoriasis-Herden scheinen sie in vivo im Stratum corneum fixiert zu sein. Sie fixieren Komplementin vitro, was man durch Komplement-Fixierungs-Tests unter Benutzung von anti C4 oder anti C3 Konjugaten beweisen kann. IF-Teste mit unterschiedliehen Kontrollen haben aufgezeigt, dab Stratum corneum als Antigen in Psoriasis-L~isionen nicht nut von IgG, sondern in der Mehrheit der Ver~inderungen auch von Komplement umgeben ist, In v611ig entwickelten L~isionen wurde das Komplement in 88 % der Biopsien festgestellt, weniger hgufig - in ungef~ihr 50 % - in sehr frfihen linearen Hautver/inderungen vorrl ~l'yp des zufiilligen K6bnerschen Ph~inomens. Diese und friihere Beobachtungell erbrlngen einen indirekten Beweis, dab Antik6tper SC an der Entstehung der Psoriaslsherde teilnehmen, und die Komplementfixierung hier von Bedeutung sein kfnnte. This study was supported by grants from the Summerhill Foundation and from the Polish Academy of Sciences No. 10.5 Offprint requests to: Dr. S. Jablonska (address see above) 0340-3696/78/0261/0123/$ 2.40
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A g r o u p of r e p o r t s o n i m m u n o l o g i c studies of psoriasis using r e d cell tagging [ 1 - 1 0 ] a n d I F [11, 12] m e t h o d s i n d i c a t e that the f o r m a t i o n of psoriatic p l a q u e s involves a n a u t o i m m u n e m e c h a n i s m . E s s e n t i a l l y all n o r m a l adult sera have s t r a t u m c o r n e u m (SC) a n t i b o d i e s a n d these do a p p e a r to fix to the SC a n t i g e n in psoriatic scales in vivo. H o w e v e r , they d o n o t a p p e a r to b i n d in vivo to the h o r n y layer of the n o r m a l skin [ 1 1 - 1 3 ] . Since the SC a u t o a n t i b o d i e s occur in virtually all h u m a n sera they can h a r d l y b e c o n s i d e r e d a b n o r m a l i m m u n e r e s p o n s e s n o r can they b e ascribed to a n u n u s u a l a n t i g e n i c stimulus. W h i l e the n o r m a l bacterial flora has b e e n i m p l i c a t e d as the a n t i g e n i c s t i m u l o u s [4] this r e m a i n s to b e p r o v e n . T h e n o r m a l SC a n t i g e n fails to react with the u n i v e r s a l SC a u t o a n t i b o d i e s a p p a r e n t l y b e c a u s e it is s e q u e s t e r e d b o t h b y its a n a t o m i c l o c a t i o n in a n d a b o v e the s t r a t u m g r a n u l o s u m a n d b y the n o n reactive b i o c h e m i c a l f o r m in which it n o r m a l l y occurs. T h e findings set forth in this p a p e r deal with a n indirect c o m p l e m e n t I F m e t h o d as well as the c o n v e n t i o n a l indirect I F m e t h o d for d e m o n s t r a t i n g SC a n t i b o d i e s in in vitro test systems o n n o r m a l skin, with direct I F tests for in vivo fixation o f I g G a n d c o m p l e m e n t in psoriatic scales a n d with c o m p a r i s o n s of the two for e s t i m a t e s of free SC a n t i g e n . T h e findings suggest a possible p a t h o g e n i c effect of the o b s e r v e d i m m u n o l o g i c events.
Material and Methods IF complement studies were performed in 60 active, fully developed psoriasis cases and in 30 linear fresh lesions of unintentionalK6bner phenomenon type. Controls have been performed on 15 normal human biopsies. Some experiments have been carried out on the skin of 5 monkeys. Skin Biopsy Specimens. Specimens of psoriatic lesions were taken during the active stage of the disease. Normal skin biopsy specimens served a s controls; specimens were screened to select those which gave a suitably strong indirect IF staining reaction. Almost all normal skin specimens tested contained some SC antigen; the few which appeared to be negative had to be rejected as controls. Specimens were either fresh frozen or frozen after being held in Michel's transport medium and washed in the indicated buffer [ 14]. Comparisons between IF reactions with direct frozen skin specimens and specimens held in Michel's transport medium indicated that the SC antigen, the fixed IgG and the fixed complement remained unchanged in biopsy specimens in the meditma [15]. Sera and Conjugates. Fresh normal human sera afforded a source of both SC antibodies and of human complement. Normal human sera were screened to select those which had above average (40-160) titers of SC autoantibodies as detected in a standard indirect IF test system or titrated for complement in a complement IF test for SC antibodies. Labeled antibodies to human IgG and to human complement component C4 and C3 were prepared and characterized as described previously [16,17]. The characteristics of the conjugates were as follows: C-427 Goat antihuman IgG: - Total protein 12 mg/ml, total fluorescein 85.4 ~tg/ml, molar F/P 2.9, antibody 8 units/ml, final dilution for IF staining - I : 32, final antibody concentration 1/4 unit/ml. C-452 Goat antihuman C4 : - Total protein 9.9 mg/ml, total fluorescein 45 ~tg/ml, molar F/P 1.9, antibody 4 units/ml, final dilution for IF staining - 1 : 16, final antibody concentration 1/4 units/ml. C-413 Goat antihuman C3 : - Total protein 11.2 mg/ml, total fluorescein 68.5 ~tg/ml, molar F/P 2.4, antibody 8 units/ml, final dilution for IF staining - 1 : 32, final antibody concentration 1/4 unit/ml. Conjugates absorbed with human sera for nonspecific staining controls were used at the following dilutions: C-427 - 1 : 16, C-452 - 1 : 8, 1 : 8, C-413 - 1 : 16. Serum Studies by Indirect IF Tests. The complement indirect IF staining method for SC antibodies was carried out by allowing complement to fix at the same time that the SC antigen-antibody corn-
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plexes and by treating with labeled anticomplement reagent. Both anti C3 and anti C4 conjugates served to reveal the SC antibody reactions. Sera with SC antibodies were diluted in tris buffer with 000665M CaCI2 and MgC12for complement indirect IF tests. They were selected for studies of biopsies on the basis of their reactivity in chessboard type titrations with the two step complement IF tests. Sera heated to 56~ for 30 min or diluted in a tris buffer with EDTA served as negative controls. Standard indirect IF tests [ 16] for IgG class antibodies were performed in parallel with these complement IF tests using heated sera as positive controls.
Biopsy Studies. The methods used included direct IF tests for in vivo fixation of IgG, C3 and C4, and the comparison of direct and indirect IF staining for IgG and complement. Standard incubation was for 30 rain and washing for 10 rain after serum treatment and for 30 min after conjugate treatment. Comparisons of indirect IF and complement indirect IF staining with fresh and heat inactivated normal sera on normal skin sections serve as a check on the reactivity of the SC antibodies, of the complement and of the conjugates. In IF tests in which all controls give the expected reactions the following determinations were made: a) whether IgG and C4 or C3 have bound to the SC in vivo at the site of the lesions in a pattern comparable to that obtained by indirect IF staining (presumed to be in vivo bound autoantibodies and complement) and b) whether only some or essentially all of the SC antigen sites which are reactive with SC antibodies were saturated with the in vivo bound IgG and C4 by comparisons of indirect and direct IF test reaction patterns. Microscopy and Reading. An American Optical Company model 10 microscope equipped with a darkfield condenser with a toric lens and an HBO 200 mercury vapor light source was used for most of these studies. In some experiments readings were made with both the UV and BV types of illumination as specified in the methods of Kawamura et al. [18], but most experiments were read with only the UV type of illumination using a Schott UG-1 primary of exciter filter and no secondary filters.
Results Table 1 summarizes the experimental format used and Figures 1 and 2 depict the I F reactions obtained respectively with normal skin and psoriasis lesion biopsy specimens. Absorbed anti-IgG conjugate (free of antib'odies) failed t o give positive staining reaction either in normal or psoriatic skin as indicated in the first column of Table 1 and portrayed in Figures l a and 2 a (no serum) and in Figure 2 b (with serum). The same held true of the absorbed anti C4 conjugate (Fig. l e , 2 e and 2f) thus indicating that nonspecific staining (NSS) due to Fc binding did not play a role in the SC staining in these cases. Direct I F staining for IgG and C4 consistently gave negative I F reactions on normal skin as shown in Figures 1 b and 1 f. As had b e e n found in previous studies [12] psoriatic lesions consistently give positive direct IF staining for IgG (Fig. 2c) and usually also for C4 though somethimes only weak or negative reactions occur as in Figure 2 g. In most psoriatic biopsy specimens the direct I F staining for IgG give an irregular linear staining pattern with dots, dashes and commas such as the one shown in Figure 1 c. This psoriatic pattern is easily recognized morphologically. In addition to this pattern some fresh lesions contain homogenously stained cell-like structures such as those shown in Figure 2. On the other hand more advanced psoriatic lesions give more diffuse staining reactions in direct IF tests for IgG, C4, C3 (and other Ig), particularly over dermal papillae. Figure 2c illustrates strongly positive I F staining of diffuse structures and of amorphous masses in the cornified layers. A d j a c e n t to the densely stained areas some psoriatic pattern can also be seen. Indirect IF staining with SC antibodies using both heat inactivated serum (Fig. 1 c) and fresh serum (Fig. 1 d) gave positive staining of the cornified layer. Importantly, complement indirect IF staining gave negative reactions with heated serum (Fig. 1 g) but positive IF staining was obtained
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Fig. 1 a - h . Frozen sections of a normal human skin biopsy specimen. These were treated in the first step either with phosphate buffered saline (PBS) or with SC antibodies in the form of a 1 : 10 dilution of fresh or heated inactivated (56 ~ for 30 rain) normal human sera with a SC antibody titer of 1:40 and in the second step with conjugated anti IgG (a to d) or anti complement component C4 (e to h). These conjugates were either absorbed free of their specific antibodies or were diluted to 1/4 unit/ml. • 250. a Treated with fresh serum with SC antibodies and secondly with absorbed anit IgG conjugate. Note negative IF in horny layer, b Treated with PBS and anti-IgG conjugate with 1/4 unit/ml. Note that this direct IF staining reaction also yields a negative SC reaction on normal skin sections. r Treated with heat inactivated normal serum with SC antibodies and anti-IgG conjugate. Note positive indirect IF staining of the stratum granulosum and corneum, d Treated with fresh normal serum with SC antibodies and anti-lgG conjugate. Note again the positive indirect IF reaction comparable to Figure 1c in the horny layers, e Treated with fresh serum with SC antibodies and absorbed anti C4 conjugate. Note negative IF staining in the SC. f Treated with PBS and anti C4 conjugate diluted to i/4 unit/ml. Note that this direct IF staining procedure also gives a negative SC reaction, g Treated with heat inactivated serum with SC antibodies and anti C4 conjugate as for Figure 1 f. Note that the heated serum gives a negative complement indirect IF reaction in the horny layers, h Treated with fresh normal serum with SC antibodies and anti C4 conjugate as for Figure I f. Note positive IF staining both in the strata granulosum and corneum; this serum had complement fixing antibodies to epidermal nuclei which fail to react in the standard indirect IF test. (See Fig. I d)
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Fig. I e - h
with fresh serum containing SC antibodies (Fig. 1 h). The histologic distribution of SC antigen varies with the type of skin biopsy specimen examined. In most normal skin specimens the "SC antigen" occurs in the granular layer and at least in the lower parts of the cornified layer. This is particularly evident in Figures 1 c and 1 d. The serum used for these studies also contained a complement fixing A N A as may be seen in Figure 1 h. Both conventiox/al indirect IF with an anti-IgG conjugate and complement indirect IF staining with an anti-C4 conjugate gave the expected positive reactions on sections of a psoriatic lesion as depicted respectively in Figures 2d and 2h and indicated in Table 1. The intensity of the psoriatic pattern staining in the direct and indirect IF tests with IgG or complement afford a measure of the amount of free SC antigen. The amount of free SC antigen in biopsies in which the direct and indirect tests yielded a similar intensity and distribution of a psoriatic pattern as "none". This suggests that the SC antibodies have saturated the antigen in vivo or that in vivo complement fixation has gone to completion. Each of the 60 fully developed psoriatic lesions examined had in vivo deposits of IgG in the SC in a typical psoriatic staining pattern. Most (53 of 60) also had
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Fig. 2 a-h. Frozen section of a skin biopsy specimen of a fully developed psoriatic lesion. The cornified layer tends to desquimate both as seen on these sections and in vivo. Section from which the horny layer is lost are not suitable for IF studies of this type. The reagents used for the treatment of these sections are the same as those used for the normal skin sections depicted in Figure 1. That is, either PBS or heat inactivated or fresh normal serum with SC antibodies were used in the first step and either absorbed or unabsorbed anti IgG (Fig. 2 a to d) or anti C4 (Fig. 2 e to h) conjugates served as second step reagents. • 600. a Treated first with PBS and secondly with absorbed anti lgG conjugate. Note negative reaction in the SC. This as welt as the reaction depicted in Figure 2b serve as nonspecific staining (NSS) controls, b Treated first with fresh normal serum and then with absorbed anti IgG conjugate. Note again the negative reaction in horny layer. This as well as the NSS test shown in Figure 2 a rule out Fc binding of conjugates, c Treated first with PBS and then with anti-IgG conjugate diluted to 1/4 unit/ml. Note strongly positive direct IF staining reaction for IgG (DIF-IgG) in two adjacent focal areas, one in the upper and one in the mid stratum corneum region.d Treated with fresh normal serum and secondly with the anti-IgG conjugate for an indirect IF (IIF) test with SC antibodies. Note
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Fig. 2e-h positive IF reaction in two adjacent loci in the SC comparable to the ones shown in Figure 2c. This indicates that the SC antigen sites are either completely or almost completely saturated with IgG presumably due to in vivo bound SC antibodies, e Treated with PBS and absorbed anti C4 conjugate. Note negative NSS reaction in the SC except for a trace staining of scattered particles. These resemble nuclear staining in some NSS tests of this type. f Treated with fresh normal serum and secondly with absorbed anti C4 conjugate. Note again negative reaction except for scatter particle staining. This is a form NSS which can easily be distinguihed from the more linear, "'psoriatic" pattern seen on direct and indirect IF staining, g Treated with PBS first and then with anti C4 conjugate diluted to 1/4 unit/ml for DIF-C4 staining. Note single focus of weakly positive staining on the left side of the SC. About half of the psoriasis lesions examined gave such reactions also upon direct IF staining for C3 or C4. C3 tends to give more positives than C4. h Treated with fresh normal and secondly with anti C4 conjugate for a complement indirect IF (CIIF) staining with SC antibodies. Note positive staining in the horny layer. Since this is significantly stronger than the reaction shown in Figure 2g this specimen was rated as being partly satured with respect to C4 on the SC antigen sites. DIF for complement and CIIF staining of psoriatic scales tend to reveal more cytoid structures which may be inflammatory cells
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Table 1. Direct, indirect and complement IF plus nonspecific staining tests of human stratum corneum First step diluent and human sera
Second step conjugates used and tissues examined
Buffered saline (PBS) + 4% bovine albumin (BSA) Normal serum (NHS) heated with stratum corneum antibodies (SCAb) NHS fresh with SCAb and complement (C)
No antibodies
With antibodies
Absorbed anti-IgG*
Anti IgG 1/4 Unit/m!
Anti C4 1/4 Unit/ml
normal
psoriasis
normal
psoriasis
Absorbed anti C4
- *
-
-
+, + +, + + +
-
+, + +, + + +
(not needed)
(not needed)
+ + +
+ + +
-
+, + +, + + +
- *
-
+ + +
+ + +
+ + +
+ + +
* - = no fluorescence; if IF is positive = nonspecific staining Interpretations: If the indirect tests with IgG and complement (C) are significantly greater than the direct tests with IgG or C (+, + +), then only partial in vivo saturation of SC is reported. If IF in indirect tests with IgG and C appear the same as the direct tests with IgG and C (+ + +), then apparent in vivo saturation of the SC antigen is reported. If IF in indirect tests with IgG or C are positive and in the direct tests is negative, then SC antigen is reported as all free
Table 2. Direct IF findings in psoriatic and normal skin biopsy specimens Character of skin lesions
Fully developed psoriatic lesions Fresh linear psoriatic lesions of K6bner type Normal skin specimens
No. of cases
Direct IF staining reactions
Indirect IF staining
In vivo fixed Complement IgG C3 or C4
with IgG
with complement
+
-
+
-
+
-
+
60
60
0
53
7
60
0
54
6
30
25
5
15
15
30
0
30
0
15
0
15
0
15
14
1
14
1
in v i v o d e p o s i t s of c o m p l e m e n t in a similar p a t t e r n . T h e s e p r e s e n t e d in the m o r e a d v a n c e d lesions w i t h p r o m i n e n t i n f l a m m a t o r y cell staining (Fig. 2 h) and s o m e t i m e s with b a n d s of staining at t h e d e r m a l e p i d e r m a l j u n c t i o n . M o s t , b u t n o t all of K 6 b n e r t y p e lesions y i e l d e d p o s i t i v e direct I F staining ( 2 5 / 3 0 ) I g G ; o n l y half of t h e m had d e m o n s t r a b l e c o m p l e m e n t deposits. Since t h e p o s i t i v e direct I F staining p a t t e r n s f r e q u e n t l y p r e s e n t e d as i s o l a t e d l o c i it m a y well be that t h e n e g a t i v e findings w e r e d u e to s a m p l i n g e r r o r s t h o u g h this r e m a i n s to be d e t e r m i n e d . I n d i r e c t and c o m p l e m e n t i n d i r e c t I F staining tests y i e l d e d p o s i t i v e r e a c t i o n s with all of t h e p s o r i a t i c lesions e x a m i n e d and w i t h all b u t o n e of the n o r m a l skin b i o p s y s p e c i m e n s .
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Discussion The findings of SC antibodies set forth in this report as well as those reported previously [1-13] are of two types, notably those which show that they are true complement fixing autoantibodies [ 1-5,7,10,11] and the observations which indicate that these SC antibodies react in vivo in virtually all typical lesions of psoriasis, in a significant proportion of cases of eczema, and occasionally in other dermatoses [4,6, 8,9, 11,12]. It may be presumed from these findings that the primary events which make it possible for the SC antibodies to react in the SC include not only some break in the dermal-epidermal barrier but also a biochemical change from its normal form which is non-reactive in vivo to an abnormal reactive form. It remains to be determined just what the relative import of these two factors is and what the nature of the biochemical changes may be.
Autoantibody Nature of SC Antibodies and Their Antigen. Since all sera contain SC reactive factor(s) one may question whether they are antibodies. If we define antibodies as immunoglobulins which give specific and sensitive reactions with their homologous antigen(s) in serologic tests, then reported observations on SC antibodies relevant to each of the components of this definition may be summarized as follows: a) Immunoglobulin nature of the SC antibodies is shown by mixed agglutination on tissue sections [8] and by indirect IF tests [ 11] since both methods employ anti-IgG antibodies as indicators. b) The specificity of SC antibodies for the stratum eorneum can be demonstrated morphologically by immune adherence [1], mixed agglutination [8] and by immunofluorescence [11]. The specificity of the SC antibodies for purified soluble SC antigens has been demonstrated by indirect hemggglutination methods [7]. c) The sensitivity of the SC antibodies is demonstrated by the high titers obtained with selected sera. Titers as high as 16,384 have been reported by indirect hemagglutination [7]. Indirect IF titers usually do not exceed 160 [11]. d) At least one SC antigen has been isolated and purified. It is a carbohydrate with an e~ linked glucose determinant [5,10,12]. e) The serologic tests with which these SC antibodies can be demonstrated include not only the above mentioned immune adherence, mixed agglutination, indirect hemagglutination and indirect IF tests but also complement fixation by immune adherence [1] and now also the complement IF system described in this report. These observations serve to identify the SC antibodies as true antibodies. Their in vitro reactivity with horny layers of the skin of antibody producers [3,11] affords documentation of their autoantibody nature.
In vivo Reactivity of SC Antibodies. Mixed agglutination revealed the presence of IgG and rheumatoid factor in psoriatic lesions [8]. Extracts of psoriatic scales contained IgG, IgM, IgA and C3. Acid elution of well washed psoriatic scales yielded SC antibodies, rheumatoid factor and C3 [9]. The initial and present studies on complement IF reveal in psoriatic scales and in certain other dermatoses IgG [ 11,12], IgA, IgM (unpublished) and at least in some cases C3 and C4 deposits in the cornified layers. These appear to be bound in vivo to the site of SC antigen. These various observations point to in vivo reactions of SC antibodies in the carrified layer in psoriasis. Such reactions occur in most if not all cases of psoriasis
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and occasionally in other dermatoses that have been studied thus far [12]. The association of psoriatic staining patterns seen occasionally in other dermatoses with psoriasiform changes in the histologic picture remains to be determined. The passive agglutination studies and other studies of Krogh and his associates [4,6-10] indicate that in vivo reactions of SC antibodies may entail unexpected mechanisms. That is, if the SC antibodies do play a role in psoriasis it may be way of first changes in SC antigens and then increasing permeability of the epidermis to the serum protein. If this be true then the increased passage of SC antibodies and complement, coming from the capillaries and fixing in vivo in a biochemically altered stratum corneum may contribute to the self perpetuating nature of the disease.
Passage of SC Antibodies to the SC. The available evidence indicates that in the normal state amino acids can pass through the epidermis to the granular layer but large protein molecules cannot under normal conditions. The tritium labeling studies of Fukuyama and Epstein show dearly that amino acids not only permeate but are incorporated into proteins in the granular layer [19-21]. However, minor trauma, as well as a variety of dermatoses permit serum proteins to pass through to the cornified layer though they usually fail to fix to the SC antigen sites as is seen in most cases of psoriasis [8,9,11,13]. It can be presumed that in psoriasis the stratum corneum must be changed by some unknown event which makes it an antigenic substrate for the reaction with SC antibodies, and the fixation of complement may induce chemotaxis of leukocytes. It is conceivable that the attraction of leukocytes to the SC, notably the squirting papillae suggested by Pinkus [22,23] may be responsible for the observed in vivo "saturation" of the SC antigen sites with IgG (presumably SC antibodies) and not infrequently also with complement. IF findings tend to support the interpretation of Pinkus and to afford one possible explanation.
Comparisons of SC Antibody and Pemphigus Antibody Reactions The appearance of SC antibodies in all normal sera and the inability of the hidden SC antigen in the normal skin to react in vivo serve to distinguish these antibodies from the rare disease specific antibodies of pemphigus which can react in vivo with accessible antigens in normal skin as indicated in Table 3. That is, studies on the sera and normal skin biopsy specimens of pemphigus patients with intercellular antibodies serve to demonstrate that insofar as IgG molecules can pass beyond the basement lamina in vivo binding can take place in the apparently normal skin [24]. This stands in contrast to the SC antibodies which fail to give demonstrable IF reactions beyond the strict confines of frank psoriatic lesions. Yet, as indicated in Table 3, both are circulating antibodies predominantly of the IgG class and both bind to their respective layers of the skin in vivo in their respective lesions. On the one hand the observed participation of leukocytes in the evolution of psoriatic lesions may be due to the in vivo fixation of complement in psoriatic scales and to the leukotactic factors released as a result of C3 and of C 5 - C 6 reactions in the complement cascade. Since some primary mechanism such as trauma must be responsible for the passage of SC antibodies, complement and other sermn proteins into the horny layer and some biochemical changes must convert the SC antigens
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3. Comparisons of pemphigus antibodies with stratum corneum antibodies
Antibodies
Occurrence in sera
Complement fixation
In vivo fixation of IgG
in vitro
in vivo
in lesion
adjacent to lesion
Pemphigus antibodies
pemphigus sera only
not fixed (25)
variable (25-27)
yes (24)
yes (24)
SC antibodies
all human sera
fixed (2 and this report)
variable - positive (this report) - in most active cases
yes (11)
no (I 1)
to a reactive form, it seems probable that the role of the SC antibodies is that of a participating or contributing factor rather than the single causative factor, whereas pemphigus antibodies appear to play a primary role in the pathogenesis of that disease [28,29]. The concepts of a u t o i m m u n i t y of Witebsky [30] and Burnet [31] appear to encompass the relation of pemphigus antibodies to the bullous eruptions they appear to produce. O n the other hand, the relationships observed between SC antibodies and psoriatic lesions can best be explained by concepts of the type set forth by Boyden [32] and more recently by G r a b a r [33] in the form of the "transporteur" hypothesis.
References
1. Krogh, H. K., T6nder, O.: Adherence of erythrocytes to stratum corneum of skin tissue sections. Int. Arch. Allergy 34, 170-180 (1968) 2. Krogh, H. K.: Role of complement in the adherence of erythrocytes to stratum corneum. Int. Arch. Allergy 34, 397-408 (1968) 3. Krogh, H. K.: Antibodies in human sera to stratum corneum. Int. Arch. Allergy 36, 415-426 (1969) 4. Krogb, H. K.: The occurrence of antibodies to stratum corneum in man. Int. Arch. Allergy 37, 649-659 (1970) 5. Krogh, H. K.: The antigen involved in immune adherence with stratum corneum and human serum. Int. Arch. Allergy 38, 78-92 (1970) 6. Krogh, H. K., T6nder, O.: Subcorneal pustular dermatosis. Pathogenetic aspects. Brit. J. Derm. 83, 429-434 (1970) 7. Krogh, H. K., Maeland, J. A., T6nder, O.: Indirect haemaggtutination for demonstration of antibodies to stratum eorneum of skin. Int. Arch. Allergy 42, 493-502 (1972) 8. Krogh, H. K., T6nder, O.: Immunoglobulins and antiimmunoglobulin factors in psoriatic lesions. Clin. Exp. Immunol. 10, 623-634 (1972) 9. Krogh, H. K., T6nder, O.: Antibodies in psoriatic scales. Scand. J. Immunol. 2, 45-51 (1973) 10. Maeland, J. A., Krogh, H. K., T6nder, O.: Heterogeneity of antigen from callus of man. Int. Arch. Allergy 46, 619-628 (1974) 11. Beutner, E. H., Jablonska, S., Jarzabek-Chorzelska, M., Maciejowska, E., Rzesa, G., Chorzelski, T. P.: Studies in immunodermatology. VI. IF studies of autoantibodies to the stratum corneum and of in vivo fixed IgG in stratum corneum of psoriatic scales. Int. Arch. Allergy 48, 301-323 (1975)
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12. Jablonska, S., Chorzelski, T. P., Jarzabek-Chorzelska, M., Beutner, E. H.: Studies in immunodermatology. VII. Four compartment system studies of IgG in stratum corneum and of stratum corneum antigen in biopsies of psoriasis and control dermatoses. Int. Arch. Allergy 48, 324- 340 (1975) 13. Krogh, H. K.: Antibodies to straum corneum in man. In: Immunopathology of the skin: Labeled antibody studies, p. 402-414, E. H. Beutner, T, P. Chorzelski, S. F. Bean, R. E. Jordon (Eds.). Stroudsburg, Pa: Dowden, Hutchinson and Ross 1973 14. Michel, B., Milner, Y., David, K.: Preservation of tissue fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and buIlous diseases. Preliminary report. J. invest. Derm. 59, 449-452 (1973) 15. Hale, W. L., Beutner, E. H.: Comparison of two buffer solutions as transport media for immunofluorescent (IF) studies of epithelial tissue with in vivo bound gamma globulins. ASM Meetings, April 1975 (Abstr.) 16. Beumer, E. H,, Nisengrad, R. J.: Defined immunofluorescence in clinical immunopathology. In: Immunopathology of the skin: Labeled antibody studies, p. 197-247, E. H. Beutner, T. P. Chorzelski, S. F. Bean, R. E. Jordon (Eds.). Stroudsburg, Pa: Dowden, Hutchinson and Ross 1973 17. Diaz Gill, G. A., Beutner, E. H., Eisenberg, R.: Quantitative studies of immunofluorescent staining. VI. Defined complement immunofluorescent staining for antinuclear antibodies in systemic lupus and other collagen diseases. Int. Arch. Allergy 45, 868-879 (1973) 18. Kawamura, A., Mizuoka, K., Matuhasi, T., Fukuoka, Y.: Methods used in Japan for FTA-ABS tests (fluorescent treponemal antibody test). In: Immunopathology of the skin: Labeled antibody studies, p. 424-445, E. H. Beutner, T. P. Chorzelski, S. F. Bean, R. E. Jordon (Eds.). Stroudsburg, Pa: Dowdon, Hutchinson and Ross 1973 19. Fukuyama, K., Epstein, W. L.: Synthesis of RNA and protein during epidermal cell differentiation in man. Arch. Derm. 98, 75-79 (1968) 20. Fukuyama, K., Epstein, W. L.: Comparative autoradiographic study of keratohyalin granules containing cystine and histidine. J. Ultrastr. Res. 51, 314-325 (1975) 21. Fukuyama, K.,Epstein, W.L.: Heterogenous proteins in keratohyalin granules studied by quantitative autoradiography. J. invest. Derm. (In press) 22. Pinkus, H.: Psoriasiform tissue reactions. Aust. J. Derm. 8, 31-35 (1965) 23. Pinkus, H.: Lichenoid tissue reactions. Arch. Derm. 107, 840-846 (1973) 24. Beutner, E. H., Chorzelski, T. P., Jordon, R. E.: Autoimmunity in pemphigus and bullous pemphigoid. Springfield, Ill. : Charles Thomas 1970 25. Jordon, R. E., Sams, W. M., Diaz, G. A., Beutner, E. H.: Negative complement immunofluorescence in pemphigus. J. invest. Derm. 57, 407-410 (1971) 26. Cormane, R. H., Chorzelski, T. P.: "Bound" complement in the epidermis of patients with pemphigus vulgaris. Dermatologica (Basel) 134, 463-466 (1967) 27. van Joost, Th., Cormane, R. H., Pondman, K. W.: Direct immunofluorescent study of the skin on occurrence of complement in pemphigus. Brit. J. Derm. 87, 466-474 (1972) 28. Jarzabek, M.: Passive transfer of pemphigus with human sera. Doctoral Thesis (in Polish), Warsaw, Poland 1970 29. Beutner, E. H., Chorzelski, T. P. : Experimental studies on autosensitization in bullous diseases and on transfer of pemphigus. In: Immunopathology of the skin: Labeled antibody studies, p. 330-353, E. H. Beutner, T. P. Chorzelski, S. F. Bean and R. E. Jordon (Eds.). Stroudsburg, Pa: Dowden, Hutchinson and Ross 1973 30. Witebsky, E.: Ehrlich's side-chain theory in the light of present immunology. Ann. N. Y. Acad. Sci. 59, 168-181 (1954) 31. Burnet, F. M.: The clonal selection theory of aquired immunity. Cambridge U~aiv.Press, London (1959) 32. Boyden, S.: Pathology. Autoimmunity and inflammation. Nature 201, 200-201 (1964) 33. Grabar, P.: Hypothesis. Autoantibodies and immunological theories: An analytical review, Clin, Immun. Immunopath. 4, 453-466 (1975) Received September 27, 1977
