ELSEVIER Journal of Demnxtological Science 8 (1994) 194-202

Autoantibodies

JOURNALOF

Dermatological Science

to annexins: a diagnostic marker for cutaneous disorders? *

Boris C. Bastian*a, Bernadette NuD”, Jiirgen R6mischb, Michael Krausb,

Eva-B. BrGckera

‘Department of Dermatology, University of Wiirzburg, Josef-Schneider-Str. 2, D-97080 W&burg, Germany bBehringwerke AG Research Lrrboratories, Marburg, Germany

Received 23 February 1994; revision received 27 April 1994; accepted 12 May 1994

Abstract

Annexinsflipocortins are a group of structurally related calcium and lipid binding proteins which have been im- plicated as mediators of the anti-inflammatory action of corticosteroids. Autoantibodies against annexin-l have been reported in association with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and their presence has been hypothesized as the reason for the steroid resistance phenomenon. In this study we investigated IgG- and IgM-autoantibodies against annexin-1,-2,-3,-4,-5 and -6 in sera of 221 patients with skin disorders and 114 healthy blood donors with newly established ELISAs. Patients were clustered into 5 groups according to their diagno- sis: autoimmune diseases, psoriasis, leg ulcer, malignant melanoma, and miscellaneous diseases. Autoantibodies directed against each annexin were detectable in all investigated groups, in the control group as well as in the disease groups, without displaying any significant correlation to any of the disease states. The homogenous distribution of annexin-autoantibodies throughout the control group and all the disease groups studied, do not support the implica- tion of annexin-autoantibodies in pathophysiological states and make them an unlikely candidate for use as a diagnos- tic marker.

Keywords: Annexins; ELISA; Autoimmune diseases; Skin diseases; Lipocortins

1. Introduction

*This work was presented in part at the annual meeting of the European Society of Dermatological Research in London, April 1992 and at 8th International Congress of Immunology in Budapest. August 1992.

Abbreviations: AAC, autoantibody; ANA, anti-nuclear antibody; ARA, American Rheumatism Association; AX, an- nexin; BSA, bovine serum albumin; CS, corticosteroids; HRP, horseradish peroxidasc, PBL, periphereal blood lymphocytes; OPD, o-phenylendiamine; RA, rheumatoid arthritis; SLE, sys- tcmic lupus erythematosus; TBS, tris-buffered saline.

*Corresponding author, Tel.: 49 931 201 2701; Fax: 49 931 201 2700.

0923-1811/94iS07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0923-1811(94)00336-D

Annexins (AX), previously known as lipocor- tins, are a family of structurally related, unglycosylated proteins [I]. They have been demonstrated in a number of species and tissues, with each cell type apparently presenting a characteristic AX pattern [2,3]. Particularly when located intracellularly, they may represent a con- siderable proportion of the cell protein. Despite the fact that AX lack a leader sequence, traces of them have been seen to be present in body fluids

B.C. Bastion et al. /J. Demratol. Sci. 8 (1994) 194-202 195

and on cell surfaces. The association with eitra- and intracellular surfaces and elements of the cytoskeleton may be related to the calcium- binding properties of AX [4,5]. Although, thus far, no clear physiological function has been able to be ascribed to these proteins, their significance as po- tential anticoagulants and their possible participa- tion in cytodynamic and ionic transport processes has been investigated [6]. They have also been discussed as mediators of the glucocorticosteroids (CS) by directly inhibiting the pro-inflammatory enzyme phospholipase A2 (PLA2) [7,8], but recent studies have demonstrated that this effect only oc- curs at very low substrate concentrations unlikely to occur in vivo [9, lo]. Nevertheless, an increase in AX-1 synthesis after administration of CS has been clearly demonstrated in a number of cells [ 11,121. In addition several other investigators

found a direct anti-inflammatory action of AX-1 itself or certain peptides of the protein [ 13- 181.

Soon after Hirata et al. first described lipomodulin, which is synonymous to AX-l, this same group found that sera from patients suffering from rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) decreased the in- hibitory action of lipomodulin on the arachidonic acid release by PLA2 in vitro [19]. This effect disappeared on depleting the sera of IgM, but not after preabsorption of IgG. The findings of the presence of IgM-AAb to lipomodulin in patients with RA and SLE suggested a role in the patho- genesis of rheumatic diseases. Subsequently, a fur- ther study by Goulding et al. revealed raised IgM-AAb to AX-1 in patients with active disease [20]. Speculating that the AX-I-AAb might be the cause of the ‘steroid resistance’ phenomenon,

Table 1 Composition of the diagnosis subgroups

n

bale n Age Age Age ANA-pos ANA-neg RA Female Male Female

Psoriasis Psoriasis vulgaris Psoriasis arthropathica

20 17 3

13 7 49.1 47.3 52.5 0 0 0 13 4 47.5 47.3 48.0 0 0 0 0 3 58.5 - 58.5 0 0 0

Autoimmune-Diseases Dermatomyositis Lupus Erythematodes (systemic) Lupus Erythematodes (discoid) Scleroderma (systemic) Scleroderma (circumscribed) Pemphigus Bullous pemphigoid Cicatrial pemphigoid Dermatitis herpetiformis M. behcet

27 5 3 4 4 2 3 3

4 23 55.1 62.7 53.0 13 I 4 57.8 68.0 55.3 2 0 3 35.4 - 35.4 3 0 4 47.5 - 47.5 2 0 4 56.8 - 56.8 4 1 1 54.8 70 39.6 1 0 3 68.2 - 68.2 1 1 2 72.3 78.0 69.4 0 0 1 53.1 - 53.1 0 0 1 56.0 - 56.0 0 1 0 33.0 33.0 - 0

0 1 0 0 0 0 0 0 0 0

Leg ulcer 19 12 7 69.5 65.8 76.0 0 0

Melanoma 36 11 25 48.3 50.2 47.5 stage I 16 2 14 46.7 54.5 45.6 stage II 6 1 5 54.8 71.0 51.6 Stage III 14 8 6 47.4 46.5 48.6

Miscellaneous 119 64 55 47.6 45.9 49.5 6 3

Controls 114 n.d. n.d. nd. n.d. nd. n.d. n.d. n.d.

RA, rheumatoid arthritis; n.d., no data available.

1% B.C. Bastion et al. /J. Demuztol. Sci. 8 (1994) 194-202

monitoring of AX-I-AAb during steroid medica- tion was recommended. Subsequently, further studies found raised titres of AX- 1 -AAb in inflam- matory bowel diseases [21] and in patients with psoriasis arthropathica and sero-negative arthritis [22] whereas studies on patients with asthma revealed no raised AX-l-AAb titres [23,24]. In this study we asked whether, among a heterogeneous panel of dermatological patients, distinct subsets could be characterized which have raised autoan- tibodies to annexins. We used solid phase ELISAs and investigated the patterns of AAb against 6 dif- ferent AXs in the sera of 221 patients suffering from skin disorders and 114 healthy blood donors as controls.

2. Materials and methods

2.1. Patients The blood samples were taken in the morning.

The sera were kept frozen at a temperature of -40°C. 221 in- and out-patients from the Depart- ment of Dermatology of the Wiirzburg Medical School were retrospectively placed in one of the following diagnosis groups on the basis of their medical history: autoimmune diseases (n = 27), psoriasis (n = ZO), chronic leg ulcer (n = 19), melanoma (n = 36), and miscellaneous (n = 119). Table 1 shows the composition of the individual diagnosis groups. The group ‘miscellaneous’ com- prised cases of herpes simplex, zoster, eczema, epi- dermal and nevoid tumours, hair diseases, allergic drug reactions, chronic urticaria and acne. Fur- thermore the patients sex, age and available data on the titre of anti-nuclear antibodies (ANA) (titre on HEP2cells > 1: 160/ < 1:160) were documented.

As a control group sera from 114 apparently healthy blood donors were used. The serum sam- ples were collected anonymously and no informa- tion on gender and age was available. According to the information of the blood bank there was no significant disequilibrium of gender, the mean age was estimated at 35 years. AX-AAb values below the 95th percentile of the control group were defin- ed as normal.

2.2. Determination of annexin-autoantibodies The titres of AX-AAb were determined by ap-

plying a series of new enzyme immunoassays following the sandwich principle [25]. Microtitra- tion plates were coated separately with 125 ~1 of purified AX, apart from AX-1 and -2, which were only available as a mixture, at a concentration of 5 &ml in 0.001 M sodium acetate buffer, pH 5.5. After incubation overnight the plates were washed with TBS and blocked with 1% BSA solution for 1 h, washed again with TBS and dried. Samples were diluted 1: 10 in TBS, containing 3% BSA (w/v) and 0.5% tween 20 (w/v). One-hundred ~1 of each diluted sample were incubated for 1 h at room temperature per annexin coated well. For control of non-specific binding a pool of sera from healthy blood donors was used in all experiments. The microtitre plates were washed 3 times using en- zygnost washing buffer (Behringwerke AG, Mar- burg, Germany) and the wells were incubated each with 100 ~1 of either anti-human IgG- or IgM- HRP conjugate in TBS, 1% BSA, 0.5% Tween 20, for another hour at room temperature. Subse- quently, the microtitre plates were washed again and 100 ~1 of substrate solution (Enzygnost OPD substrate solution, Behringwerke AG, Marburg, Germany) were added per well. After 30 min in- cubation at room temperature the reaction was terminated with 100 ~1 of 0.5 N H#O,+ The ab- sorbency was determined at 490 nm (reference 630 nm) in an ELISA plate reader (Dynatech, Guem- sey, UK). For comparison the absorbency values were as relative absorbance related to the 95%- interval of the log-normal distribution of absor- bancies as determined in 114 sera from healthy blood donors:

rel.abs. = absorbance in ELISA

absorbance at 95’1~interval of normal distribution

Samples with absorbancies values above this in- terval were regarded as positive. Special care had been taken to avoid unspecific binding of AAb due to serum matrix effects or rheumatoid factors [25].

2.3. Statistical methods Examination with respect to normal distribution

B.C. Bastian et al. /J. Dermatol. Sci. 8 (1994) 194-202 191

I.6 N=20 N=27 N=l9 N=M N=l19 N-114 of AX-AA\, within the whole group of 221 pa- tients and the 114 control persons using the probit- system showed a log-normal distribution of all AX-AAb values. Therefore, the following statistical calculations were carried out using the logarithms of the AX-AAb values.

The difference between individual variables within the various groups was established using the Student’s t-test. Dependency between 2 vari- ables in several possible groups was tested using a 2-factor covariance analysis. Already in the first series of calculation a clear-cut dependency on the patients age became obvious. To correct for the age bias each AX-AAb value was converted to an age value of 50 years using the correlation coeffic- ient of age and the AX-AAb of the whole group as a corrective factor. Corrective factors were deter- mined individually for each AX and for both sexes according to the gender-specific age dependency demonstrated.

To provide a clearer picture of the plethora of data from the different AX and immunoglobulin classes investigated in this work representative values for all AX-AAb antibodies in one immuno- globulin class were used in the figures. This was possible because the AX-AAb levels exhibited a high correlation within one immunoglobulin class (P C 0.001, see results). Consequently, 2 cumula- tive factors were calculated for the IgG- and IgM- AX-AAb levels using a factor analysis in order to provide a clearer picture. Factor analysis is used to reduce information. Factors are new variables which are calculated from the original data by ad- justed addition [26]. Apart from the advantage of the concentration of the information on 2 vari- ables, these factors are independent from one another. Thus, the interpretation of the results is not disturbed by minor deppendencies. These values were used in the Figs. 1 and 2 as relative values representing the AX-AAb levels of the individual immunoglobulin classes. The corresponding graphs calculated from the original data of the in- dividual AX-AAb differed in no respect from the data shown in Figs. 1 and 2. Nevertheless, a direct comparison of the absolute level of both Ig-classes is not possible in these diagrams due to the use of this procedure. The statistical data described in the text were all calculated using the original data of the individual AX-AAb.

3.0 -

2.5 -

2.0 -

1.5 - 0

L” 00

0

1.0 - 0

0.5 - t 0

0.0 - 4.5 B -

t -1.0 - D

. 0

8

: 0

i

-1.5

I

0 0

-2.0

-2.5

0

AD UC MM MC BD A Ps

[EM N=20 N=27 N=19 N=M N=119 N=ll4 2.5 0 B 2.0 1 0 0 00

1 0

1.5 0

I.0

t

B

0.5 0

-1.5 0

-2.0 L 0 0

5.5

B ps AD UC MM MC BD

Fig. 1. Factor-analysisa of the IgG (a) and IgM type AAb to AX (b) in the diagnosis subgroups and healthy blood donors. PS, psoriasis; AD, autoimmune disease; UC, leg ulcer; MM, melanoma; MC, miscellaneous; BD, healthy blood donors. The numbers above the groups represent the number of patients in each subgroup.

*The values on the y-axis are the representative values de- rived from the factor analysis [15]. They reflect the distribution of the IgG (a) and IgM type (b) AAb against all annexins in- vestigated and are used to render the results clearer. The pat- tern of the individual types of AAb in each immunoglobulin class showed no significant change in comparison to the pattern shown in the figures.

198 B.C. Bastion et al. /J. Dematol. Sci. 8 (1994) 194-202

3. Results

A consideration of the total patient group with regard to the range of AX-AAb detected showed that AAb may be formed, in principle, against any of the AX investigated. The mean values of the in- dividual AX-AAbs, however, exhibited differences (Table 2). In the case of IgG-AAb, the IgG-AX-4- AAb were highest, IgG-AX-5-AAb lowest. In the case of IgM-antibodies, the highest level was ex- hibited by IgG-AX-1/2AAb, the IgG-AX-5-AAb remaining the lowest.

If a 95th percentile of the values of the normal group were taken as the threshold value between ‘normal’ and ‘raised’, 63 patients (29.7%) showed raised levels in one of the 10 AX-AAb, 17 (8.0%) showed 2 raised AX-AAb levels, and in 7 patients (3.3%) 3 different raised AX-AAb levels could be demonstrated.

Using a variance analysis of the individual AX- AAb values, a highly significant correlation among the AX-AAb levels in the individual im- munoglobulin classes - IgG and IgM - could be established (P ~0.001). On the basis of this sharp distinction between the behaviour of IgG- and IgM-AX-AAb, factor analysis provided 2 representative factors for the 2 immunoglobulin classes. In order to present a clearer picture, these 2 factors were used in Figs. 1 and 2 to represent the pattern of the AX-AAb of the 2 immunoglobulin classes (see Materials and methods).

3.1. Age and sex dependency A clear correlation was found between age and

AX-Mb pattern in the 221 patients. In the group as a whole, there was a highly significant decrease in all AX-AAb of the IgM-type with increasing age (P < 0.001 to P < 0.00001). The decrease was continuous in male patients, whereas AX-AAb titres in women over 60 years of age dropped abruptly (Fig. 2b). This effect was particularly clear for IgM-AX-l/2-AAb (data not shown). AX- AAb of the @G-type rose slightly with increasing age (Fig. 2a), being statistically significant only for IgG-M-4-AAb.

In general, women had much higher IgM-AX- AAb levels than men (P < 0.05-0.0001).

3.2. Infruence of diagnosis A comparison of the individual diagnosis

1.5

8 1.0

‘j 2 0.5

6 =o.o

-0.5

-1 I I , I f 1 I 50 40 50 60 10 60 00

age tyerrsl

2.0 , B

1.5 Women KI

Ia

Man Cl 1.0

s g 0.5 a T x 0.0

2 -0.5

-1.0

-1.5 30 40 50 60 70 60 90

ago [yrrrsl

Fig. 2. Representative values of the factor analysisa of IgG (a) and IgM type (b) AAb dependent on the age of the 221 pa- tients. The numbers below the bars represeni the upper range of age in this subgroup. 'See Fig. 1.

groups in the variance analysis of the AX-AAb showed clear differences, particularly for the IgM antibodies in the ulcus cruris group, which, on the whole, were distinctly lower than the groups auto- immune diseases, melanoma, and miscellaneous (P c 0.02-0.001). No differences could be establish- ed between the ulcus cruris patients and those with psoriasis. If one considers the different age distri- butions within the groups (Table l), a clear

B.C. Bastian et al. /J. Dennatol. Sci. 8 (1994) 194-202 199

Table 2 Logarithms and standard deviations of the AX-Mb levels in the groups investigated

Psoriasis

n = 20

Autoimmune disease n = 21

Leg ulcer

n= 19

Melanoma

n = 36

MisceIIaneous Controls

?I=119 ?I= 117

Ax-m-IgG AX-bIgG AX4IgG AX-SIgG AX-6-IgG AX-I/2-IgM AX-3-IgM AX4IgM AX-S-IgM AX-6IgM

1.57 l 0.21 1.64 f 0.35 1.51 f 0.31 1.55 f 0.36 1.85 f 0.20 1.82 zt 0.35 1.35 f 0.38 1.31 f 0.33 1.54 f 0.27 1.68 f 0.32 1.72 f 0.37 1.81 zt 0.31 1.65 + 0.42 1.65 * 0.38 1.63 zt 0.26 1.66 l 0.31 1.47 f 0.33 1.46 l 0.31 1.69 f 0.32 1.71 zt 0.32

1.71 f 0.42 1.57 f 0.27 1.55 l 0.35 1.71 f 0.40 1.57 f 0.30 1.61 zt 0.35 1.93 f 0.27 1.84 zk 0.29 1.77 f 0.26 1.53 f 0.42 1.40 l 0.28 1.46 l 0.40 1.58 f 0.28 1.62 f 0.26 1.62 f 0.32 1.54 f 0.341 1.88 f 0.32 1.84 f 0.30 1.42 f 0.33 1.69 f 0.24 1.64 f 0.34 1.54 f 0.33 1.83 f 0.27 1.71 zt 0.29 1.32 f 0.33 1.55 f 0.26 1.48 EIZ 0.31 1.54 f 0.34 1.82 f 0.32 1.74 f 0.29

1.66 t 0.30 1.66 zt 0.27 1.88 zt 0.29 1.62 zt 0.26 1.90 Et 0.30 1.80 zt 0.27 1.63 ztz 0.28 1.85 zrz 0.27 1.62 jz 0.28 1.77 f 0.30

diminution of the differences between the groups is obvious. To exclude a possible bias due to heter- ogeneity of age, in a separate statistical analysis all values were adjusted to the value of a 50-year-old test person by using the gender-specific regression coefficient of age dependency and then compared once again (see Materials and methods). Addi- tionally, the increases in the regression graphs of age and AX-AAb within the different diagnosis groups were compared. Both tests showed an

Fig. 3. Autoantibodies to AX expressed in relative absorban- ties (r&l) (see Materials and methods) of the 15 ANA-positive and 19 ANA-negative patients.

almost complete disappearance of the differences between the groups when the levels were adjusted for age.

The comparison of the IgG-AX-AAb showed no clear differences in the various groups.

In contrast, a comparison of the 5 diagnosis groups and the group of normal persons showed that normal individuals had a considerably higher AX-AAb level. This applied for both the IgG and IgM immunoglobulin classes, with IgG-AX-j- AAb, IgG-AX&AAb, and IgM-AX&AAb in particular, showing the clearest differences. Again, the potential bias of the different ages within the groups was controlled and the differences within the IgM antibodies were seen to be much smaller; the alterations in IgG values remained unchanged or became even clearer. For age adjustment of the control group, the mean age of the blood donor group was estimated at 35 years (see Materials and methods). Figs. la and b show the Ig-factors from the factor analysis within the individual diagnosis groups and from the blood donor group. Graphs plotted for the 10 different AX-AAb (data not shown) exhibited no fundamental differences from the Ig-factors shown in the Figs.

The t-test as a means of comparing ANA- positive (6.3%) and negative patients (9.5%) show- ed higher AX-AAb values on the whole for ANA- positive patients (Fig. 3). This dependency was clearest for IgG-AX-4-AAb (P = 0.016) and IgC-AX-l/2-AAb.

200 B.C. Bastion et al. /J. Dermatol. Sci. 8 (1994) 194-202

4. Discussion

The discovery of a lipid-binding CS-inducible protein (lipocortin) at the beginning of the 1980s’ offered a possible explanation for the anti- inflammatory effect of steroids [27]. The demon- stration of AAb against lipocortin-VAX-1 and their potential for abolishing the inhibition of arachidonic acid release from lipids in vitro, led to the hypothesis of a causal link between the steroid resistance in individual patients with auto- immune/inflammatory diseases and the ap- pearance of AX-AAb. AX-I-AAb were tirst demonstrated in patients with RA and SLE [19,20].

In the course of the last decade, 7 further human anti-inflammatory proteins of the same type have been discovered and have been summarized as AX-l-8 1251. Several AX, or peptides derived from their structure, have been shown to exhibit various levels of anti-inflammatory activity in in vitro experiments dependent on their affinities to membranes; only a few have been tested in vivo thus far [ 1 l-181. Nevertheless, the data on the direct anti-inflammatory action of AX or AX- peptides is conflicting and several groups had not been able to reproduce their effect [15,28,29]. The role of the AX in the physiology of inflammation still remains unclarified and it is neither known which protein of the AX-family is the major actor in this context nor how it exerts its hypothesized effects. It therefore appears logical to include more members of the AX-family in investigations of AX-autoantibodies. Obviously, it could be of great diagnostic value, if a certain pathophysiological state can be demonstrated to be linked to the presence of AX-AAb. Conse- quently, it seems useful to initially screen a rather broad palette of different disease states to reveal otherwise hidden strong associations to certain pathological circumstances. Thus we examined AAb against AX-l to -6 in the sera of patients with various skin disorders in comparison to apparently healthy donors.

To our knowledge this study represents the first investigation on AX-AAb which includes other AX than AX-l. Nevertheless, the fact that our ELISA cannot distinguish AAb from AX-l and

AX-2 limits the ability to compare our data on AX-l with other studies on AX-l in the way that positive values can not be traced to either AX-l or AX-2-AAb. However, negative values in our tests clearly state the absence of AX-l and AX-2 anti- bodies.

The principle result of our study is that any clear association or pattern among the diseases studied is completely lacking. Facing the fact that even ap- parently healthy people and people with minimal skin disease, such as minor acne or nevi, can have high titres of AX-AAb, makes it difficult to think of any disease-specific pattern beyond the scope of the diseases investigated. Furthermore, cases with RA and SLE which are described to display elevated titres of AX-I-AAb showed no significant alterations in their AX-AAb profiles in com- parison to the control group. These findings give rise to the question whether AX-AAb plays any role - generally and regardless of any specific ill- ness - in pathophysiology, or whether they repre- sent an epiphenomenon.

One further major finding is, that obviously, AAb cannot only be raised against AX-l/2 but against any of the AX studied. AAb to AX-3, AX- 4, AX-S, and AX-6 both of IgG- and IgM-type can be detected in each of the groups investigated. There is a highly significant correlation between falling IgM-AX-AAb titre (but not IgG) and in- creased patient age. This applies for all the AX tested. Goulding et al., however, found no connec- tion between age of the patient and AX-I-AAb, a fact which may be related to the relatively homogenous age distribution of their patient group [20]. In contrast to male patients, significantly higher titres of IgM-AX-AAb could be observed in female patients under 60 years of age. These decreased abruptly after the age of 60. The fall in IgM-AX-Mb was more continuous in men. The sex-dependent progression of this titre would suggest that the appearance and regulation of AX and their AAb may be dependent on the in- fluence of sexual hormones.

The positive association between the presence of ANA and AX-AAb might indicate that the forma- tion of AX-AAb are an expression of a generally raised autoreactivity.

To date, the significance of AX-AAb has been

B.C. Bastian et al. /J. Dermatol. Sci. 8 11994) 194-202 201

discussed under the premise that the (or at least one) physiological function of AX is the regulation of (anti-) inflammatory processes - a question which is still the subject of controversial discus- sion. If, however, one considers the other biologi- cal properties of AX which have been described, the influence of the relevant AAb on hemostatic, cytodynamic or development/differentiation pro- cesses could be of some significance [6,30-331.

The clarification of the role of AX-AAb depends upon a knowledge of the eventual biologi- cal role of annexins. Future investigations should therefore include as many proteins of this family as possible.

References

111

Pl

[31

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[91

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