Clin. exp. Immunol. (1992) 90, 422-427

Autoantibodies against cyclophilin in systemic lupus erythematosus and Lyme disease A. KRATZ*t, M. W. HARDING*, J. CRAFT+, C. G. MACKWORTH-YOUNG§ & R. E. HANDSCHUMACHER* Departments of *Pharmacology, tEpidemiology and Public Health, and TMedicine, Yale University School of Medicine, New Haven, CT, USA, and §Department of Medicine, Westminster Hospital, London, UK

(Acceptedfor publication 6 August 1992)

SUMMARY A binding protein, were detected in sera of 29 of 46 a cyclosporin cyclophilin, Autoantibodies against (63%) patients with systemic lupus erythematosus and 14 of 40 (35%) Lyme disease patients. The antibodies are directed against the denatured form of both the major and minor isoform of cyclophilin and can be demonstrated in Western blots. Some first-degree relatives of lupus patients also express these antibodies. They are specific for cyclophilin and are not the consequence of hypergammaglobulinaemia. Four monoclonal 1gM antibodies from a patient with lepromatous leprosy also bound to cyclophilin. The generation of these antibodies may be of special interest because they are against a protein involved in the control of the immune system not known to be directly associated with DNA or RNA. Keywords cyclophilin autoimmunity systemic lupus erythematosus Lyme disease

INTRODUCTION The success of cyclosporin A (CsA) in organ transplantation [1] has led to clinical trials in the treatment of autoimmune diseases [2]. Knowledge of the cellular and molecular mechanisms by which immunosuppression and toxic side effects of CsA are mediated is evolving. Cyclophilin-18 (CyP-18) is a small (mol. wt 17 737) cytosolic protein with high affinity and stereospecificity for CsA binding [3] that is abundant in both lymphoid and non-lymphoid mammalian tissues. Fisher and co-workers [4] and Takahashi and colleagues [5] have shown that CyP- 18 is identical to a peptidyl-prolyl cis-trans isomerase and thus may function to facilitate protein folding; this activity is selectively inhibited by CsA. Zuker and his co-workers have demonstrated that the NinaA gene product, a protein associated with a visual transduction mutant in Drosophila, shows a high homology to CyP-18 and is responsible for the folding of rhodopsin [6]. Several recent reports have identified additional members of the cyclophilin family of proteins [7,8]. Evidence ofeffects on the assembly of NFAT, the transcription factor for IL-2 [9], and the folding of collagen [ 10] and transferrin [ 11] are consistent with a role in protein folding. The most specific evidence for a molecular target is the finding that the cyclophilin-cyclosporin complex is a potent inhibitor of the Ca2+-dependent phosphatase activity of calcineurin, a result consistent with the general selectivity of CsA for Ca2+-modulated signal transduction [12].

Although the evolutionary conservation of the CyP- 18 structure has made it difficult to generate antibodies, we report that some patients with systemic lupus erythematosus (SLE) and Lyme disease spontaneously produce antibodies against this intracellular protein. This report extends our preliminary description of this phenomenon [13], as well as the recent report of Harigai and colleagues [14]. MATERIALS AND METHODS

Sera Sera were obtained at Yale-New Haven Hospital from healthy individuals or patients diagnosed as having SLE [15], Lyme disease, colon carcinoma, and multiple sclerosis. A panel of 101 first-degree relatives of 21 lupus patients seen at the Hammersmith Hospital Lupus Clinic was kindly provided by Dr M. J. Walport. They consisted of 13 fathers, 19 mothers, 34 brothers, 26 sisters, four sons, four daughters and one granddaughter. All sera were initially tested in a dilution of 1: 100 by ELISA and when appropriate at 1: 40 for immunoblotting. Approval for use of sera for these studies was obtained from the Human Investigations Committee of Yale University School of Medicine.

Cyclophilin CyP-18 was purified to homogeneity from bovine thymus as previously described [3] except that a final purification was achieved on a CM 300 HPLC preparative column instead of isoelectric focusing. A comparison of the activity of the major

Correspondence: Dr R. E. Handschumacher, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, CT 06510, USA.

422

Autoantibodies against cyclophilin and minor (N-terminal acetyl derivative of the major isoform) bovine thymus isoforms in ELISA and Western blots gave identical results. ELISA Linbro 96-well plates (Flow Laboratories, McLean, VA) were coated with 0-1 ml of a 10 pg/ml solution of CyP-18 in 10 mm sodium phosphate buffer, pH 6 0. The acidic pH of the coating buffer was essential to assure maximum adsorption of CyP-18. After incubation overnight at 40C, remaining protein binding sites were blocked by incubation with 0-1 ml of a 0-1 % solution of bovine serum albumin (BSA) in phosphate-buffered saline (PBS) overnight at 40C. Plates were washed three times with PBS containing 0-05% Tween 20 (PBS-T) prior to use. Unless otherwise noted, sera from healthy controls or patients were diluted 1: 100 in RPMI-1640 containing 10% fetal bovine serum (FBS) and 0-1 ml aliquots were added to quadruplicate wells. After incubation for 90 min at 37TC, plates were washed three times with PBS-T followed by iincubation for fo 90mm 90 min with alkaline phosphatase conjugated goat goat anti-human IgG antiserum (Sigma, St Louis, MO) diluted 1: 1000 in PBS-T. After three washes with PBS-T, binding of the antiglobulin conjugate was determined by measuring p-nitrophenyl phos-

wishedthralklinesphosphatase conjuged

phate

banti-hun

(Sigma) hydrolysis at 20C after 45 mm inaTiterek.MC

atafter 410minin nm.CmetiSphetro-pa)hotometro(Flowy Laborato tionexperimentswere perFormed Labyratinating1theappromprti-

tion experiments were performed by incubating the appropriately diluted serum with an equal amount of a 10 pg/ml CyP- 18 solution. Positive and negative internal controls were included on every plate.

Western blotting

Electrophoresis of pure CyP-18 was performed in 15% SDSpolyacrylamide gels (37-5:1 acrylamide: bis acrylamide) [16]. The protein was transferred to nitrocellulose (BA-85, Schleicher and Schuel, Keene, NH) in a Transblot cell (Bio-Rad, Richmond, CA) at 100 mA for 105 mm in transfer buffer containing 25 mM Tris pH 8&8, 192 mm glycine, 01% SDS and 20% methanol [17]. Fixation of the protein was achieved with 20% ethanol and 10% acetic acid. The nitrocellulose was washed three times with PBS-T, and the remaining protein binding sites blocked by incubation of the nitrocellulose filters in PBS-T containing 5% Bovine Lacto Transfer Technique Optimizer (BLOTTO) [18] with shaking overnight at 370C. Patient sera were diluted 1:40 in PBS-T containing 5% BLOTTO and ultracentrifuged for 2 h at 100 000 g. Incubation with the CyP blot was performed for I h at 37°C with shaking. After three washes with PBS-T containing 5% BLOTTO, filters were incubated with '25l-labelled Protein A (NEN Research Products, Boston, MA) (2-3 x 105 ct/min/ml) for I h at room temperature. Filters were washed three times with PBS-T containing 5% BLOTTO, and three times with PBS-T, air dried and exposed to X-ray film (Kodak XAR- 1) at - 70°C.

Monoclonal antibodies MoAbs from hybridomas derived from peripheral blood and splenic lymphocytes of patients with SLE have been previously described [19]. MoAbs derived from peripheral blood lymphocytes of a patient with lepromatous leprosy were also used in this study. The characteristics and specificity of these MoAbs have been described in detail [20]. Statistical analysis Statistical analysis was performed on an IBM PS-2/50 computer, using PC-SAS (SAS Institute Inc., Cary, NC). For multiple comparisons, a general linear model was chosen. The procedure described by Bonferroni [21] was used to test which groups differed from the normal control group at the P < 0-001 level.

RESULTS

Detection of CyP-18 autoantibodies in patients with SLE and Lyme disease by ELISA In a group of 43 normal human controls screened in this study fratatbde gis y-18 nA1 LS for autoantibodies against CyP-18, none gave an A4t0 ELISA reading greater than 0u3, a value that is 3 s.d. above the mean. In other related studies of 280 normal controls (W. Wong et al., unpublished observations), less than 5% of the ELISA A410 values exceeded 0 3. Initial pilot studies had shown the presence of autoantibodies against CyP- 18 ofthe IgG, but not of the IgM istp naot4 ainswihSEadLm ies dt isotype in about 40 patients with SLE and Lyme disease (data not shown). total of 185 sera from a variety of patients were analysed ~~~~~~~~~~A for autoantibodies against CyP- 1 8 ofthe IgG isotype by ELISA. Of these, 58 were obtained from 46 patients with SLE, 40 from oegv

patients with Lyme disease, nine from patients with multiple sclerosis, 35 from patients with histologically confirmed colon cancer, and 43 from normal controls. An additional 101 sera were from first-degree relatives of 21 SLE patients. ELISA data from both the patients and normal controls are shown in Fig. 1. Of the 46 patients with SLE, 29 (63%) had anti220 200,

100-u 080 @ g< 0 60 L

.

040 020

t

LC-Partigen radial immunodiffusion plates (Behring Diagnostics, Somerville, NJ) were used to determine the IgG level in patients and normal human sera. The sera were diluted in saline, and plates read after 72 h.

'

000

hNumal (n=43)

IgG determination

423

cancer

sclerosis

disease

(n =9) (n =40) (n=35) A=0 09 A=0 13 A=0-17 A=0 31 s.d.=020 s.d.=0 23 s.d.=0-07 s.d.=0 1I 1. Prevalence of spontaneous autoantibodies to CyP

SLE (n=46) A=0 44) s.d.=027

in normal ~ controls ~ ~ ~ ~Fig. and in patients with colon cancer, multiple sclerosis, Lyme

disease and SLE. Each data point represents the average of quadruplicate ELISA values from a serum sample. An ELISA reading > 0 3 is considered positive as described in Results.

A. Kratz et al.

424

v

CyP antibodies as measured by ELISA. In 40 Lyme disease patients, 14 (35%) were found to have autoantibodies against cyclophilin. In contrast, only three of 35 patients (8-6%) with colon cancer had a borderline elevation of their ELISA reading. Three of nine (333%) sera from patients with multiple sclerosis showed minimal elevation of ELISA readings. Multiple sera (two or three per patient) from five SLE-patients indicated that antibody levels did not vary by more than 30% over a period of 4 years (data not shown). Only the SLE and Lyme disease patients differed significantly from the normal control group (P < 0-00 1).

Specificity of the autoantibodiesfor Cyp-18 To determine if elevated ELISA readings were the result of nonspecific binding to BSA or plastic, a representative group of sera were assayed with plates coated only with BSA. In general, there was no correlation between non-specific binding and binding to. plates coated with CyP-18 (Fig. 2). Confirmation of the reactivity to CyP- 18 was seen with Western blots performed with selected sera. Positive blots with CyP- 18 were only seen in patient sera that had positive ELISA values and were roughly proportional to these readings (Fig. 3).

0 17

0.00

0 00

X

X

-

0-27

0 44

I

w

0 54

ELISA readings Fig. 3. Immunoblots of cyclophilin using sera from normal human controls and patients with SLE. The same amount of CyP (10 pg) was loaded in each lane of an SDS-polyacrylamide

gel. After electrophore-

Comparison of total IgG and cyclophilin autoantibody levels Since autoimmune patients often have hypergammaglobulinwere aernia,IgG concentrations in the sera sera of SLE aemia, SrLEI patients were determined using a radial immunodiffusion assay (Fig. 4). Further evidence for the specificity of response was seen in the lack of correlation between anti-CyP ELISA values and IgG concentrations in the sera.

sis, transfer to nitrocellulose was performed as described in Materials and Methods. The nitrocellulose was cut into strips, which were incubated with control and patient sera. Duration of exposure during auodigphwsienclfralbot(2ay)

Anticyclophilin antibodies bind the denaturedform of the protein Earlier studies have shown that rabbit antisera against CyP generally react only against denatured forms of CyP (Harding MW, Sarris A, Jiang T-R, Aftab D, and Handschumacher RE, unpublished results). The corresponding properties of human antisera were examined. Preincubation of sera (A4,o=0 15 to 1 2) at 1: 100 dilution with native CyP-18 (10 jg/ml) for 90 min at 370C failed to inhibit the binding of antibodies to CyP-18 adsorbed in the plastic well of the ELISA assay. However, preincubation of the same sera under identical conditions with

l

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v

b

2

ao

0°91 07

00 6p 05 (n W

0 10

2

3

4

5

i

i

i

-

02 al_

6

7

8

9

10 11I

122113 14

4000

30000 2c0Y C-P

0 90

0 80

PLATES BSA PLATES

-

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3

4 |

aU ° 50

0 40

range

2

-) 060 (I)

Normal

1000

-

|

10

11

12

13 14

* | * * * * * | | | | | | |

Fig. 4. Comparison of the CyP autoantibody reactivity and serum IgG concentrations in a spectrum of patients with SLE. ELISA values and IgG levels were obtained as described in Materials and Methods.

R

CyP-18 (10 yg/ml) that had been denatured by boiling for 10

-

Ii 0-30

5 6 7 8 9 |Patient No.

0-200.10

A RB CC A

D0K E

F G H

J

K L M N 0 P Q

Patient Fig. 2. Comparison of CyP specific to non-specific BSA responses in the ELISA. Plates coated with Cyp before blocking with BSA were run at the same time and under the same conditions as plates coated only with BSA (see Materials and Methods).

mmn reduced the ELISA reading by approximately 50% in each sample (Fig. 5). These data suggest that CyP-18 autoantibodies recognize epitopes that are exposed by solid phase adsorption of CyP- 18 or by heat denaturation but are not accessible in the native conformation of the molecule.

425

Autoantibodies against cyclophilin

Table 1. Serological features of 12 CyP-autoantibody positive relatives

2-

M No preincubotion

With native CyP

M With denatured CyP

|

0 | el

ELISA A410

Family

1

0-392

Mother

0-332

Mother

en

0 330 0-380 0467

Brother Sister Mother

0-372

Half-brother

0-342 0-359 0-483 0-655 0-350 0-364

Mother SisterFather

1:640

Mother

-

|

' 5 6 7 8

Prevalence of spontaneous CyP-18 autoantibodies in relatives of SLE patients Sera from 101 first-degree relatives of 21 SLE patients were examined for CyP-1 8 autoantibodies. Twelve relatives of seven patients had positive CyP- 18 autoantibody titres. In total, seven of 21 families (33%) had at least one seropositive family member. Analysis of this group showed that nine relatives were female, three were male. Two of these relatives had some of the clinical manifestations of SLE, but did not meet the diagnostic criteria for the disease [15]. One patient had a photosensitive rash, while the other presented with Raynaud's syndrome and a history of deep venous thrombosis. Two other relatives did meet the diagnostic criteria of SLE. No correlation between CyP- 18 autoantibodies and cardiolipin antibodies, circulating C l q binding complexes, cross-reactive anti-DNA antibody idiotypes or C4 allotypes was detected (Table 1) [23,24].

ANA

3 ;

Patient No Fig. 5. Anticyclophilin antibodies are directed against the denatured form of the protein. ELISA plates were coated with CyP as described in Materials and Methods. Sera diluted 1:100 were preincubated with 10 ug CyP/ml that was denatured by boiling for 10 min.

Relation to patient

SisterSister

-

-

-

-

-

-

-

-

-

-

1:160

X

ANA, antinuclear antibody titre > 1:160 is positive; ENA, antibodies to extractable nuclear antigens, e.g. Ro, La, RNPs, Sm; DNAb, DNA binding antibody measured by the Farr assay; ACA, anticardioli-

Monoclonal IgM antibodies with public idiotypes bind CyP-18 Supernatants of human-human hybridomas derived from SLE patients and one leprosy patient were also screened by ELISA. No CyP-18 reactive antibodies were identified among 24 monoclonals derived from SLE patients. However, four reactive IgM monoclonals (8E7, 4G7, 4E3, and 8B 1I) were identified among seven hybridomas derived from the leprosy patient (Table 2). The characteristics of these monoclonals have been described [20] and include binding to ssDNA, mitochondria and Mycobacterium leprae and staining of mitochondria and cytoskeletal cellular components as determined by immunoflourescence analysis. Monoclonals 4G7 and 8E7 cross-react with five different anti-idiotype specific rabbit antisera, and 4E3 crossreacts with four anti-idiotypes; such findings suggest the expression of one or more public idiotypes by these CyP- 18 binding MoAbs [25,26].

Clone

A410

ssDNA

M. leprae

Mitochondria

4G7

0-48

+

+

+

Idiotype reactivityt Rld8E7, Rld4G7

Rld4D5, RldTH9 RID16/6 -

-

1:160

Antigen reactivity*

-

-

1:160

ELISA

0-40

ACA

-

Table 2. Characteristics of leprosy-derived MoAb that bind cyclophilin

8E7

DNAb

ENA

+

Rld8E7, Rld4G7,

RIdTH9, Rld16/6 8B11

1-09

-

+

+

4E3

0-33

+

+

+

* Data taken from [20]. t Data taken from [25,26].

Rld8E7, Rld4G7 Rld4D5 RID16/6

Rld405

A. Kratz et al.

426 DISCUSSION

Several features of this autoimmune response to CyP- 18 encourage further investigation of this phenomenon. The cyclophilin-related proteins and FK506 binding protein, which also binds rapamycin [27-30], represent two structurally unrelated species that participate in a newly emerging element of signal transduction that may involve a catalytic role in the folding and unfolding of proteins [6,31] or the formation of ligand receptor complexes with other proteins such as calcineurin [12]. At this time there is no evidence that the antibodies discussed in this report affect these regulatory mechanisms that may control immune responsiveness. The fact that CyP- 18 is a cytosolic protein and that the antibodies react only with denatured CyP-18 argues against this possibility. However, membrane-bound forms of cyclophilin have recently been documented in mammalian cells and are inferred from the mRNA sequences obtained from humans, Drosophila and Neurospora [32-35]. Although autoantibodies against CyP-18 can be found in both the 'classical' autoimmune disease SLE as well as in Lyme disease which also has some autoimmune characteristics, this does not indicate that these antibodies in any way contribute directly to the disease process. In general, the potential pathogenic effect of antibodies in SLE is not considered to be directly via inhibition of the activity of their target proteins; rather they are thought to cause symptoms indirectly consequent to precipitation of antibody-antigen complexes [36]. Molecular mimicry, the process whereby the immune system mounts a response to an epitope on an invading microorganism which is also found on a host protein, has been proposed as one of the initiating mechanisms of autoimmunity [37,38]. Of the four MoAbs found in this study to bind to CyP- 18, three (4G7, 4E3, and 8BI 1, Table 2) bind to antigen(s) on a foreign organism, Mycobacterium leprae. The fourth, 8E7, carries an idiotype which Mackworth-Young has detected in the sera of patients with leprosy, lupus and Lyme disease [39]. These data would be consistent with the hypothesis that autoantibodies to CyP- 18 arise in response to environmental antigens that mimic epitopes on autoantigens. Although the same autoantibodies arise in these diseases involving polyclonal B cell activation, the independence of anti-CyP autoantibodies from hypergammaglobulinaemia argues against the assumption that their presence is simply a result of the elevated immunoglobulin levels. After submission of this manuscript, an independent report appeared describing the presence of autoantibodies to CyP of the IgG class in 40% of patients with SLE [14], a slightly lower rate than reported here. Harigai and co-workers were able to demonstrate the presence of IgM autoantibodies in 20% of their patients, however none of our patients showed antibodies of this isotype. Further studies will be needed to determine whether these differences are methodological or due to variations in the populations under study. Our finding of anti-CyP antibodies in first degree relatives of SLE patients is in agreement with earlier studies indicating that they have an increased risk of developing SLE [40]. A positive family history of SLE can be found in 4% of cases. It is unclear whether this genetic predisposition can be explained by shared V-region genes [41]. Prospective studies of the relatives of the SLE patients would be useful to determine whether those with autoantibodies against CyP-18 develop SLE more frequently.

The predominance of female positives among the relatives of our patients could be an interesting point, considering the higher incidence of lupus in females.

ACKNOWLEDGMENTS The authors thank Waichi Wong and Dr Kevin P. High for valuable advice and for helpful suggestions. We also thank Dr John Marsh, Dr Peter Heald, Dr Timothy Vollmer, Dr R. S. Schwartz, Dr D. Duggan and Professor M. J. Walport for the kind gift of sera and reagents, Dr Elizabeth Claus for statistical advice and Joyce Nepiarsky for her excellent secretarial assistance. This work was supported by ACS 67H, GM 40660, CA 08341, Al 26853 and the Austrian Ministry of Science.

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