Journal of Autoimmunity (1992) $443-452

Antibodies to Mycobacterial65 kDa Heat Shock Protein in Systemic Sclerosis (Scleroderma)

Maria Giovanna Danieli, Marco Candela, Anna Maria Ricciatti, Roberta Reginelli, Giovanni Danieli, Irun R. Cohen* and Armando Gabrielli Istituto di Clinica Medica dell’Universitk di&acona, Ancona, Italy and *Department of Cell Biology, Weizmann Institute of Science, Rehovot, Israel (Received 31 December 1991 and accepted 27 February 1992)

It has been reported that immunity to the 65 kDa heat shock protein of tuberculosis (MT-hsp65) not only accompanies rheumatoid arthritis (RA), but may also be characteristic of chronic inflammation. We now report serum antibodies to MT-hsp65 in 47% of systemic sclerosis (SSc), 38% of primary Raynaud’s phenomenon (PRP) and 5% of systemic lupus erythematosus (SLE). Antibody levels were higher in patients with active or progressive SSc and correlated with the degree of skin fibrosis. Thus, immunity to MT-hsp65 appears in SSc and is not limited to RA. However, it does show some degree of specificity beyond chronic inflammation: PRP patients have a higher reactivity than do SLE patients. Mycobacterium

Introduction

Systemic sclerosis (scleroderma) (SSc) is a chronic disorder characterized by fibrotic and inflammatory changes in the skin and internal organs [ 11. The etiology and pathogenesis are not understood in detail, but autoimmunity is thought to be involved in the microvascular modifications and in the excessive fibroblastic activity with abnormal deposition of type I and type III collagen, which are constant features of SSc. Several immunological abnormalities have been described which include both the humoral and cellular arms of the immune response. Antibodies to intraCorrespondence to: Maria Giovanna Danieli MD, Istituto Generale, Torrette di Ancona, 60020, Italy. Fax: 39-71-888 972.

Clinica Medica Generale,

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Maria Giovanna Danieli et al.

Table 1. Characteristics phenomenon

Group

(PRP)

of patients with systemic sclerosis (SSc), primary Raynaud’s and systemic lupus erythematosus (SLE) and of normal controls

Number of subjects

M/F

ssc

53

5148

PRP

36

lo/26

SLE

36

3133

Normal controls

42

8134

Mean age (years _+SD) (range)

Mean disease duration (years + SD) (range)

51&11 (19-77) 43+15 (20-71) 34k12 (18-64) 46+ 19 (25-65)

6.8k4.1 (0.5-20) 5.3k6.7 (0.5-30) 5.5 +3.5 (1-14)

cellular antigens have been found in over 95”/, of SSc patients [2] and a large proportion shows enhanced immune responsiveness to type I and IV collagens and to laminin [ 1,3]. Changes of cellular immunity include reduced numbers of circulating T lymphocytes and decreased lymphocyte proliferation to mitogens [4,5]. Heat shock proteins (hsp), highly conserved in evolution, are synthesized in large amounts when cells are exposed to heat or to other environmental stress [6]. However, some hsp molecules are expressed in the absence of stress and play important physiological roles in maintaining normal cell function [7]. Increased levels of antibodies to hsp have been demonstrated in several autoimmune diseases [&lo]. The significance of hsp immunity is not completely clear, but it is thought to be relevant to the etiology and pathogenesis of autoimmune disorders because of the high homology between bacterial and human hsp molecules and because of the importance of hsp in cell physiology [ 111. We now demonstrate the presence in SSc patients of circulating antibodies reacting with the M. tuberculosis 65-kDa heat shock protein (MT-hsp65). The serum levels of these antibodies correlate with the clinical activity of scleroderma and with the extent of skin fibrosis. Since Raynaud’s phenomenon can precede the appearance of full-blown scleroderma, some patients with Raynaud’s phenomenon not associated with a connective tissue disorder (primary Raynaud’s phenomenon; PRP) were also included in the study. We compared the response of the patients with SSc and PRP with those of patients with systemic lupus erythematosus (SLE). Patients and methods Patients

Serum samples from 53 patients with SSc, from 36 patients with PRP, and from 36 SLE patients were examined. The characteristics of these patients are reported in Table 1. The diagnosis of SSc was made according to the ACR criteria [ 121. Pulmonary, oesophageal, articular (osteolysis), cardiac and renal involvements were present in

hsp-65 immunity in systemic sclerosis

445

44.6,64.2, 16.0,5.3 and 5.3% of the cases, respectively. The patients were classified according to the extent of skin involvement as described: type I, involvement of the hands up to the wrists; type II involvement of the forearms; type III, fibrosis starting at the trunk [ 11. No patient was receiving steroids or immunosuppressive drugs at the time of the investigation. Patients were considered to have active disease when progression and/or inflammation were present. Progression was defined as deterioration in the function of visceral organs during the 9-month period preceding study, and inflammation as the presence of any three of the following: ESR > 40 mm/h, gammaglobulins> 1.6 g/d&positive CRP and ANA> l/160. Fifty of the 53 (94.3%) patients with SSc suffered from Raynaud’s phenomenon with a mean disease duration of 10.9f6.3 years (range 0.5-30 years) and with a variable degree of severity. Raynaud’s phenomenon was detected by an abnormal cold provocation test, the results of which were documented using a strain gauge pletismograph. The diagnosis of primary PRP was based on the absence of large vessel obstructive disease, arterial disease, signs or symptoms suggestive of connective tissue disorders, or history of drug intake known to trigger the phenomenon. Thirty-six patients in whom SLE was diagnosed according to ACR criteria were also investigated as a control disease group [ 131. Four of them (11%) had Raynaud’s phenomenon. Serum samples were collected from patients regardless of the activity of the disease or the therapy. Forty-two sera from healthy subjects, matched for age and sex, were collected from the local blood bank and constituted the normal control group.

Antigens Recombinant MT-hsp65, MT-hsp70, the 180-188 peptide of MT-hsp65 and a control E. cola’preparation containing the pEX2 plasmid without the hsp65 gene (pEX2) [14,15] were kindly provided by R. van der Zee and J. D. A. van Embden, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands. The synthetic link protein peptide of rat proteoglycan was composed of 16 amino acids, 134-149 in the published sequence of the link protein [ 161. Bovine serum albumin (BSA) was purchased from Sigma Chemical Co. (St Louis, MO, USA). Enzyme-linked

immunosorbent assay

Antibodies were assessed using an enzyme-linked immunosorbent assay (ELISA). All the antigens, hsp and peptides, were diluted to a concentration of 2 ug/ml in 0.02 M carbonate buffer (pH 9.6) and 100 ~1 was added to each well of polystyrene microtiter plates (Nunc, Denmark). After overnight incubation at 4°C the plates were coated with Dulbecco’s phosphate buffered saline (pH 7.8) containing 0.5% Tween 20 (PBS-T) and 1% BSA for 30 min at room temperature to block nonspecific ligands. After washing, 100 ul of test serum, diluted 1:lOO in PBS-T containing 0.176 BSA, was added in duplicate to antigen coated wells. After 45 min incubation with rocking at room temperature, the plates were washed with PBS-T,

446 Maria Giovanna Danieli et al.

50

100

200

Reciprocal

of

sewm

400

800

dilution

Figure 1. Titration curve of antibody reactivity against MT-hsp65 in 5 systemic sclerosis (SSc) patients, 5 primary Raynaud’s phenomenon (I’RP) patients and 5 normal controls. Data are expressed as the ELISA ratio (see Patients and methods for details). -BSSc patients; - q - PRP patients; -+controls.

and 100 ~1 of peroxidase conjugated goat anti-human IgG, IgA and IgM antibody was added to each well, at a 1: 10,000 dilution in PBS-T containing 0.1 y0 BSA. The plates were incubated for an additional 45 min and, after thorough washing, 100 ~1 of ABTS was added to each well. After incubation for 15 min, absorbance was determined at 417 nm on a Multiskan plate reader (Titertek, Flow Laboratories, Milano, Italy). Results are expressed as a ratio of the absorbance value of the test serum to the mean of three known negative sera. A ratio value greater than 2 SD above the mean of the ratio obtained in normal control sera (n = 42) was considered positive. Statistical

analysis

Statistical analyses were performed on a Personal IBM Computer with the Statistical Programs for the Social Sciences. Data are expressed as the mean f 1 SD. Statistical significance was determined using Student’s t-test and the Chi-square (x2) test. All reported values are two-tailed; P values less than 0.05 were regarded as significant. Results

Figure 1 shows a titration curve for normal control and patient sera tested by ELISA against MT-hsp65; subsequently all sera were assayed at a dilution of 1: 100. When tested against MT-hsp65, the meanf SD absorbance ratio of sera from patients with SSc (1.76 + 1.03) was significantly greater (P< 0.001) than that of sera from control subjects (0.89kO.33) (Figure 2). The absorbance values of sera

hsp-65 immunity in systemic sclerosis

447

:

. b

3.

.

--

-

.

. .

.. . . . . ..

. . ! :.

A

i

: .:. .:. ri* .i .

2

2.

.

t

-m-

_ . .

.

::

.:. .t. : :: * .:. . ..

l.*

.*.

i

. .:. . ..

-i

Y :: I .

N

ssc

PRP

SLE

Figure 2. Antibody titer to MT-hsp65 in 42 normal subjects (N), 53 patients with systemic sclerosis (SSc), 36 with primary Raynaud’s phenomenon (PRP) and 36 with systemic lupus erythematosus (SLE). The data are expressed as the ELISA ratio. Bars show group means. The broken line indicates the mean + 2SD of normal control values.

from patients with PRP (1.47 _t 0.81) was higher than controls (P < O.OOl), and not significantly lower than those obtained with sera from patients with SSc. Using an absorbance ratio of 1.55 as a cut-off (2SD above the mean value of the healthy controls), 25 of 53 sera (47.1%) from SSc patients displayed elevated levels of antibodies to MT-hsp65 (Figure 2). Sera from PRP patients demonstrated elevated antibody levels (Figure 2) in 14 of 36 (38.8%) patients. In contrast, only two patients with SLE (5.5%) showed antibody levels slightly above the cut-off line. The mean value of the SLE group (0.81) 0.32) did not differ from that of controls and was not influenced by the presence of Raynaud’s phenomenon, by the clinical activity of the disease, or by the type of treatment. The two SLE patients with levels of circulating antibodies to MT-hsp65 above normal had active disease with renal involvement, and one of them suffered from Raynaud’s phenomenon. When the state of SSc was taken into consideration, the highest MT-hsp65 reactivity was found in patients with active and progressive systemic disease

448

Maria Giovanna Danieli et al.

Table 2. Levels of antibodies against MT-hsp65,

MT-hsp70 andpEX2 SSc, PRP and SLE and in normal controls

inpatients

with

ELISA ratio for antigens (% incidence of positive antibodies) Number of subjects

Group

MT-hsp65

MT-hsp70

pEX2

53

1.76+

1.53kO.92

1.19kO.43

24

(47%) 2.22* 1.15*

(7%) 1.89& 1.22

(0%) 1.21+0.39

29

(75%) 1.39 * 0.75*

(16%) 1.22k0.39

(0%) 1.16+0.50

16

(24%) 2.40* 1.29*

(0%) 1.74+0.80

(0%) 1.30f0.76

37

(75%) 1.49+0.76*

(12%) 1.44kO.97

(0%) 1.17kO.47

PRP

36

(35%) 1.47+oJ31*

(5%) 1.7650.46

(0%) 1.36+0.40

SLE

36

(38%) 0.81 + 0.32

(0%) 1.4lkO.42

(0%) 1.22+0.27

42

(5%) 0.89kO.33

(0%) 1.55 + 0.73

(0%) 1.09kO.32

ssc All patients Active Inactive

SSc SSc

Progressive

SSc

Nonprogressive

Normal

controls

SSc

1.03*

(0%)

(0%)

(0%)

*P