Journal of Autoimmunity (1995) 8, 267-277

E. Bar-Meir*, S. S. Teuber†‡, H. C. Lin§,  I. Alosacie§, G. Goddard*||, J. Terybery§,N. Barka§, B. Shen§, J. B. Peter§, M. Blank*, M. E. Gershwin†, and Y. Shoenfeld*

Correspondence should be addressed to: Y. Shoenfeld, Department of Medicine B, Research Unit of Autoimmune Diseases, Chaim Sheba Medical Center, Israel 52621.

||Supported also by grants given by the Chief of the Ministry of Health and Welfare Israel.

There are estimated to be over 1.5 million women with silicone breast implants (SBI) in the United States. Throughout the world an additional 150,000 women have undergone implantation every year [1-4]. Silicone was once thought to be biologically inert [5], However, numerous reports have been published concerning women who developed pathology after receiving silicone breast implants [6-25]. Guerrero et al. have recently published a review in which they summarized the literature; they report 293 patients with rheumatic symptoms after receiving gel-filled breast implants [26]. Of these 293 patients, 56 had definite connective tissue disease (of which 38 had scleroderma) and 15 patients had a possible connective tissue disease. The remaining 222 patients with gel-filled implants had constitutional symptoms, signs or laboratory abnormalities which did not fit a classic rheumatic disease. The over-presentation of scleroderma relative to other autoimmune disease in patients with implants is a fairly consistent finding [27]. Another interesting point has been the effect of explantation on the clinical course. In some cases of connective tissue disease, removal of the implants led to clinical improvement in spite of the usual chronic course of autoimmune diseases. The improvement was not only seen clinically but also evident by serology: titers of diverse autoantibodies regressed and some even vanished [23]. Several studies have shown higher frequencies of autoantibodies in subsets of implant patients. To address these issues further, we have performed a large blind control study to determine the frequency of a broad range of autoantibodies. Using a panel of 20 different autoantigens, we report that women with implants are more likely to exhibit autoantibody production.

250 woman were evaluated: 116 women with implants and 134 female controls. Of the 116 implant patients, 104 women had silicone gel-filled implants., three had saline-filled implants, three had a polyurethane-coated implant, and six had a double lumen implant (Table 1). The age of the control group was 43.6 ± 7.9 years. The age of the patients ranged from 26 to 66 years (mean: 45.7 ± 8.3 years). Breast prostheses were in place for a mean period of 15 ± 5.6 years (range: 4 to 30 years).

One hundred and eleven subjects ware Caucasians, three were African-American, and two were Hispanic. Three patients (2.5%) had prostheses placed during reconstruction after mastectomy for breast cancer, four (3.5%) after mastectomy for fibrocystic breast disease, and 109 (94%) for augmentation purposes alone. None had previously known autoimmune disease prior to implantation. Thirty patients had histories of past or current implant ruptures (Figure l (a)). The patients' complaints were fatigue (n=25), myalgias (n=19), fibromyalgia (n=4), recent onset of arthralgias (n=40), morning stiffness (n=10), decreased memory (n=1), polyarthritis (n=7), and Raynaud's phenomenon (n=6) (Figure l (b)). Ten subjects developed the following syndromes: autoimmune sensorineural hearing loss (n=1), antiphospholipid syndrome (n=2), cutaneous polyarteritis nodosa (n=l), primary biliary cirrhosis (n=1), episcleritis (n=l), systemic lupus erythematosus without renal involvement (n=l) (by American College of Rheumatology (ACR) criteria), Sjogren's syndrome (n=l), rheumatoid arthritis (n=l), and scleroderma (n=l). Twenty-one patients had significant capsular contracture upon breast examination (i.e. one or both implants were very firm to palpation).

The control group consisted of sera from 134 healthy women of comparable age, also living in the same geographic area, with no history of inflammatory arthritis or other autoimmune disorders, and no history of collagen injections or silicone prostheses.

The sera of all 250 women were tested for 20 autoantibodies in a blind fashion by ELISA assays. To compensate for non-specific binding of immunoglobulins to the plastic, the optical density obtained in the absence of antigen was subtracted from that obtained in wells coated with antigen. Fifteen sera from normal subjects were also included in every ELISA plate assay for the purpose of quality control. A known positive standard was also included. Sera were considered to contain antibodies if their ELISA optic density (OD) values were greater than three SD above the mean for the control sera.

Antibodies to histone (H2AH2B), SSA (Ro), SSB (La) and cardiolipin were tested as previously described [28]. Antibodies to RNP, ribosomal-phosphate (HPRPP) and to Sm were detected using Enzyme ImmunoAssay Diagnostic Kits (EldarTech, Israel).

Antibodies to GBM were examined by ELISA using Non-Collagen-l-Antigen (NC1), (Specialty Laboratories, Santa Monica, USA), as follows: 96-well ELISA plates were coated with 100 µl of 1:250 (NCI in Guanidine Tris buffer, pH 7.3 (6M Guanidine HC1 with 0.05 m Tris) per well, and were kept in a humid chamber at 4°C overnight. The plates were then washed five times with 200µl of 0.05% Tween-PBS blocked by 150µl of 1% bovine serum albumin (BSA) in PBS-Tween for 1 h at 37°C, then washed and covered with 1:200 serum dilutions for 1 h at room temperature. The plates were then washed again and reacted with alkaline phosphatase and substrate.

Anti-TG antibodies were detected as described [29). Briefly, human TG was prepared from autopsy material by ammonium sulphate precipitation of a saline thyroid extract with Sephycryl S-300 gel filtration (Pharmacia, Piscataway, New Jersey, USA). The antigen was kindly provided by Dr N. Rose, Baltimore, USA. Plates were coated with 50 µl of 1 µg/ml TG in 0.05 m carbonate/bicarbonate buffer, pH 9.6, then incubated for 22 h in a humid chamber at 4°C, washed five times with 0.05% Tween-PBS and with 150 µl 5% bovine serum (BS) in PBS, per well, and then incubated 1 h at 37ºC. The plates were washed again, and 1:200 serum dilutions in 2% BSA in Tween-PBS were added and the plates were incubated at 37 °C for 1 h. Anti-human Ig conjugated to alkaline phosphatase and substrate, were added as described above.

Antibodies to brain gangliosides (GM1 and GD1 a), to Scl-70 and to Jo-1 antigens, phosphatidyl-serine, and to collagen type I, II and IV were examined by ELISA kits in Specialty Laboratories, Santa Monica, California, USA. Antibodies to myeloperoxidase and proteinase-3 were tested as previously described [30, 31]. Representative positive samples were also examined by an additional alternative method (immunoblot, immunofluorescence, etc.) to confirm positivity.

Differences in the frequency of autoantibody levels were determined by chi-squared analysis.

The incidence of 15 out of the 20 autoantibodies tested on SBI subjects in comparison to healthy controls reached statistical significance (Table 2). The difference in the incidence of the following 12 autoantibodies in SBI patients in comparison to matched controls reached a statistical significance of P<0.01: SSA, SSB, Sm, RNP, scl-70, collagen type I collagen type II, H2AH2B, sulfatide, MPO, HPRPP-ribosomal phosphate, and CL.

The difference in the incidence of the following three autoantibodies in SBI in comparison to matched controls reached a statistical significance of P<0.05:collagen type IV, phosphatidyl serine, GM. The incidence of the number of autoantibodies among patients is shown in Table 3. It is interesting to note that some women had 7, 8, 9 or 11 autoantibodies: three out of these seven patients had evidence of an autoimmune disease (systemic lupus erythematosus, seronegative rheumatoid arthritis, antiphospholipid syndrome) and one patient had a non-specific disease (small joint arthralgias, tender proximal interphalangeal joints with some synovial thickening). The absolute mean ± SD of the ODs of the positive seras for the individual autoantibodies in the study group were found to be higher than the positive ODs recorded in the control group (Table 2).

A total of 20 patients were positive to either type I, II or type IV collagen antibodies. Of the 14 patients with anti-collagen I autoantibodies, six patients also had anti-collagen II and anti-collagen IV autoantibodies. Of the remaining eight patients with anti collagen I autoantibodies, seven had anti-collagen IV autoantibodies. Only one patient had anti-collagen I autoantibodies with no anti-collagen II or anti-collagen IV autoantibodies.

Table 2. Statistical evaluation of autoantibody frequency. A
statistical analysis showing the number of positive patients com-
pared with the number of positive controls. The mean ± SD of the
positive OD values is also shown for each

*P <0.01 Student's t-test compared with controls; **P <0.05 Student's t-test
compared with controls.

Table 3. Incidence of polyreactivity

Figure 2. Correlation between the number of autoantibodies in a patient and the number of years the implants were in place

Of the 10 patients with anti-collagen II autoantibodies, only four patients did not have anti-collagen I autoantibodies. Of the 15 patients with anti-collagen IV autoantibodies, only two patients did not have anti-collagen I autoantibodies. No correlation was found between the number of autoantibodies in a patient and the number of years the implants were in place (Figure 2).

In our study, women with silicone breast implants have a higher incidence of autoantibodies than healthy women without breast implants. Other investigators have previously assessed the presence of eight of the 21 autoantibodies analysed by us in SBI patients [26]. Five of the autoantibodies, anti-Sm, anti-RNP, anti-Ro, anti-La and anti-scl-70, were evaluated in more than 150 SBI patients. These autoantibodies were all found in a much smaller number of SBI patients than in our study. The remaining three autoantibodies (anti-thyroglobulin, and-GBM (NCl),and anti-cardiolipin) were studied in a very small number of patients (one or two);therefore, no comparison with our results can be made. It should be pointed out that the group of patients assessed for each autoantibody in the different studies was not the same as that screened for the other autoantibodies. The reason for this is that in most of the studies done, either a large number of patients were investigated for a small panel of autoantibodies, or a small number of patients were surveyed for a large panel of autoantibodies.

Teuber et al. [33] have shown that autoantibodies to both types I and II collagen are found in patients with silicone breast implants. The higher frequency of antibodies to type I collagen in comparison to other collagen antigens may be a result of differences in tissue exposure to the different collagens: type I collagen would be major component of the scar tissue around the implant, while type II found in the hyaline cartilage [3, 4]. For most patients, antibodies to type II collagen may be secondary to the response to type I collagen; there is strong sequence homology between the two collagen types, and antibodies to several collagen types frequently occur together. In Teuber et al.’s work, 12 out of the 16 patients that were positive for anti-collagen antibodies reacted against type I collagen or denatured type I collagen, and seven patients reacted against native type II collagen. Only three patients had reactivity to type II collagen alone. Our results are in accordance with this study: of the 20 patients with autoantibodies against native type I, II or type IV collagen, 14 patients had antibodies against type I. Of the 10 patients with anti-collagen II autoantibodies, only four patients did not have anti-collagen I autoantibodies. Of 15 patients with anti-collagen IV autoantibodies, only two patients did not have anti-collagen I autoantibodies.

Anti-scl-70 antibodies were found in higher incidence in SBI patients. In our study, of the 13 patients that had anti-scl-70 antibodies, three had an autoimmune disease ( one each with systemic scleroderma, systemic lupus, or the antiphospholipid syndrome), three had non-specific autoimmune complaints (morning stiffness and arthralgia), and one woman had morning stiffness and cold, blue hands and legs in the morning. It is interesting to point out that no patient with Raynaud's phenomenon had anti-scl-70 antibodies. However, this finding is consistent with literature: anti-scl-70 antibodies are found only in a small percentage of patients with local scleroderma of CREST syndrome [35, 36].

To our knowledge, this is the first study in which 20 autoantibodies were evaluated in a large number of patients (n=116 by the same laboratory and method (ELISA), with a comparable large control group (n=134 women). The incidence of each of the autoantibodies in the control group ranged from 0.5 to 7%.This is in accordance with our previous screening of 5000 healthy women [37]. Yet, it should be emphasized that the ‘positives’ in the SBI group were higher in OD comparison than the control ones (see Table 2). For each of the positive autoantibodies in the SBI group, representative samples were also screened in an additional system (e.g. immunoblot for RNP and Sm or immunofluorescence for proteinase-3, GBM, etc.) to confirm positivity. The significantly higher incidence of 15 autoantibodies in women with silicone breast implants suggests that silicone may exert an adjuvant effect. This is further strengthened by observations of investigators who have noted hypergammaglobulinemia following silicone injection and that silica (silicon dioxide) may have adjuvant effects[32]. The presence of autoantibodies in these patients may possibly indicate future development of an autoimmune disease. Such a conclusion is important since many of the patients harbored several autoantibodies (polyreactivity), e.g. 20% of the patients had four autoantibodies and 8% had six autoantibodies. To date only 10 of the SBI patients (out of 116) have a definite autoimmune disease. The small number of women with autoimmune disease could be explained by the relatively short follow up time (14.6 ± 13 years). Another strong possibility is that the autoantibodies are produced as a result of exposure of the immune system to the silicone gel and may not represent an ominous sign of a developing autoimmune disease. Nonetheless, the data are of concern. The question why some women develop autoantibodies following exposure to silicone, or even overt autoimmune disease, is unresolved. It is probably due to individual factors such as a specific genetic predisposition towards autoantibody production and perhaps the presence of significant inflammation around the implant. A prolonged follow-up period is necessary to determine the role and significance of the autoantibodies in individual patients.

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