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Serum antinuclear antibodies in women with silicone breast implants. Cuéllar ML, Scopelitis E, Tenenbaum SA, Garry RF, Silveira LH, Cabrera G, Espinoza LR. ABSTRACT. OBJECTIVE. Recent evidence suggests that immunologic abnormalities are not uncommon in individuals with silicone breast implants. The purpose of our study was to evaluate in a consecutive manner, the prevalence of autoimmunity as assessed by the presence of antinuclear antibodies in a larger number of patients with silicone breast implants. METHODS. Antinuclear antibody (ANA) testing using an indirect immunofluorescence technique was performed on 813 individuals with silicone breast implants. All subjects except for 3 transsexual males, were female. The overwhelming majority, over 99%, were white. The average age of the subjects was 46. 2, with a range of 17 to 72 years. RESULTS. ANA positivity was found in 244 of 813 individuals (30%) using a mouse kidney substrate; and in 470 of 813 (57.8%) using a HEp-2 cell line. The most common immunofluorescent pattern found using HEp-2 was speckled, present in 341 (72.5%) individuals, followed by homogeneous pattern in 113 (24%), nucleolar in 63 (13.4%), and 5 (1.06%) were anticentromere. Anti-dsDNA antibodies measured by an ELISA assay were found in 6 of 71 patients (8%). Rheumatoid factor and C-reactive protein were found above healthy controls in less than 10% of cases studied. The high prevalence of ANA found in patients with silicone breast implants agrees with similar observations by others. The finding of anticentromere and nucleolar patterns has great interest and relevance. These fairly distinct ANA patterns are most commonly seen in the idiopathic form of scleroderma and related conditions. CONCLUSION. These findings suggest that ANA positivity is relatively common in individuals with silicone breast implants, and may support the existence of autoimmune mechanisms in the pathogenesis of the clinical manifestations seen in this population. (J Rheumatol. 1995; 22:236-40) From the Section of Rheumatology, Department of Medicine, LSU Medical Center at New Orleans, LA, USA. M.L. Cuéllar, MD, Research Fellow; E. Scopelitis, MD, Associate Professor; S. Tenenbaum, MS; R. Garry, PhD, Professor of Microbiology and Immunology; L.H. Silveira, MD, Assistant Professor; G. Cabrera, MD, Fellow in Rheumatology; L.R. Espinoza, MD, Professor and Chief, Section of Rheumatology. Address reprint requests to Dr. M.L. Cuéllar, Section of Rheumatology, LSU Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112-2822. Submitted February 24, 1994 revision accepted August 9, 1994. A variety of clinical manifestations have been described in association with silicone breast implants1-4. Many patients exhibit ill defined connective tissue disorders (CTD). They predominantly complain of malaise, fatigue, lymphadenopathy, arthralgia, and myalgia. These may or may not be accompanied by serologic evidence of autoimmunity. The term human adjuvant disease has been used to refer to the disorder of these patients5. Others may experience distinct but less frequent CTD that include scleroderma and its variants, systemic lupus erythematosus, rheumatoid arthritis (RA), and other rheumatic disorders1-4, 6-10. The etiopathogenesis of this association has generated considerable debate and controversy in the past few years. Several reports describe remission of the clinical symptomatology in over half of the patients, and serological improvement with a decrease of antinuclear antibodies (ANA), following removal of the silicone implant material. These support a causal relationship between the silicone implant and the clinical manifestations11-16. The pathogenic mechanisms underlying this association have not been elucidated, although accumulated evidence suggests the presence of underlying autoimmune mechanisms. Hypergammaglobulinemia and rheumatoid factor (RF) were reported early on. Most recently ANA have been found in over half of the patients17-19. Most studies, however, have included only a small number of patients and/or a highly selected population. The main objective of our study was to evaluate the prevalence of autoimmunity as assessed by the presence of ANA in a large number of patients with silicone breast implants. MATERIALS AND METHODS Patients. From January, 1991 to October, 1993, 813 patients were seen. These patients had been referred for evaluation of rheumatic complaint to the Section of Rheumatology, LSU Medical Center. Within 24 h of drawing blood, sera were processed for autoantibody testing: fluorescent anti- nuclear antibody, anti-centromere, anti-dsDNA, anti-RNP, anti-SSA, anti- SSB, and anti-Sm antibodies, anti-Scl-70, and Pm-Scl. Some patients were also tested for RF, and C-reactive protein (CRP). ANA determination. Indirect immunofluorescence (IIF) using both mouse kidney and a HEp-2 cell line as substrates was performed. Fluorescence intensity was graded on a scale of 0 (none) to 4 (brilliant)20. For mouse kidney, ANA were considered positive at a 1:64 dilution (> 1 + intensity) and HEp-2 ANA at a 1:40 dilution (>2 + intensity)21. During the same period 264 women, with a mean age of 45 years (range 21-69) were referred to our laboratory for ANA testing. One hundred forty-two had a diagnosis of fibromyalgia, while the remaining 122 had soft tissue rheumatism, including tendinitis, bursitis, and back pain. This group served as a control population. ANA positivity was found in 7.6%, with speckled pattern present in 96%. None had distinct ANA. There were 37 women, aged 40-44, and in this group ANA positivity was found in 7 out of the 37 (18. 8%). Anti-SSA (Ro), SSB (La), Sm, RNP, Scl-70, and Pm/Scl antibodies were tested using a modified Ouchterlony double diffusion technique22, using human spleen extract (American Biochemical, Inc.) as the source of SSA (Ro) antigen and rabbit thymus extract (Pel-Freeze Biologicals, Rogers, AK) as the source of the other antigens. Prototype sera with known antibodies were used as reference. Anti-dsDNA antibody determination. Anti-dsDNA antibodies were measured by a standard IIF technique, using Crithidia luciliae slides (Sanofi Diagnostics Pasteur, Inc. Chaska, MN), FITC conjugated goat antihuman globulins, and Evans blue counterstain23. Sera were screened at an initial dilution of 1:10. ELISA for dsDNA testing. According to the method described by Gharavi, et al 24, an ELISA for dsDNA testing was also used. Microtiter plates were coated with poly-L-lysin 50 µg/ml in phosphate buffered saline (PBS) for 2 h, washed with PBS and coated with calf thymus DNA 100 µg/ml in DNA buffer (Tris 10 x, EDTA 1 mM pH 7.4) overnight at 4°C. DNA was digested with S1-nuclease 0.05 M Nacl, 0.1 mM ZnCl2, and 5% glycerol pH 4.6, 60 min at 37°C. Plates were washed and then blocked with 10% adult bovine serum in PBS 60 min at room temperature. For the standard curve, a known strong positive anti-dsDNA serum in dilutions beginning 1:100 to 1:12,800 in ABS was used. Samples of patients and controls were diluted with ABS 10%, and after incubation, conjugate (goat antihuman IgG 1:1000) was added. Optical density (OD) values greater than 3 standard deviations (SD) above the mean of a group of 15 controls were considered positive. RF and CRP determination. RP was detected by latex agglutination (Behring Diagnostics, Inc. Somerville, NJ) and positive samples quantitated by ELISA (Sigma Diagnostics, St. Louis, MO). CRP was detected by turbidimetry (Behring Diagnostics, Somerville, NJ) with a normal value of 0.6-1.5 mg/dl. RESULTS
Patients. This study included 813 patients; all were white except for 2 Hispanics and 1 black. Three patients were transsexual males; 810 were women. The average patient age was 46.2 years, with a range of 17 to 72 years. Patients' symptoms varied and included extreme fatigue, diffuse muscle pain, flu-like illness, fever, and hair loss. Other patients had symptoms referable to more specific conditions which include RA, scleroderma, lupus-like syndrome, Raynaud's phenomenon (RP), livedo reticularis, and angioneurotic edema25. ANA determination. Most patients, 470 (57.8%), were positive for ANA using HEp-2 cells as substrate, and 244 (30%) patients were positive with mouse kidney as substrate. The most common pattern observed was speckled and fine speckled, 341 (72.5%), homogeneous in 113 (24%); nucleolar pattern was observed in 63 (13.4%) and mitochondrial and cytoplasmic in 35 (7.4%) and 18 (3.8%), respectively; 5 (1.06%) were anticentromere. Initial testing in our laboratory had given a higher anticentromere antibody prevalence, but further analysis of the anticentromere sera showed that only 5 of the original sera were true anticentromere antibodies. All of the remaining sera gave a fine, dense speckled pattern that was initially confused with anticentromere antibodies26. The specificity of the anticentromere antibodies was confirmed by immunoblotting (data not shown, courtesy of Dr. E. Tan, Scripps Clinic, La Jolla, CA), (Tables 1 and 2). Several patients had more than one pattern, the homogeneous-antichromosal combination being the most common27. Nucleolar pattern was observed with speckled and homogeneous pattern in 55 and 23 % of cases, respectively. ANA profiles. Only a few patients exhibited seropositive ANA profiles. Two patients were anti-dsDNA positive when IIF with Crithidia luciliae was performed, 3 were positive for anti-SSA and SSB, one for anti-RNP, one for anti-Scl-70, and 2 patients for Pm/Scl antibodies (Table 3). dsDNA testing. When an ELISA assay was used for anti dsDNA antibodies, 6 of 71 patients tested 3 SD above the median of 15 control subjects (OD 0.418) (Figure 1). Table 1. Frequency of ANA in 813 silicone breast implant recipients
1+, 2+ = fluorescence intensity, mk = mouse kidney. Table 2. Frequency of ANA pattern of 470 positive ANA sera of patients with silicone breast implants
*Homogeneous/speckled pattern was seen in 2% of the cases. Table 3. Autoantibody specificity of 470 positive ANA sera of silicone breast implant recipients
IIF = indirect immunofluorescence; ID = double immunodiffusion. Fig. 1. ELISA for dsDNA antibodies in patients with silicone breast implants and healthy controls. Values above 0.418 OD (3 SD above the mean in 15 healthy subjects) were considered positive. Table 4 summarizes available data on ANA testing from several published reports. RF and CRP. RF and CRP were found in about 10% of patients tested. Similar results have been found by others. (Data not shown). DISCUSSION
In the United States, silicone breast implants were first used in 1962, and to date, about one million women have undergone breast augmentation with silicone implants28. The use, however, of biological medical silicone products for several medical and cosmetic purposes is much more widespread29. When first introduced, the notion that silicone implants did not appear to elicit a biological response found great appeal. In recent years, however, evidence has mounted suggesting that silicone products may not be totally biologically inert and may actually induce a variety of tissue and immunological reactions. In vivo degradation of silicone has been demonstrated29. A nuclear magnetic resonance spectroscopy study in animal models has shown the formation of metabolic active products including silica following injection and silicone gel implant placement30. Table 4. ANA findings in different series of patients with silicone breast implants
n = immunofluorescence; rat 1 = rat liver; WB = western blot. The implant generally used, until recently was a silicone shell filled with silicone gel. Both components are a heterogeneous combination of polymers of varying chain lengths and a configuration of polydimethylalloxane (silicone), mineral fillers, catalyst, and solvent residuals31. Local tissue reactions were recognized first. Silicone migration to a variety of tissues and lymphadenopathy with foreign body granuloma formation have been well described32. The presence of macrophages, fibroblasts, and lymphocytes is characteristic in this type of foreign body response. Furthermore, experimental animal models show that the chemical components present in the implant, when considered individually or as extracts, are not inert, as reflected by the degree of the animal's granulomatous response and induction of cells derived from the immune system32. Whether the immune system directly participates in the pathogenesis of the observed inflammatory reactions has been difficult to prove. Recent accumulated evidence in experimental animal models, however, supports an active role for the immune system in the pathogenesis of the reactions observed. Kossovsky, et al33, first demonstrated the immunogenicity of silicone-protein complexes. In their model, they suggest that protein-silicone interaction yields an immunogenic moiety. These protein-silicone complexes in animals can evoke an inflammatory reaction consistent with a delayed hypersensitivity response. Results obtained by Nairn, et al further support the theory of an active role for the immune system in the pathogenesis of the inflammatory reactions observed34. They compared the immune adjuvanticity effects of silicone gel with that of Freund's adjuvant; using bovine serum albumin as the test antigen in rats. Their results showed that silicone gel is a potent immunological adjuvant, compared to both complete and incomplete Freund's adjuvant. Similar observations have been made in humans. The presence of antibodies to silicone elastomers was recently demonstrated in the sera of 2 patients with ventriculoperitoneal shunts35. The 2 patients described experienced intense inflammatory reactions at the sites of silicone implants and silicone coated sutures. More interesting, as compared with controls and patients without inflammatory reactions, their serum samples contained IgG that bound to the tubing to a greater extent. More recently, an observation of scleroderma-like esophageal disease in 6 of 8 children breast fed from mothers with breast implants was reported. Abnormal motility, decreased lower esophageal sphincter pressure and mild to moderate chronic inflammation were demonstrated; serologic abnormalities such as positive ANA (nucleolar pattern) were also noted in 3 of them36. These observations seem to indicate that silicone gel may trigger autoimmune reactions in patients with implants. In addition to the reported clinical findings suggestive of CTD, a series of immunological abnormalities including lymphocyte subsets and immunoglobulin isotopes37, abnormal levels of soluble interleukin-2 receptor (sIL-2R)38, and ANA with the same specificities as seen in the distinct CTD all have been reported39-42. In the latter, Claman, et al42 carried out a cross sectional survey in 150 women, of whom 131 had implants. Four groups were studied. Group 0 consisted of 19 healthy volunteer women without breast implants, Group 1 38 healthy volunteer women with silicone breast implants, Group 2 82 women with implants who had various symptoms, and Group 3 11 women with implants who had autoimmune disease. Scleroderma was overrepresented in Group 3 (6 of 11). ANA were determined on HEp-2 cells, and a positive test was indicated by 1 + or more fluorescence at 1:256. ANA were positive in 0% of Group 0, 18% of Group 1 (p <0.05 versus Group 0), 26% of 2, and 64% of Group 3. Our findings confirm, and expand these abnormal immunological findings in a large number of patients with silicone breast implants. The data indicate a high prevalence of ANA in individuals with silicone breast implants regardless of the clinical presentation. The high incidence of antinucleolar and anticentromere antibodies, highly specific for the scleroderma related group of disorders, have particular relevance. Anti- centromere antibodies were found in 5 of 813 (0.61 %) silicone breast implants. This figure is several times higher than that reported in normal individuals. Fritzler, et al19 only found 2 patients positive for anticentromere antibodies in 2500 (0.08%) healthy controls studied. Only one patient with anticentromere antibody had RP. The remaining 4 patients with anticentromere antibody had ill defined CTD characterized by livedo reticularis (one), chronic fatigue (2), and the remaining one oligoarthritis fever, and hair loss. Sili cone breast implant related disorders should be added to the list of CTD associated with the presence of anticentromere antibodies43. The low frequency of Pm/Scl seen in this study and also noted by Press, et al18 suggests that other nucleolar antigenic specificities account for the nucleolar pattern reactivity. Anti-dsDNA antibodies and RF were also found in a small proportion of patients in agreement with previous observations17,18. There were a few reported studies with lower incidence of ANA in silicone breast implant patients. These studies, however, differ from the others in patient selection, smaller number of patients, high incidence of ANA in the control population, and a higher serum dilution than the recommended 1:40 for ANA on HEp-2 cells20. In conclusion, our findings demonstrate a high prevalence of ANA positivity in patients with silicone breast implants. This provides further support for the existence of autoimmune mechanisms in the pathogenesis of the clinical manifestations seen in this population of patients. Controlled prospective studies are needed, however, to evaluate the exact role of silicone in the induction of these serological and clinical abnormalities. The role, if any, of genetic factors including HLA-DR antigenic determinants deserves evaluation. Studies of this nature may contribute to newer insights into the pathogenesis of and therapeutic approaches to these and related disorders. ACKNOWLEDGMENT
The authors appreciate the assistance of Julie Basco in the preparation of the manuscript. REFERENCES
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