ALESSANDRA DELLAVANCE, VILMA S.T. VIANA, ELAINE P. LEON, ELOISA S.D.O. BONFA, LUÍS E.C. ANDRADE, and PAULO G. LESER

ABSTRACT.

Objective. Autoantibodies to lens epithelium-derived growth factor (LEDGF) depict a distinctive nuclear dense fine speckled (DFS) pattern in the indirect immunofluorescence antinuclear antibody assay (IIF-ANA). Definition of the clinical spectrum associated with anti-LEDGF antibodies has been evolving over the last decade. We investigated the frequency, clinical spectrum, and immunologic specificity of the DFS pattern in a general clinical laboratory routine.

Methods. All serum samples entered for IIF-ANA determination within a 2 year period were examined for the DFS pattern. Positive samples with consistent clinical information were studied further by IIF with isotype-specific conjugate and immunoblot analysis.

Results. Among 13,641 ANA-positive samples, 5081 (37%) presented the DFS pattern. Within a 6 month nested period, there were 650 samples with DFS pattern, and consistent clinical data were available for 81 of these. DFS reactivity was mainly due to IgG. Most samples (86%) presented titer ≥ 1/640. Eighty of the 81 DFS samples reacted with a 75 kDa band that comigrated with the band elicited by the standard anti-LEDGF serum. Antibodies that were affinity-purified from the 75 kDa band reproduced the DFS pattern on IIF-ANA. The clinical spectrum associated with DFS reactivity included autoimmune diseases (39%) and an array of nonautoimmune conditions (61%). Among the autoimmune patients, over half presented evidence of autoimmune thyroiditis.

Conclusion. Anti-LEDGF/p75 antibodies are a common finding among ANA-positive individuals with no evidence of rheumatic autoimmune disease, and should be regarded as a low specificity finding even when in moderate or high titer. (J Rheumatol 2005;32:2144-9)

Key Indexing Terms:

ANTINUCLEAR ANTIBODIES
AUTOANTIBODIES
AUTOANTIGENS
AUTOIMMUNITY

Supported in part by Research Funds of the Brazilian Society of Rheumatology.

A. Dellavance, MSc; P.G. Leser, MD, PhD, Fleury Research Institute; V.S.T. Viana, MSc; E.P. Leon, MSc; E.S.D.O. Bonfa, MD, PhD, Rheumatology Division, Universidade de São Paulo; L.E.C. Andrade, MD, PhD, Rheumatology Division, Universidade Federal de São Paulo.

Address reprint requests to Dr. P.G. Leser, Fleury Research Institute, Rua Gal. Waldomiro de Lima 508, São Paulo, SP, 04344-171, Brazil. E-mail: Paulo.leser@fleury.com.br

Accepted for publication June 27, 2005.

The diagnosis of autoimmune rheumatic diseases (ARD) is largely based on the combination of clinical, serologic, and radiographic findings. For many of these diseases a set of such findings has been carefully chosen as classification criteria by the American College of Rheumatology. Autoantibodies are highly prevalent in many of the ARD and have been proposed as part of the classification criteria for some ARD, including systemic lupus erythematosus (SLE), systemic sclerosis, Sjögren's syndrome, and inflammatory idiopathic myopathy^1-4. The standard test for antinuclear antibody screening is the indirect immunofluorescence assay (IIF-ANA). The sensitivity of this test has been improved due to the replacement of rodent tissue substrates by human tumor cell lines, such as HEp-2 cells. However, this improvement influenced the specificity of the test, since an increasing proportion of healthy individuals and of patients with noninflammatory clinical conditions has been shown to be ANA-positive⁵.

Although the ANA titer observed in healthy subjects tends to be lower than that observed in patients with ARD, this is a relative guideline, since many exceptions do occur. The fluorescence pattern may also be of relative help since some patterns are associated with autoantibodies that are selective for specific ARD. Some examples are the centromeric pattern that is associated with anticentromere antibodies and with the limited form of systemic sclerosis⁶ as well as the homogeneous nuclear pattern that is associated with anti-native DNA/nucleosome antibodies and SLE⁷. On the other hand some IIF-ANA patterns are associated with autoantibodies that display less stringent association with ARD and may be observed also with the sera from healthy individuals and patients with other unrelated diseases. This is the case for the nuclear discrete speckled pattern associated with antibodies to p80-coilin, which may be found in several autoimmune conditions but also in patients with no evidence of autoimmunity⁸. The same may be extended for IIF-ANA patterns associated with the Golgi system⁹ and the mitotic apparatus¹⁰.

A novel IIF-ANA pattern has been reported as a dense fine speckled (DFS) nuclear pattern associated with antibodies to a 75 kDa protein¹¹. The target antigen, initially designated DFS-70, was later shown to be the transcription coactivator p75^12,13, also termed lens epithelium-derived growth factor (LEDGF)¹⁴. A characteristic feature of the DFS pattern is that the chromosome plate of metaphase cells is nicely stained in a dense fine speckled fashion. This contrasts sharply with most fine speckled nuclear patterns that usually do not stain the metaphase chromosome plate. Antibodies to LEDGF/p75 were originally reported in patients with interstitial cystitis¹¹, atopic dermatitis^13,15, asthma¹³, Vogt-Koynagi-Harada syndrome¹⁶, and Sjögren's syndrome¹³.

In routine IIF-ANA screening with HEp-2 cells we have observed that the DFS pattern is rather common. However, our laboratory is not dedicated to patients with the clinical conditions initially reported to be associated with the DFS pattern. This observation raised the possibility that the clinical and immunological associations of the DFS pattern might be broader than originally reported. Thus we investigated the frequency of the DFS pattern in a nonbiased general laboratory to determine the clinical spectrum and the autoantibody specificity associated with this ANA pattern.

MATERIALS AND METHODS

Serum samples and clinical characterization. All serum samples screened for ANA from January 2001 to January 2003 at Fleury Clinical Pathology Laboratory were analyzed by 2 observers and classified according to the immunofluorescence pattern. From August 2002 to January 2003, all samples yielding a DFS pattern were forwarded to 2 medical consultants to elicit clinical information. Clinical characterization of a patient was considered to be reliable when contact with a physician in academic practice could be made for a definitive diagnosis. All the DFS samples for which clinical characterization was possible were further probed against whole HEp-2 cell extract by immunoblot. In all immunologic assays, serum from healthy volunteers was used as negative control and sera with known reactivity to Sm/RNP and SSA/Ro were used as positive controls. A prototype anti-LEDGF/p75 serum (associated with the DFS pattern) was kindly provided by Dr. Carlos Casiano (Loma Linda University, Loma Linda, CA, USA).

Indirect immunofluorescence. Sera were tested both in commercial HEp-2 cell slides (Kallestad^TM; Bio-Rad, Redmond, WA, USA) and in custom-made HEp-2 cell slides. All serum samples were diluted 1:80 in 0.15 M NaCl, 10 mM phosphate buffered saline (PBS), pH 7.4, and incubated with HEp-2 cells for 20 min at room temperature in a moist chamber. After washing twice in PBS for 10 min, cells were incubated with anti-human IgG goat immunoglobulin labeled with fluorescein isothiocianate (Sigma, St. Louis, MO, USA) for 20 min at room temperature in a moist chamber. After washing twice as before, slides were assembled with buffered glycerol pH 9.5 and cover slips. Similarly, IgM and IgA-specific conjugates were used for determining antibody isotypes associated with the DFS pattern. ANA titer was determined using 2-fold dilutions of the serum. Analysis was performed by 2 independent observers using a Zeiss Axio-sp fluorescence microscope under ´400 magnification.

Counterimmunoelectrophoresis. All serum samples were tested in a standard counterimmunoelectrophoresis assay against dog spleen extract¹⁷. Reference sera derived from the Centers for Disease Control (Atlanta, GA, USA) standards for anti-SSA/Ro, anti-SSB/La, anti-Sm, and anti-U1-RNP were used for identification of precipitin lines.

Immunoblotting. All DFS serum samples with consistent clinical characterization were analyzed by immunoblot against HEp-2 cell crude extract. HEp-2 cell cultures at 80%–90% confluence were washed in PBS and removed from tissue flasks with a rubber spatula, washed 3 times in PBS, and resuspended in PBS containing protease inhibitors, 1 mM PMSF, and 2 µg/ml leupeptin/aprotinin. Total cell extract was prepared by 3 freeze/thaw cycles followed by sonication, and the total protein content of the sample was determined by Lowry colorimetric assay¹⁸. HEp-2 cell extract was processed in 12.5% polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) and transferred to nitrocellulose membrane as described¹⁹. Nitrocellulose membranes were blocked with 5% skim milk in PBS (M-PBS) for 2 h at room temperature and probed with sera diluted 1:100 in M-PBS containing 0.05% Tween 20 for 1 h at room temperature. Normal human serum, ANA-positive controls, and the prototype anti-LEDGF/p75 serum were used as controls. Strips were then washed extensively in 0.05% Tween 20 in PBS (T-PBS) and incubated with alkaline phosphatase-conjugated goat anti-human IgG (Sigma) for 1 h at room temperature. After washing, the reaction was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).

Affinity purification of antibodies associated with the DFS pattern. The procedure was based on the method described by Smith and Fisher, with minor modifications²⁰. After incubation with a representative DFS serum, the right and left edge longitudinal strips of a nitrocellulose membrane were cut and processed in standard immunoblots. The 70–80 kDa region labeled by the DFS serum was removed across the remaining nitrocellulose membrane and briefly incubated with 0.1 M glycine, 0.15 M NaCl, pH 2.8. The eluate was immediately neutralized by addition of 1 M Tris pH 8.0. The affinity purified antibody was then concentrated in Amicon PM30 filters and used straight in IIF-ANA assays.

This study was approved by the Committee on Ethics in Research at the University of São Paulo.

RESULTS

From January 2001 to January 2003 a total of 30,728 serum samples were screened for IIF-ANA. The majority of the 13,641 positive samples presented antinuclear reactivity (Figure 1A and 1B). The 2 dominant nuclear patterns were the fine speckled and the DFS (Figure 1C). Interestingly, samples with the fine speckled pattern had predominantly low titer (Figure 1D), whereas samples with the DFS pattern presented a more even titer distribution (Figure 1E). Out of a total of 7733 serum samples screened during the clinical data search period (August 2002 to January 2003), 2168 were ANA-positive at 1:80 screening dilution, and 30% (650) of these presented the DFS pattern. Reliable clinical information from the attending physician was obtained for 81 of these 650 samples. The immunofluorescence reactivity depicted by the 81 DFS sera was reproducible in commercial and custom-made HEp-2 cell slides, and was mainly due to IgG antibodies (Figure 2). The majority (92%) of serum samples presented exclusively the IgG DFS isotype, whereas 4 sera displayed IgM antibody concomitantly. Two other sera had exclusive IgM binding. IgA DFS reactivity was uniformly absent. The intensity of DFS reactivity was low (≤ 1/160) in 11 (14%), intermediate (1/320) in 28 (35%), and high (≥ 1/640) in 42 (51%) of the samples. Among the 81 sera selected on the basis of the DFS pattern on IIF-ANA, 80 presented an immunoblot reactivity estimated as 75 kDa and comigrating with the band elicited by the standard anti-LEDGF/p75 serum (Figure 3). Additionally, antibodies affinity-purified from the 75 kDa band reproduced the DFS staining pattern on IIF-ANA. Antibodies affinity purified from other regions of the nitrocellulose sheet yielded no relevant IIF-ANA reactivity (data not shown).

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Figure 1. From January 2001 to January 2003, 30,728 samples were assayed for ANA. A and B. Among 13,641 positive samples, 91% yielded a nuclear IIF-ANA pattern. C. The predominant patterns among the 12,413 samples with a nuclear IIF-ANA pattern were fine speckled and DFS. The majority of samples with fine speckled pattern presented low titer reactivity (D), whereas DFS samples presented a rather even titer distribution (E).

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Figure 2. Indirect immunofluorescence on HEp-2 cells depicting the typical DFS nuclear pattern. Representative DFS serum diluted 1:100. DFS staining covers the whole interphase nucleus with some heterogeneity in density and intensity. During mitosis, DFS staining migrates to the condensed chromosome masses. Cells in telophase (T) show dense staining of chromosome masses. Cells in metaphase (M) show intense dense staining of the chromosome plate.

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Figure 3. Immunoblot with HeLa cell total extract. Lane 1: normal human serum. Lanes 2, 4, 5, 6: representative sera associated with DFS pattern. Lane 3: prototype anti-LEDGF/p75 serum.

In counterimmunoelectrophoresis, 2 DFS sera were shown to be reactive with SSA/Ro protein. These 2 sera were from patients with SLE and undifferentiated connective tissue disease (UCTD), respectively. No serum reacted with SSB/La, Sm, or U1-RNP. Three other DFS samples presented low titer (≤ 1/80) rheumatoid factor and were from patients with rheumatoid arthritis, UCTD, and urinary tract infection, respectively. The remaining 76 DFS samples had no other associated autoantibodies.

The spectrum of diseases and clinical conditions presented by the patients was broad and heterogeneous (Table 1). Interestingly, the clinical conditions originally associated with the DFS pattern, namely, interstitial cystitis and atopic dermatitis, were observed in only one and 3 patients, respectively. Autoimmune diseases were diagnosed in only 30 patients (39%) and the remaining (61%) presented nonspecific musculoskeletal complaints and miscellaneous conditions. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) data were available for 36 of the 42 patients with so-called nonspecific presentation (diffuse pain, gynecologic syndromes, and miscellaneous conditions), and slightly abnormal results were present in only 12 of these patients: one had elevated CRP (0.8 mg/dl, reference range < 0.5 mg/dl) and 12 had Westergren ESR ranging from 13 to 35 mm (mean 19 mm). Among the patients with autoimmune disorders, autoimmune thyroiditis was the most frequent, responding for almost half the cases. Remarkably, no other relevant clinical condition was observed in these patients with hypothyroidism. Women represented 88% of the DFS-positive patients. The average age was 35 years (range 6–75 yrs).

Table 1. Clinical presentation of the 81 patients whose sera displayed the dense fine speckled pattern in IIF-ANA assay with HEp-2 cells.

By analyzing the laboratory databank we could verify that the followup immune response associated with the DFS staining pattern was rather stable. Forty DFS-positive patients had blood drawn on 2 or more occasions, within an interval of 23.7 ± 14.4 months (range 1–48 mo, median 33.5). The number of tests per patient was 3 ± 1.4 (range 2–6). In all these 40 patients there was no change in the IIF-ANA pattern, and in 37 (92%) of them the ANA titer was the same or changed only one-fold. Only 2 patients presented an increase in ≥ 2-fold dilutions in successive tests. Antibody fluctuation from 1/320 to 1/160 and further to 1/640 was observed in another patient.

DISCUSSION

We analyzed the frequency of the DFS nuclear pattern in the bulk ANA routine of a general clinical laboratory and determined the clinical spectrum and the autoantibody specificity associated with this ANA pattern. We found that the DFS and the traditional fine speckled patterns were the major IIF-ANA patterns in the ANA screening routine. While the traditional fine speckled pattern was observed predominantly in low titer, the DFS pattern was observed equally in low, intermediate, and high titer. In addition, several samples presented persistent high titer DFS reactivity for several years. These findings emphasize the relevance of the DFS pattern in the clinical laboratory routine and the need for clarification of the associated autoantibody specificity and clinical significance. Among a subset of DFS sera selected sequentially we found that all but one contained antibodies reactive to a protein with electrophoretic mobility around 75 kDa. Antibodies affinity purified from this region of the nitrocellulose immunoblot displayed the original DFS nuclear pattern. The immunoblot reactivity common to all 80 DFS sera was shown to be identical to the one displayed by the prototype anti-DFS-75 serum¹¹. Together, this evidence strongly argues for the identification of this collection of sera in the DFS-75 system. The target antigen of the DFS-75 system has been identified as the nuclear transcription coactivator p75^12,13, also designated lens epithelium-derived growth factor (LEDGF)¹⁴. LEDGF/p75 is a nuclear protein identified as a survival factor that promotes growth and resistance to cell death induced by a wide spectrum of environmental stress factors including heat shock, oxidative stress, and serum deprivation²¹. LEDGF/p75 is a highly charged proline-rich protein with a predicted molecular weight of 60 kDa. Multiple sites for posttranslation phosphorylation, glycosylation, and amidation are compatible with the observed aberrant SDS-PAGE migration at around 75 kDa. LEDGF/p75 has also been shown to be a DNA-binding protein²¹, which may explain the rather dense distribution of the autoantibody staining in the interphase nucleoplasm and in the metaphase chromosome plate, as can be appreciated in IIF-ANA.

Ochs, et al originally associated anti-LEDGF/p75 antibodies with a restricted set of diseases, including atopic dermatitis (30%), interstitial cystitis (9%), and asthma (16%)^11,13. The clinical spectrum associated with anti-LEDGF/p75 antibodies has been extended to patients with Vogt-Koynagi-Harada syndrome¹⁶, Sjögren's syndrome¹³, and alopecia areata²². In our series of 81 DFS-positive patients the above diagnoses were a minority. Indeed, we have found that anti-LEDGF/p75 antibodies were rather frequent in the context of a general laboratory ANA routine and that the clinical association was quite heterogeneous, including patients with diverse systemic and organ-specific autoimmune diseases, nonspecific arthralgia, and asymptomatic subjects. Nonspecific arthralgia and hypothyroidism stand out as the most frequently associated conditions in this series. With the timeframe limitation of a cross-sectional study in mind, we were stringent in admitting clinical data only from patients for whom the physician confirmed the clinical status. In addition, 40 patients persistently presented the DFS pattern at repeat determinations within 23.7 ± 14.4 months (median 33.5 mo) before the clinical evaluation. Although these considerations argue for the consistency of the observed clinical associations, it should be noted that slightly abnormal acute phase reactant results were present in some of the patients with nonspecific clinical conditions, which might indicate undiagnosed inflammatory processes in a fraction of these patients. Most DFS-positive patients in our series were female, as reported^15,23; however, anti-LEDGF/p75 antibodies may also be observed in men, as described by Daniels, et al²⁴.

The discrepancy in the clinical associations observed in our study and in previous reports is probably due to serum selection bias. While sera examined in previous studies were obtained from sera collections of laboratories dedicated to autoimmune, allergic, and ophthalmologic diseases, the present series was derived from nonselected sera from a general laboratory ANA routine. Therefore, a broader array of clinical conditions was sampled in our study compared to reports from Ochs, et al^11,13 and Yamada, et al¹⁶. The observation that anti-LEDGF/p75 autoantibodies were not associated with a restricted set of diseases may indicate the cellular function of this particular autoantigen. Given that the LEDGF/p75 protein is upregulated by increased oxidative stress²¹, it is possible that anti-LEDGF/p75 autoantibodies represent a response to increased expression of the protein in the context of diverse chronic inflammatory conditions.

The high frequency of anti-LEDGF/p75 antibodies in a general ANA routine, as we observed, is probably a reflection of the relatively high frequency of this autoantibody specificity in the general population. This would be in accord with recent findings showing the presence of anti-LEDGF/p75 antibodies in healthy subjects. Yamada, et al detected anti-LEDGF/p75 antibodies in 5.4% of 650 blood donors¹⁶, while Watanabe, et al described these antibodies in 11% of 597 healthy hospital workers²³. In the latter study, anti-LEDGF/p75 antibodies represented 54% of the ANA reactivity in healthy subjects with a positive ANA test²³. That our findings in a Brazilian sample were similar to the observations in Asian subjects argues against ethnic restriction for this ANA pattern. The high frequency of the DFS pattern in a general laboratory ANA routine highlights the importance of correct identification of this ANA pattern and elucidation of its clinical significance. It is relevant that 80 out of 81 samples associated with the DFS nuclear pattern were reactive with anti-LEDGF/p75 in immunoblotting. Therefore, although not sufficient for the definition of anti-LEDGF/p75 antibodies, the DFS nuclear pattern seems to be highly suggestive of this autoantibody specificity.

We demonstrate that anti-LEDGF/p75 antibodies are very common among ANA-positive patients examined in a general clinical laboratory and have a wide spectrum of associated clinical conditions. The observation that over half the anti-LEDGF/p75-positive individuals had no defined disease is striking because they usually presented persistently moderate to high DFS-ANA titer. As a practical corollary we suggest that a positive ANA test with a DFS nuclear IIF-ANA pattern should be regarded as a finding of unremarkable specificity even when in moderate or high titer.

ACKNOWLEDGMENT

We thank Cleonice Bueno and Francisca V. Souza for technical assistance. We also thank Dr. Carlos Casiano, Loma Linda University, Loma Linda, California, USA, for the kind donation of standard anti-LEDGF/p75 serum.

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