Identification of retroviral DNA sequences in serum of cutaneous forms of lupus erythematosus patients

ARTICLE

Auteur(s) : Janusz PROKOP¹, Pawel P. JAGODZINSKI²

¹Department of Dermatology.
²Department of Biochemistry and Molecular Biology
University of Medical Sciences, 6 Świecickiego St., 60-781 Poznań, Poland

Article accepted on 09/04/2003

Lupus erythematosus (LE) is an autoimmune disease of unknown etiopathogenesis that can be divided into cutaneous and systemic forms of LE [1]. The cutaneous forms of lupus erythematosus are discoid LE (DLE), discoid disseminated LE (DDLE) and subacute cutaneous LE (SCLE) [2].

It has been observed that occupational exposure, drugs, chemicals, food, viruses and other infectious factors might result in profound changes of the immune system [3, 4]. Alterations in the immune system include the production of autoantibodies with different specificities, changes of T cell function, oncogene expression as well as failure of phagocytosis [5]. Three major mechanisms might cause the initiation and promotion of LE: increased amounts of nuclear autoantigens as well as abnormal presentation of them, T-cell-dependent stimulation of B cells for the biosynthesis of antinuclear antibodies (ANA), damage of tissues mediated by anti-double stranded DNA (anti-dsDNA) antibodies and immune complexes [5-7].

It has been reported that increased apoptosis or the decreased efficiency of removal of apoptotic cells may contribute to the formation of necrotic cells and the production of antibodies directed against nuclear antigen [8]. The nuclear autoantigens were also found in the surface blebs of apoptotic cells and were accessible to immune cells [8-11]. It has been observed that the transformation of the cutaneous form of LE to systemic lupus erythematosus (SLE) is associated with the elevation of anti-dsDNA antibody production and increase of tissue damage [12, 13]. The cationic anti-dsDNA antibodies may bind to heparan-sulfate, which is a major constituent of glomerular basement membranes, and result in the development of renal disease [12,13].

It has been suggested that expression of certain endogenous retroviral components may mimic the nuclear autoantigens and result in production of cationic anti-dsDNA autoantibodies [14]. Human endogenous retroviruses (HERV), that generally do not undergo through an extracellular phase, constitute 0.1% to 0.6% of the human DNA genome [15]. Expression of human endogenous or exogenous viral components and improper CD4⁺T cell dependent B cell activation may result in breakdown of self-tolerance and induction of LE related autoimmune disease [14, 16]. It has been reported that expression of HERV proteins may correlate with development of autoimmune diseases including SLE [17, 18].

In the present study, we attempted to determine the relationship between production of ANA, anti-dsDNA, anti-single stranded DNA (anti-ssDNA) antibodies and the presence of HIV-1 pol sequences in DNA isolated from serum of DLE, DDLE and SCLE patients.

Materials and Methods

Patients

The investigated groups included DLE (n = 85), DDLE (n = 51) and SCLE (n = 22) patients diagnosed according to revised criteria of the 1982 American Rheumatism Association [1, 2]. Control samples were obtained from age and sex matched normal healthy volunteers. The patients of investigated groups and healthy volunteers were HIV-1 and HIV-2 negative.

Antinuclear antibodies detection and determination of circulating immune complexes (CIC) concentration

The presence of ANA in the serum of LE patients was determined by a test which uses mitotic human epithelioid cells (Hep-2) as a substrate (Immuno Concepts, Sacramento, USA) [19].
The presence of anti-dsDNA and anti-ssDNA in the serum of LE patients was detected by a staining test of the kinetoplast within the organism Crithidia luciliae (Immuno Concepts Sacramento, USA) [20].
The concentration of CIC in peripheral blood serum samples was determined by enzyme linked immunosorbent assay (ELISA) utilising monoclonal anti-C1q antibody (DRG International Inc. Mountainside, USA). Results were expressed as μg/ml equivalent to heat-aggregated IgG. CIC concentration values higher than 40 μg/ml in the anti-C1q method were regarded as abnormal.

Isolation of DNA from serum of LE patients and healthy individuals

Serum samples were mixed with polyethylene glycol (PEG) and 2.5 M NaCl solution and then were centrifuged at 4 °C [21]. The supernatants were discarded and pellets were resuspended in buffer, containing 10 mM Tris-HCl (pH 8. 3), ethylenediaminetetraacetic acid (EDTA), 0.2% sodium dodecyl sulphate (SDS) and 50 μg/ml of proteinase K. Samples were incubated over night at room temperature and the mixture was extracted with phenol: chloroform: isoamyl alcohol method [21]. The aqueous phase containing nucleic acids was collected, mixed with equal volume of cold isopropanol and centrifugated. The DNA pellets were washed by cold 70% ethanol and dried at room temperature. To decompose the RNA traces, the DNA samples were incubated for 24 hr at 37 °C with 1 ml Rnase (5 mg/ml) and dissolved in 0.1 M NaOH solution.

Dot blot hybridisation

The DNA samples were applied to Hybond-N filters (Bio-Rad, Mnchen, Germany) 30 μg per dot and washed with SSC buffer (3 M NaCl; 0.3 M sodium citrate, pH 7.0). Filters were kept for 6 hr at 42-43 ⁰C in prehybridization solution (50% formamide, 6xSSC, 5xDenhard’s, 0.1% SDS, and 1 mg/ml salmon sperm DNA) prior to hybridization with a ³²P-labeled single stranded DNA probe (196 bp).
The probe sequence corresponds to the HIV-1 pol fragment: (5’-ATA AAC AAT GAG ACA CCA GGG ATT AGA TAT CAG TAC AAT GTG CTT CCA CAG GGA TGG AAA GGA TCA CCA GCA ATA TTC CAA AGT AGC ATG ACA AAA ATC TTA GAG CCT TTT AGA AAA CAA AAT CCA GAC ATA GTT ATC TAT CAA TAC ATG GAT GAT TTG TAT GTA GGA TCT GAC TTA GAA ATA GGG CAG CAT AGA A-3’) [21]. This HIV-1 pol fragment corresponds to conservative sequence of retroviruses genome and the similarity between this probe and corresponding HERV sequences was found to be in the range of 60-88% [21]. The hybridisation was conducted for 24 hr at 65 ⁰C. Then filters were washed three times, dried and exposed to the film (DuPont NEN Company, Boston, USA) (Fig. 1). The DNA isolation and hybridisation procedures were performed in triplicate.

Statistical analysis

The statistical significant of the association between positive results of the dot blot hybridization and the presence of ANA, anti-dsDNA and anti-ssDNA antibodies was evaluated by the v-square, Yates-corrected chi-square or Fisher’s exact test. The strength of the association was assessed by

Φ = √χ²/N  [22]

Results

The presence of antinuclear antibodies in serum of LE patients

Our studies revealed that in the group of DLE patients (n = 85): 19 (22.4%), 11 (12.9%), 5 (5.9%) were positive for ANA, anti-dsDNA and anti-ssDNA antibodies respectively. In the same group, 66 (77.6%) patients exhibited lack of ANA antibodies in serum (Table I). In the group of DDLE patients (n = 51), ANA, anti-dsDNA and anti-ssDNA antibodies were found in 15 (29.4%), 8 (15.7%), 5 (9.8%) subjects, respectively. We also observed that in this group 36 (70.6%) patients did not produce the ANA antibodies (Table I). The determination of antibody production in the group of SCLE patients (n = 22) showed that 17 (77.3%), 7 (31.8%) and 4 (18.2%) were respectively positive for ANA, anti-dsDNA and anti-ssDNA antibodies (Table I). We were not able to detect ANA antibodies in 5 (22.7%) SCLE patients. We found that most of ANA positive individuals exhibited the presence of anti-dsDNA and anti-ssDNA antibodies (Table I). We also determined that ANA, anti-dsDNA and anti-ssDNA antibodies in the groups of DLE, DDLE and SCLE patients mainly belonged to the IgG class.

Table I. ANA, anti-dsDNA and anti-ssDNA antibodies in the groups of DLE, DDLE and SCLE patients.

The correlation between ANA, anti-dsDNA and anti-ssDNA antibodies and the presence of HIV-1 pol DNA sequences in the serum of LE patients

We attempted to determine the relationship between the production of ANA, anti-dsDNA, anti-ssDNA antibodies and the presence of homologous HIV-1 pol sequences in DNA isolated from the serum of cutaneous form of LE patients.
We found that 47.0% of ANA^-, 78.9% of ANA⁺, 81.8% of anti-dsDNA and 100% of anti-ssDNA positive DLE patients exhibited a strong reaction with pol probes (Table II). In the group of DDLE patients, we observed that 47.2% of ANA^– subjects exhibited a positive reaction with pol sequences (Table II). In the same group, we also found that 80.0% of ANA⁺, 100% of anti-dsDNA and 100% of anti-ssDNA positive patients of DDLE exhibited a presence of HIV-1 pol sequences (Table II). In the group of SCLE patients 20.0% of ANA , 82.4% of ANA⁺, 100% of anti-dsDNA and 100% of anti-ssDNA exhibited the presence of HIV-1 pol sequences (Table II). In the control group we did not identify homologous HIV-1 pol sequences as well as ANA, anti-dsDNA and anti-ssDNA antibodies.

Table II. Correlation between the biosynthesis of ANA, anti-dsDNA and anti-ssDNA antibodies and the presence of HIV-1 pol sequence.

In the group of SCLE patients we observed the greatest strength of association (Φ) between the presence of anti-dsDNA, anti-ssDNA antibodies and a positive result of dot blot hybridisation, with Φ being equal to 0.837 and 0.800 (Table III). However, in the groups of DDLE and DLE patients, strength of association between production of anti-dsDNA and anti-ssDNA antibodies and a positive result of dot blot hybridisation was respectively two to four times lower than in the group of SCLE patients (Table III).

Table III. Correlation between the presence of HIV-1 pol sequences and the biosynthesis of ANA, anti-dsDNA and anti-ssDNA antibodies in DLE, DDLE and SCLE patients.

Measurement of CIC concentration in serum of LE patients

We found that the average concentration of CIC was higher in ANA⁺ than ANA^–  blood samples of DLE, DDLE and SCLE patients (Table IV). The concentration of CIC in ANA⁺ serum samples, which were also positive for anti-dsDNA, anti-ssDNA antibodies and dot hybridisation were found to be in the range of 42.8-58.1 μg/ml (Table IV).

Table IV. Average concentration of CIC in-groups of DLE, DDLE and SCLE patients.

Discussion

The increase of apoptosis and decrease of clearance of apoptotic cells might be responsible for the elevation of genome DNA debris in peripheral blood plasma of patients with various forms of lupus erythematosus [8-11].
We attempted to determine the correlation between the biosynthesis of ANA, anti-dsDNA and anti-ssDNA antibodies and the presence of HIV-1 pol sequences in DNA isolated from serum of DLE, DDLE and SCLE patients.
It has been reported that the presence and expression of HERV sequences in the human genome might trigger various autoimmune diseases [18]. The complete pol sequences are presented in the genome of retroviruses and encode viral enzymes such as reverse transcriptase, protease and integrase [23]. The presence of homologous HIV-1 pol sequences in DNA isolated from the serum of SLE patients has been described and may support the role of certain HERV viruses in the etiopathogenesis of SLE [17, 21].
Our results revealed a significant correlation between the biosynthesis of anti-dsDNA, anti-ssDNA antibodies and the presence of homologous HIV-1 pol sequences in DNA debris isolated from the serum of SCLE patients. In contrast, the same studies conducted in groups of DLE and DDLE patients revealed a lower correlation between anti-dsDNA, anti-ssDNA antibodies and the presence of pol sequences than in SCLE patients. We also observed that ANA⁺ serum samples, which were positive for anti-dsDNA, anti-ssDNA antibodies and pol sequences exhibited a higher concentration of CIC than other serum samples of LE patients.
Our findings suggest that the presence of pol sequences might be responsible for expression of viral components and induction of an improper immune response in the cutaneous form of LE patients. These results are similar to those of Herrman et al. [21], who reported that DNA isolated from serum of SLE patients contained homologous HIV-1 pol sequences.
Antibodies against retroviral components and particles have been detected in serum and tissues of patients with lupus erythematosus [24]. The involvement of viral sequences in autoimmune disease can be supported by the similarity of immune deregulation between lupus erythematosus patients and those infected with HIV-1 [25].
Our observations suggest that the presence of endogenous viruses or incorporation of defective exogenous viruses into the human genome might exhibit a relationship with the emergence of a cutaneous form of LE disease. The viral components mimic the various nuclear antigens and the presentation of viral antigen on the cell surface may trigger immune responses resulting in the biosynthesis of ANA, anti-dsDNA and anti-ssDNA antibodies [26]. The production level of viral components might also be one of the factors responsible for the transformation of cutaneous forms of LE to SLE [10]. Though our results show a relationship between the biosynthesis of anti-dsDNA, anti-ssDNA antibodies and the presence of homologous HIV-1 pol sequences, further studies are required to determine the antigens which may participate in the development of these autoimmune diseases.

Acknowledgements. Supported by a grant No. 6PO5B09221 from the State Committee for Scientific Research (KBN).

References

1. Wolska H, Blaszczyk M, Jablonska S. Phototests in patients with various forms of lupus erythematosus. Int J Dermatol 1989; 28: 98-103.

2. Beutner EH, Blaszczyk M, Jablonska S, Chorzelski TP, Kumar V, Wolska H. Studies on criteria of the European Academy of Dermatology and Venerology for the classification of cutaneous lupus erythematosus. I. Selection of clinical groups and study factors. Int J Dermatol 1991; 130: 411-27.

3. Love LA. New environmental agents associated with lupus-like disorders. Lupus 1994; 3: 467-71.

4. Norris DA. Pathomechanisms of photosensitive lupus erythematosus. J Invest Dermatol 1993; 100: 58-68.

5. Kalden JR. Defective phagocytosis of apoptotic cells: possible explanation for the induction of autoantibodies in SLE. Lupus 1997; 6: 326-7.

6. Casciola-Rosen L, Rosen A. Ultraviolet light-induced keratinocyte apoptosis: a potential mechanism for the induction of skin lesions and autoantibody production in LE. Lupus 1997; 6: 175-80.

7. Kalden JR, Winkler TH, Herrmann M, Krapf F. Pathogenesis of SLE: immunopathology in man. Rheumatol Int 1991; 11: 95-100.

8. Nakajima M, Nakajima A, Kayagaki N, Honda M, Yagita H, Okumura K. Expression of Fas ligand and its receptor in cutaneous lupus: implication in tissue injury. Clin Immunol Immunopathol. 1997; 83: 223-9.

9. Rosen A, Casciola-Rosen L, Ahearn J. Novel packages of viral and self-antigens are generated during apoptosis. J Exp Med 1995; 181: 1557-61.

10. Provost TT, Watson R. Anti-Ro (SS-A) HLA-DR3-positive women: the interrelationship between some ANA negative, SS, SCLE, and NLE mothers and SS/LE overlap female patients. J Invest Dermatol 1993; 100: 14-20.

11. Datta SK, Patel H, Berry D. Induction of a cationic shift in IgG anti-DNA autoantibodies. Role of T helper cells with classical and novel phenotypes in three murine models of lupus nephritis. J Exp Med 1987; 165: 1252-68.

12. Pirner K, Rascu A, Nurnberg W, Rubbert A, Kalden JR, Manger B. Evidence for direct anti-heparin-sulphate reactivity in sera of SLE patients. Rheumatol Int 1994; 14: 169-74.

13. Fillit H, Shibata S, Sasaki T, Spiera H, Kerr LD, Blake M. Autoantibodies to the protein core of vascular basement membrane heparan sulfate proteoglycan in systemic lupus erythematosus. Autoimmunity 1993; 14: 243-9.

14. Hishikawa T, Ogasawara H, Kaneko H, Shirasawa T, Matsuura Y, Sekigawa I, Takasaki Y, Hashimoto H, Hirose S, Handa S, Nagasawa R, Maruyama N. Detection of antibodies to a recombinant gag protein derived from human endogenous retrovirus clone 4-1 in autoimmune diseases. Viral Immunol 1997; 10: 137-47.

15. Leib-Mosch C, Brack-Werner R, Werner T, Bachmann M, Faff O, Erfle V, Hehlmann R. Endogenous retroviral elements in human DNA. Cancer Res 1990; 50: 5636-42.

16. Talal N, Garry RF, Schur PH, Alexander S, Dauphinee MJ, Livas IH, Ballester A, Takei M, Dang H. A conserved idiotype and antibodies to retroviral proteins in systemic lupus erythematosus. J Clin Invest. 1990; 85: 1866-71.

17. Ogasawara H, Naito T, Kaneko H, Hishikawa T, Sekigawa I, Hashimoto H, Kaneko Y, Yamamoto N, Maruyama N, Yamamoto N. Quantitative analyses of messenger RNA of human endogenous retrovirus in patients with systemic lupus erythematosus. J Rheumatol 2001; 28: 533-8.

18. Urnovitz HB, Murphy WH. Human endogenous retroviruses: nature, occurrence, and clinical implications in human disease. Clin Microbiol Rev 1996; 9: 72-99.

19. Weller TH, Coons AH. Fluorescent antibody studies with agents of varicella and herpes zoster propagated in vitro. Proc So Exp Biol Med 1954; 86: 789-94.

20. Simpson L. Behavior of the kinetoplast of Leishmania tarentolae upon cell rupture. J Protozool 1968; 15: 132-6.

21. Herrmann M, Kalden JR. PCR and reverse dot hybridization for the detection of endogenous retroviral transcripts. J Virol Methods 1994; 46: 333-48.

22. Armitage P. Statistical methods in medical research 3rd ed. Oxford: Blackwell Scientific Publications 1971.

23. Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 1990; 20: 213-22.

24. Haraguchi S, Good RA, Cianciolo GJ, Engelman RW, Day NK. Immunosuppressive retroviral peptides: immunopathological implications for immunosuppressive influences of retroviral infections. J Leukoc Biol 1997; 6: 654-66.

25. Koshino K, Tokano Y, Hishikawa T, Sekigawa I, Hashimoto TY. Detection of antibodies to HIV-1 gp41- and HLA class II antigen-derived peptides in SLE patients. Scand J Rheumatol 1995; 24: 288-92.

26. Perl A. Endogenous retroviruses in pathogenesis of autoimmunity. J Rheumatol 2001; 28: 461-64.