in F1 mice undergoing the graft-vs-host reaction - NCBI

Clin. exp. Immunol. (1987) 69, 385-393

Specificity of anti-nuclear antibodies induced in F1 mice undergoing the graft-vs-host reaction: isotypes and cross-reactivities M. KIMURA, S. IDA, K. SHIMADA & Y. KANAI* Department of Internal Medicine, and *Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

(Acceptedfor publication 19 February 1987)

SUMMARY

(C57BL/6 x DBA/2)F1 mice undergoing the graft-vs-host reaction (GVHR) produce autoantibodies after the injection of DBA/2 lymphoid cells. The anti-nuclear antibodies, including anti-poly (ADP-ribose) and anti-extractable nuclear antigens (ENA), in the sera of the autoimmune GVH F1 mice were investigated. Antibodies to double-stranded DNA, single-stranded DNA and ENA were predominantly IgG. In contrast, the autoantibodies to poly(ADP-ribose) were both IgG and IgM, although the former was predominant. These autoantibodies induced by the GVHR showed similar cross-reactivities with a number of nucleic acids to the monoclonal and some serum antinuclear antibodies derived from mice or humans with systemic lupus erythematosus (SLE). These results support the idea that GVH F, mice are a good model of human SLE. Keywords anti-nuclear antibodies GVH F1 mouse cross-reactivities

INTRODUCTION

Circulating autoantibodies including anti-nuclear antibodies (Gleichmann, van Elven & van der Veen, 1982) as well as immune-complex glomerulonephritis (Gleichmann et al., 1972) are elicited by the murine graft-vs-host reaction (GVHR) that occurs in genetically normal F1 hybrid mice on injection of parental strain lymphoid cells. Thus F. mice showing the GVHR (GVH F1) are considered to be good models of human systemic lupus erythematosus (SLE). The cellular mechanism responsible for this GVHR-induced autoimmunity has been studied extensively by Gleichmann's group, who postulated that it is due to the interaction of donor-derived alloreactive helper T cells carrying the Lyt-l +2- surface phenotype and recipient-derived self-reactive B cells (Gleichmann et al., 1984). All the autoantibodies to nuclear antigens thus far shown to be produced in GVH F1 mice are anti-double-stranded (ds) DNA (van Elven et al., 1981), anti-single-stranded (ss) DNA and antihistones (Portanova, Claman & Kotzin, 1985). Antibodies to poly(ADP-ribose) or extractable nuclear antigens (ENA) in GVH F1 mice have not yet been examined. Moreover, there have been no studies on the immunoglobulin (Ig) classes and the fine specificities of the antibodies to clarify to what extent GVH F1 mice resemble patients with SLE. In this work we first measured the changes with time in autoantibodies to dsDNA, ssDNA, Correspondence: Dr M. Kimura, Department of Internal Medicine, Institute of Medical Science, University of Tokyo. 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan.

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poly(ADP-ribose) and ENA in GVH sera by enzyme-linked imunosorbent assays (ELISA), and then studied their cross-reactivities with other nucleic acid antigens by inhibition ELISA. MATERIALS AND METHODS GVH F, mice. Female (C57BL/6 x DBA/2)F1 mice of 8-12 weeks of age were injected intravenously with 50 x 106 female DBA/2 spleen cells twice with an interval of 1 week in between. All the mice were purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan). Sera were obtained by retro-orbital puncture at 2-3 week intervals and stored at -20'C until use. Preparation of antigens. dsDNA was obtained from calf thymus DNA (Sigman, St Louis, Mo, USA), treated with nuclease S1 by the method of Stollar & Papalian (1980) and purified on a hydroxylapatite column. ssDNA was obtained by heating the purified dsDNA to 100 C for 10 min and then cooling it rapidly to 4 C. Poly(ADP-ribose) was synthesized from nicotinamide adenine dinucleotide (NAD) by calf thymus nuclei and the fraction containing DNA and RNA was digested extensively with DNaseI and RNase, and treated with phenol; poly(ADP-ribose) with more than 20 ADP-ribosyl units was purified on a hydroxylapatite column (Kanai et al., 1980). ENA was extracted from nuclei isolated from calf thymus by a modification of the method of Holman (1965) and Lerner & Steitz (1979): during extraction, an RNase inhibitor, diethylpyrocarbonate (Sigma), and a protease inhibitor, phenylmethylsulphonyl fluoride (Sigma), were included in the buffer. The synthetic polynucleotides poly(I) and poly(dT) were purchased from Sigma. ELISA. ELISA for detection of antibodies to nucleic acids was carried out by a modification of the method described by Kanai et al. (1982). Volumes of 50 p1 of a solution containing 50 ng nucleic acids in Tris-buffered saline (25 mm Tris, 140 mm NaCI, pH 7-4) (TBS) were placed in wells of polystyrene microtitre plates (Immulon 2, Dynatech, Alexandria, VA, USA) that had previously been coated with poly-L-lysine. The plates were dried overnight in an incubator at 37°C, then washed four times with TBS. All following procedures were carried out at room temperature. Residual binding sites of the wells were blocked by incubating the plates with 100 p1 of 5% fetal bovine serum in TBS (FBS-TBS) for I hr. Then the plates were washed four times with 0-05% Tween 20 in TBS (TW-TBS). Sera were diluted 1:20 with FBS-TBS, and volumes of 50 p1 of each dilution were incubated in the wells for 1 h. The wells were washed four times with TW-TBS, and incubated with 50 Sp of alkaline phosphatase-anti-mouse IgG Fc (Pel-Freez, Rogers, AR, USA) or anti-mouse IgM (p-chain specific; Sigma) for I h. They were washed again four times with TW-TBS, and incubated with 100 p1 of substrate p-nitrophenylphosphate (2 5 mm in 50 mm carbonate buffer with 2 mm Mg2+, pH 9 5) at 37 C. Antibody activity was determined by measuring the absorbance at 405 nm, and was expressed as A405 units. For ELISA to detect anti-ENA antibodies, wells of polyvinyl microtitre plates (Sumitomo, Tokyo, Japan) were coated with ENA at 2 5 pg/50 P1 in 10 mm carbonate buffer (pH 9 5) at 4°C overnight. Subsequent procedures were as for assay of antibodies to nucleic acids. Inhibition ELISA. GVH sera that showed high antibody titres were pooled and the y-globulin fraction was obtained by fractionation with half saturated ammonium sulphate. A linear increase in A405 units was observed with concentrations of 0 1-05 mg/ml, so a final protein concentration of0 4 mg/ml was used in the inhibition ELISA. Samples were incubated with various amounts of inhibitors at room temperature for 1 h, and then volumes of 50 pl of reaction mixture were placed in nucleic acid-coated microtitre plates. Residual antibody activity was assayed as described above. RESULTS

Production ofautoantibodies to nuclear antigens. IgG antibodies to dsDNA were produced in the sera of GVH F. mice, their levels being maximal 2 weeks after induction of GVHR: the antibody titre of normal F1 mice was 0-0{04 A405 unit, and that of GVH F1 mice was 0 10-045 A405 units in week 2 after GVH induction (Fig. I a). No significant production of IgM antibodies to dsDNA was observed (Fig. 1 b).

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IgG antibodies to ssDNA were also detected with maximal levels 5 weeks after induction of GVHR: their level was 0-0 10 A405 units in normal F. mice and 0-42- > 2 in GVH F. mice in week 5 (Fig. 2a). IgM antibodies to ssDNA were not produced in significant amounts (Fig. 2b). High titres of IgG antibodies of poly(ADP-ribose) were obtained 2 to 7 weeks after induction of GVHR: their level was 0-0 5 A405 units in normal F1 mice, and 0 16-0 56 A405 units in GVH F, mice in week 5 (Fig. 3a). However, the titres of IgM antibodies to poly(ADP-ribose) were high in both normal and GVH F. mice, and several GVH F. mice seemed to have fairly high levels of antibodies to poly(ADP-ribose) in weeks 11 and 13 (Fig. 3b). In addition to antibodies to nucleic acid antigens, IgG antibodies to ENA also reached maximal level in week 5 after GVH induction (Fig. 4a): their level was 0-002 A405 units in normal F. mice and 0 10-0-42 A405 units in GVH F. mice in week 5. No IgM antibodies to ENA were detected during the observation period (Fig. 4b). Inhibition ELISA. IgG anti-dsDNA antibodies were effectively inhibited by dsDNA, ssDNA and poly(dT). Poly(ADP-ribose) was also inhibitory, although its inhibition was biphasic. Poly(I) was inhibitory only at low concentrations (Fig. 5a). IgG anti-ssDNA antibodies were inhibited dose-dependently by ssDNA and poly(I). Poly(dT) seemed to be inhibitory at high concentrations, but dsDNA and poly(ADP-ribose) were not inhibitory (Fig. Sb). IgG anti-poly(ADP-ribose) antibodies were inhibited by poly(ADP-ribose), dsDNA and

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ssDNA, although higher concentrations of the two latter than of poly(ADP-ribose) were required for inhibition. Poly(I) and poly(dT) were inhibitory only at low concentrations (Fig. Sc). DISCUSSION

The validity of GVH F. mice as a model for human SLE has been based on the demonstration that they produce autoantibodies to erythrocytes, thymocytes, dsDNA (van Elven et al., 1981; Gleichmann et al., 1982), ssDNA and histones (Portanova et al., 1985), and also that they show immune-complex glomerulonephritis resulting in profuse proteinuria and ascites (Gleichmann et al., 1972). In this work we obtained confirmatory evidence that GVH F. mice are suitable models by showing that they produce antibodies to poly(ADP-ribose) and ENA. Anti-poly(ADP-ribose) antibodies were first discovered by Kanai et al. (1977) and found to be more useful for both diagnosis (Okolie & Shall, 1979) and monitoring of the clinical activity (Morrow et al., 1982) of SLE than anti-DNA antibodies. Anti-ENA antibodies are produced in a variety of human autoimmune or collagen-vascular diseases including SLE. Although we did not examine the specificities of antiENA in detail, it will be interesting to determine whether they show anti-Sm specificity because this is regarded as a specific marker of SLE (Tan, 1978). Our chronological studies on antibody production indicated that some selective mechanism was in operation in GVH F. mice that resulted in predominant production of IgG with little, if any, IgM autoantibody; this is like human SLE, especially the acute stage (Pennenbaker, Gilliam & Kunkel, 1977). This mechanism has already been implicated (van Elven et al., 1981; Portanova et al., 1985), and was confirmed in the present experiment, except in the case of anti-poly(ADP-ribose). The

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Fig. 5. Inhibition ELISA showing the cross-reactivities of IgG antibodies to (a) dsDNA, (b) ssDNA, and (c) poly(ADP-ribose). The inhibitors used were dsDNA(@), ssDNA(A), poly(ADP-ribose) (-), poly(I) (0), and poly(dT) (A). A final concentration of 0 4 mg/ml y-globulin fraction from pooled GVH FI sera was incubated with various amounts of the inhibitors and then residual binding activity was assayed.

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selective production of circulating IgG autoantibodies would contribute to the selective deposition of IgG in the renal glomeruli of these mice (Rolink, Gleichmann & Gleichmann, 1983). Conceivably, autoantigen reactive B cells are already primed by self antigens, and thus produce mainly IgG antibodies with scarcely any IgM antibodies under the strong influence of donorderived alloreactive helper T cells (Gleichmann et al., 1984). The predominance of IgG antibody production in the GVHR was also observed by Ordal & Grumet (1972) in a study on the allogeneic effect in vivo on nonresponder mice. Chronological studies indicated the early appearance and late decrease of IgG autoantibodies in GVH F1 mice (Figs 1-4). This is consistent with the findings of Portanova et al. (1985). Proteinuria became significant 6-7 weeks after the induction of GVHR and its incidence increased with time (data not shown). From our data, we can not speculate about the pathogenetic role of these autoantibodies in causing immune-complex glomerulonephritis. We investigated the cross-reactivities of these antibodies with other nucleic acids, because there are a number of reports concerning multi-specificities of serum or monoclonal antibodies derived from humans or mice with SLE (Eilat, 1982; Shoenfeld et al., 1983; Kanai & Fujiwara, 1985; Kanai et al., 1985; Schwartz & Stollar, 1985). Some inhibition curves were not typical in that inhibition was only at low antigen concentrations, was biphasic or was not dose-dependent. The reason for these unusual inhibition curves is not clear, but a similar type of unexpected inhibition experiment was reported by others (Picazo & Tan, 1975) in which addition of putative competing antigens resulted in increased antigen-binding activity of human autoimmune sera. Further studies are required to solve this problem. Many antibodies to dsDNA are cross-reactive with ssDNA, irrespective of their serum source (Koffler et al., 1971; Stollar, 1975; Ebling & Hahn, 1980). The same is true of monoclonal antibodies (Hahn et al., 1980; Tron et al., 1980) derived from mice or humans with SLE, except for a few cases reported by Jacob & Tron (1982). In the present study too, anti-dsDNA antibodies were found to be cross-reactive with ssDNA and poly(dT). The slight inhibition of anti-dsDNA by poly(ADPribose) is discussed later. Anti-ssDNA was not inhibited at all by dsDNA or poly(ADP-ribose). The fact that dsDNA did not inhibit anti-ssDNA binding has also been observed with human SLE sera (Stollar, 1975; Picazo & Tan, 1975). Poly(I) is a major cross-reactant with monoclonal anti-DNA antibodies from patients with SLE (Shoenfeld et al., 1983) as well as murine SLE (Andrzejewski et al., 1980), and this was also the case in the present experiment. Cross-reactivity of anti-poly(ADP-ribose) with ssDNA has been observed with serum antibodies from humans (Okolie & Shall, 1979; Tauchi et al., 1986) and mice (Kanai & Fujiwara, 1985) with SLE, and also with a monoclonal antibody derived from an MRL/1 mouse (Kanai et al., 1985). Marked inhibition of poly(ADP-ribose) binding by high concentrations of dsDNA was demonstrated in GVH F. mice. This was consistent with previous reports on an SLE serum (Okolie & Shall, 1979) and autoimmune MRL/1 sera (Kanai & Fujiwara, 1985). Poly(ADP-ribose), ssDNA and dsDNA may share epitopes, judging from the findings on the ternary structures of poly(ADP-ribose) and ssDNA demonstrated by Minaga & Kun (1983) and Stollar & Papalian (1980), respectively. In this context, it is noteworthy that the patterns of inhibition of both anti-dsDNA (Fig. 5a) and anti-poly(ADP-ribose) antibodies (Fig. Sc) by various analogues seem to be roughly similar, although the inhibitions by poly(ADP-ribose) (Fig. Sa) and poly(dT) (Fig. Sc) were incomplete. Interestingly, a hybridoma derived from a C3H/He mouse after injection of poly(ADP-ribose) produced a monoclonal antibody that cross-reacted with both dsDNA and ssDNA (data not shown). This is consistent with the recent findings of Sibley, Braun & Lee (1986) on monoclonal antibodies that reacted with both poly(ADP-ribose) and DNA obtained after immunization with poly(ADP-ribose). The observed production of IgM anti-poly(ADPribose) in some GVH F. mice, mentioned above, and the probability that the three nucleic acids share epitopes suggest that poly(ADP-ribose) is an in vivo immunogen in autoimmune GVH F1 mice. The possibility that the poly(ADP-ribosyl)ation reaction in cell nuclei of GVH F. mice is a cause of release of antigen remains to be studied. In this work, the same antigen specificities, IgG predominance and cross-reactivities of antinuclear antibodies reported in humans and mice with SLE were demonstrated in GVH F1 mice. Thus our work supports the idea that GVH F1 mice are excellent models of SLE in humans.

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