The influence of susceptibility factors on the immune response to DNA

Environmental Toxicology and Pharmacology 4 (1997) 295 – 298

The influence of susceptibility factors on the immune response to DNA David S. Pisetsky 1 Di6ision of Rheumatology, Allergy and Clinical Immunology, Duke Uni6ersity Medical Center, Durham, NC 27705, USA

Abstract Susceptibility to autoimmune disease results from genetic factors that determine the pattern of immune responsiveness to self as well as foreign antigens. These factors may influence the immune response to DNA, a complex macromolecule that can induce antibody responses in normal as well as aberrant immunity. In systemic lupus erythematosus, anti-DNA antibodies target conserved sites present on all DNA and appear to arise by a T dependent mechanism. In contrast, in normal humans, anti-DNA antibodies react to non-conserved sites on certain bacterial DNA and have features suggesting induction by a T independent mechanism. The activity of bacterial DNA reflects the presence of base sequence motifs centering on an unmethylated CpG core. Because of susceptibility factors in patients with systemic lupus erythematosus, bacterial DNA may drive a response crossreactive with self DNA instead of a response specific for the foreign antigen. © 1997 Elsevier Science B.V. Keywords: Anti-DNA; Systemic lupus erythematosus; Bacterial DNA; Autoimmunity; Antigenicity

1. Introduction Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by multisystem inflammation in association with autoantibody production. Susceptibility to SLE, like other autoimmune diseases, appears multifactorial and reflects the contribution of a variety of genes that together lead to a state of abnormal responsiveness of both B and T-cells. Other susceptibility factors include gender, age and racial and ethnic group. Thus, SLE occurs primarily in women of the child-bearing years, with African-Americans displaying the most severe course (Kotzin, 1996). The serological hallmark of SLE is the expression of autoantibodies to DNA (anti-DNA). These antibodies target sites on both single stranded (ss) as well as double stranded (ds) DNA and react primarily with determinants on the DNA backbone Although DNA is an intracellular molecule, antibodies to DNA are never1

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theless pathogenic and can provoke renal injury. The formation of immune complexes, either in the circulation or with DNA bound in situ in the kidney, is the most likely mechanism for lupus nephritis (Emlen et al., 1986). While pathogenic antibodies to DNA are exclusive to SLE, normal humans can produce antibodies to DNA from various bacterial species. These antibodies react specifically with foreign DNA and do not crossreact with self DNA; as suggested by their specificity, these antibodies most likely arise from infection and are directed to sequences in bacterial DNA not present in host DNA. The relationship between antibodies to self and bacterial DNA is an emerging topic of research and may illustrate how susceptibility factors convert the response to a foreign antigen into an autoantibody response. In elucidating the pathogenesis of SLE, three theories have dominated investigation on the immunology of DNA. The first being that anti-DNA production is exclusive to SLE. Indeed, as demonstrated in many clinical studies, antibodies to DNA occur prominently in the sera of patients with SLE but are rarely present

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in the sera of normal human subjects (NHS) or patients with other inflammatory diseases. Anti-DNA is therefore a valuable diagnostic marker and has been considered a criterion in the classification of patients with this disease (Pisetsky, 1992). The second theory is that DNA is poorly immunogenic. Immunization of normal animals with mammalian DNA, even when coupled to a protein carrier and administered in adjuvant, fails to induce either an appreciable antibody response or clinical disease manifestations (Madaio et al., 1984; Pisetsky, 1993). The failure to induce autoimmunity by DNA immunization contrasts with the situation with protein autoantigens where immunization of normal animals reproduces both clinical and serological features of disease (e.g. experimental allergic encephalomyelitis). The final theory is that the anti-DNA response in SLE is antigen specific and arises from self DNA driving the production of high affinity antibodies by mechanisms that resemble the response to conventional foreign antigens. The main evidence for this idea derives from studies of the molecular properties of monoclonal anti-DNA antibodies from inbred strains of mice developing lupus-like illness. These antibodies display patterns of variable region gene expression and somatic mutation consistent with selection by DNA. In particular, many of these antibodies show a high frequency of the amino acid arginine in the third complementary determining region (CDR3) of the heavy chain. Since arginine can promote interaction with DNA, these findings suggest that DNA stimulates Bcells by a receptor driven mechanism (Radic and Weigert, 1994). These somewhat divergent theories can be reconciled by postulating that SLE patients, because of susceptibility factors, have a unique responsiveness to self DNA antigen. These factors may be genetic in origin and involve genes that determine non-specific immune responses (e.g. cytokine levels and complement components) as well as genes (e.g. MHC genes) that determine the immune response to specific self antigens. These genes may interact with environmental factors that ultimately allow the response to self antigens as well as immunologically inert molecules such as DNA (Kotzin, 1996). These considerations do not exclude the possibility that, in the setting of SLE, DNA is released from cells in a more potently immunogenic form that is not mimicked by DNA preparations used for experimental immunization.

2. Immune properties of bacterial DNA Recent investigations have revolutionized the conceptualization of DNA’s immunological activities and suggest novel possibilities for the manner in which

susceptibility factors promote anti-DNA production. At the center of this new research is the recognition that DNA is immunologically diverse and that some DNA, because of structural microheterogeneity, are potent immunostimulants. Thus, DNA from bacterial sources can induce a host of immune responses including polyclonal B-cell activation, stimulation of cytokine expression and induction to antibodies to non-conserved sties on this molecule. As such, bacterial DNA can trigger innate immunity and serve functions similar to endotoxin (Pisetsky, 1996). The range of these activities suggests that bacterial DNA could provoke deleterious reactions in susceptible individuals, whether the DNA is introduced by infection or intentional administration using DNA therapeutics. These reactions include autoantibody production, generalized immune activation as well as a shift in patterns of immune responsiveness (cellular versus humoral) that could affect host defense. The immunostimulatory properties of bacterial DNA were first discovered in studies on the induction of tumor resistance in mice by an extract of Mycobacterium bovis BCG. This extract, called MY-1, was composed of DNA and RNA, although the antitumor activity was DNase sensitive. This extract was not directly cytotoxic to tumor cells but rather induced resistance through stimulation of NK-cell activity by induction of IFN-a/b and IFN-g. Subsequent studies showed that IFN-g expression by NK-cells was secondary to IL-12 released by macrophage/monocytes (Tokunaga et al., 1984; Yamamoto et al., 1992; Halpern et al., 1996). In seminal experiments, the active sequences of mycobacterial DNA were shown to bear a general motif of two 5% purines, an unmethylated CpG core and two 3% pyrimidines (Kataoka et al., 1992; Yamamoto et al., 1994). This motif can form palindromes and occurs much more commonly in bacterial DNA than mammalian DNA for two reasons. Thus, mammalian DNA shows CpG suppression, with cytosine and guanosine residues occurring in tandem much less frequently than predicted by the base composition of the DNA. Furthermore, in mammalian DNA, cytosine is commonly methylated, perhaps as a mechanism for regulating transcriptional activity (Bird, 1987; Hergersberg, 1991). The immunostimulatory CpG sequences are ubiquitous among bacterial species and potentially represent a generalized system for immune activation. The role of these sequences extends to B-cells. As shown in in vitro cultures of murine spleen cells, DNA from a variety of bacterial species can directly stimulate mitogenesis and antibody production. The sequences for B-cell activation have the same general structure as those for cytokine stimulation. Bacterial DNA can also serve as an adjuvant and function synergistically with other signals such as anti-Ig cells to stimulate cell activation (Messina et al., 1991; Krieg et al., 1995).

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While the role of bacterial DNA in triggering innate immunity is speculative, its ability to induce specific immune responses in humans is well established. Thus, sera from normal human subjects contain high titers of antibodies to DNA from certain bacterial species, including Micrococcus lysodeikticus and Staphylococcus epidermidis (Karounos et al., 1988). These antibodies occur in most individuals with a range of titers similar to that of patients with SLE. At present, it is not known whether differences in the levels of these antibodies in NHS reflects the extent of exposure to bacteria or host susceptibility factors such as genes that regulate immune responsiveness. As shown by a variety of immunochemical approaches, anti-DNA antibodies in NHS differ from lupus anti-DNA in several important respects: (1) NHS anti-DNA bind selectively to bacterial DNA whereas lupus anti-DNA bind crossreactively with all DNA; (2) NHS anti-DNA are primarily the IgG2 isotype whereas lupus anti-DNA is predominantly IgG1; (3) NHS antiDNA are overwhelmingly k light chains whereas lupus anti-DNA have a more equally balanced expression of k and l light chains; and (4) NHS anti-DNA are less dependent on ionic interactions than lupus anti-DNA (Robertson et al., 1992; Robertson and Pisetsky, 1992). The IgG2 predominance of antibodies to bacterial DNA is reminiscent of the response to bacterial polysaccharides and suggests that DNA functions as a T independent antigen during ordinary encounters with bacterial organisms. According to current models for T independent responses, antibodies are produced by Bcells that have extensive crosslinking of their receptors by repeating determinants on these antigens. These B-cells also undergo stimulation by cytokines such IFN-g. The source of the cytokines are NK- and T-cells that are stimulated directly or indirectly by bacterial products (Mond et al., 1995). Since bacterial DNA can stimulate cytokines and has a repeating structure, it can function in a T independent mode. In contrast, the anti-DNA response in SLE has features of a T dependent response as evidenced by the pattern of isotype expression. The immunogenicity of bacterial DNA has been substantiated in animal immunization experiments. Thus, under conditions in which mammalian DNA is inactive, bacterial DNA can induce significant antibody responses in normal mice. With bacterial dsDNA as the immunogen, the induced antibodies are highly specific for the bacterial antigen and resemble spontaneous responses in NHS in their lack of crossreactivity with other DNA (Gilkeson et al., 1989). Since these immunizations have been carried out with complexes of DNA with protein carriers in adjuvant, a T independent mechanism has not been detected.

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3. Implications for disease susceptibility Information generated in the past few years provides a new perspective on the immunological properties of DNA and suggests distinct pathways for anti-DNA production in normal individuals and those susceptible to autoimmune disease. While anti-DNA responses in normal individuals appear T-cell independent, those in SLE are primarily T dependent. Furthermore, the specificity of these responses differs markedly since antiDNA in NHS bind non-conserved determinants present on only certain bacterial DNA. These determinants are undoubtedly linear sequences, although they must differ from the immunostimulatory motifs which are present widely on bacterial DNA. In contrast, SLE anti-DNA bind conserved determinants that are present on all DNA. These determinants represent conformations such as helical B DNA. Table 1 summarizes these differences. Studies on the specificity of SLE antibodies to protein nuclear antigens have led to the notion that susceptibility to autoimmunity is associated with an alteration in patterns of antigen recognition. Thus, the specificity of spontaneous autoantibodies to protein nuclear antigens (antinuclear antibodies or ANA) differs from that of antibodies induced to these antigens in normal animals by intentional immunization. The most prominent difference concerns the binding to short linear amino acid sequences. In contrast to induced antibodies which bind to peptidic sequences, spontaneous ANA require larger polypeptides for stable interaction; spontaneous anti-DNA can also react with antigens from various mammalian species. These findings point to the recognition of conserved conformations as opposed to linear epitopes. Furthermore, spontaneous ANA, unlike induced ANA, can inhibit the activity of nuclear antigens that are enzymes (Tan, 1989). An explanation for the shift in binding specificity associated with SLE comes from studies on immunization of lupus mice with bacterial DNA. As previously noted, normal mice immunized with bacterial dsDNA produce a specific response to the immunizing antigen. Table 1

Properties of Anti-DNA Normal

SLE

IgG2 k Light chain restriction Bind sequences High affinity Bind ss and ds DNA Non-pathogenic

IgG1,IgG3 k+l Light chain Bind conformations Moderate affinity Bind ss and ds DNA Pathogenic

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Following the same immunizations, however, NZB/ NZW autoimmune mice produce autoantibodies that bind to mammalian as well as bacterial dsDNA. Interestingly, these antibodies cannot be induced by immunization with mammalian dsDNA, suggesting that the mitogenic activity of the bacterial antigen is necessary for this response (Gilkeson et al., 1995). To account for these observations, it has been proposed that the B-cell repertoire of autoimmune individuals differs from that of normals because of tolerance defects that allow persistence of autoreactive precursors. The variable regions of these precursors may contain sequences such as CDR3 arginines that bind DNA even in the absence of somatic mutations in other parts of the molecules. In normal individuals, these precursors would be deleted or anergized; however, in the individual genetically susceptible to disease, these precursors would remain in the repertoire and lead to crossreactive autoantibodies even when stimulated by a foreign antigen. As this review suggests, the susceptibility to SLE can be considered an aberration in the specificity to an ordinarily active foreign antigen as opposed to the acquisition of responsiveness to an ordinarily inactive self antigen. Furthermore, recent research suggests that foreign DNA can perturb immunological balance in susceptible individuals and lead to potentially adverse reactions to DNA that enters the host either from infection or administration of DNA therapeutics. The coming years should be enlightening as the impact of susceptibility factors on immune response to bacterial DNA is further defined.

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