Immunologic consequences of nucleic acid therapy.
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Animals↗
Biomedical subjects
D S Pisetsky
Publications and source records attributed to D S Pisetsky.
Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA.
To investigate the role of antigen drive in anti-double-stranded (ds) DNA production, the antibody response induced in lupus-prone NZB/NZW mice by E. coli (EC) dsDNA was evaluated. Preautoimmune NZB/NZW female mice were immunized with complexes of EC dsDNA with methylated bovine serum albumin (mBSA) in complete Freund's adjuvant; control mice received either mBSA complexes with calf thymus (CT) dsDNA or mBSA alone in adjuvant. IgG antibody responses were assessed by ELISA. Similar to normal mice, immunized NZB/NZW mice produced significant levels of anti-dsDNA when measured with EC dsDNA as antigen. Whereas normal mice produce antibodies which are specific for the immunizing bacterial DNA, NZB/NZW mice produced antibodies that bound crossreactively to CT dsDNA by ELISA. Furthermore, the induced antibodies resembled lupus anti-DNA in their fine specificity for polynucleotide antigens and reactivity with Crithidia luciliae DNA. Despite their response to EC dsDNA, NZB/NZW mice immunized with CT dsDNA failed to generate significant anti-dsDNA responses. These results provide further evidence for the enhanced immunogenicity of bacterial DNA and suggest that immune cell abnormalities in NZB/NZW mice promote the generation of crossreactive autoantibody responses when confronted with a foreign DNA.
Aging↗
The mechanism of autoantibody production in an autoimmune MRL/lpr mouse.
Rheumatoid factors (RF) and anti-DNA Abs from MRL/lpr mice have features similar to Abs directed toward foreign Ags, indicating a role of specific activation by Ags during disease. But our previous studies and analogous studies from others concentrated on a limited subset of hybridomas selected on the basis of Ag binding to well characterized target autoantigens. Thus, it has been unclear to what extent clonal expansion is restricted to identifiable autospecificities. To obtain a more complete picture of disease-associated autoantibody production, we designed the following experiment. A large number of B cell hybridomas were generated from the spleen of an MRL/lpr mouse and then analyzed for self-specificity, sequence, and clonal relationship. Surprisingly, we found that clonal expansion was limited to only a few autospecificities, implying a unique property of this response. In addition, we used Southern blotting with heavy and L chain constant region probes to screen both RF and non-RF hybridomas for membership in clones, one of which was first identified among RF hybridomas. We found no non-RF members of this clone. The size and number of mutations of this clone were sufficient for us to conclude that nonspecific (i.e., non-RF) mutant members are rapidly lost. Had an Ag other than IgG2a been driving clonal expansion, we should have seen mutants that retained spectificity for that Ag but that lost specificity for IgG2a. This observation, along with the restriction of clonal expansion to a few autospecificities, provides strong evidence that normal autoantigens themselves drive autoantibody clonal expansion.
Amino Acid Sequence↗
The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine.
MRL-lpr/lpr mice spontaneously develop various manifestations of autoimmunity including an inflammatory arthropathy and immune complex glomerulonephritis. This study examines the role of nitric oxide, a molecule with proinflammatory actions, in the pathogenesis of MRL-lpr/lpr autoimmune disease. MRL-lpr/lpr mice excreted more urinary nitrite/nitrate (an in vivo marker of nitric oxide production) than did mice of normal strains and MRL-(+/+) and B6-lpr/lpr congenic strains. In addition, MRL-lpr/lpr peritoneal macrophages had an enhanced capacity to produce nitric oxide in vitro as well as increased nitric oxide synthase activity, and certain tissues from MRL-lpr/lpr mice had increased expression of inducible nitric oxide synthase (NOS) mRNA and increased amounts of material immunoreactive for inducible NOS. Oral administration of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, prevented the development of glomerulonephritis and reduced the intensity of inflammatory arthritis in MRL-lpr/lpr mice. By using interspecific backcross mice, the gene for inducible NOS (Nosi) was mapped to mouse chromosome 11. This chromosomal localization was different from those loci that we have previously demonstrated to be linked to enhanced susceptibility to renal disease in an MRL-lpr/lpr cross. However, the chromosomal location of the NOS gene was consistent with an insulin-dependent diabetes locus identified in an analysis of nonobese diabetic (NOD) mice. These results suggest that elevated nitric oxide production could be important in the pathogenesis of autoimmunity, and that treatments to block the production of nitric oxide or block its effects might be valuable therapeutically.
Administration, Oral↗
Interleukin-2 receptor levels in the sera of rheumatoid arthritis patients treated with methotrexate.
OBJECTIVE: To evaluate the association of the level of soluble serum interleukin-2 receptor (sIL-2R) with disease activity and response to therapy in patients with rheumatoid arthritis (RA). METHODS: The sIL-2R levels of 148 patients with refractory RA were determined by enzyme-linked immunosorbent assay. This parameter was correlated with other clinical observations obtained during a prospective, randomized, placebo-controlled trial of methotrexate, sponsored by the Cooperative Systematic Studies of Rheumatic Diseases consortium. Using statistical modeling, the usefulness of sIL-2R as a measure of disease activity and a predictor of outcome was evaluated. RESULTS: The mean sIL-2R level in all RA patients was markedly elevated compared with that in normal control subjects, and decreased significantly during the trial. There was no correlation of the sIL-2R level and the joint pain/tenderness count either at study entry or study end. There was a significant correlation of the sIL-2R level and the erythrocyte sedimentation rate, both at study entry and study end. A multiple linear regression model showed that treatment with methotrexate, but not the sIL-2R level or the change in sIL-2R level, predicted a change in joint count. A stepwise multiple logistic regression model defined no significant predictive information for outcome for the level of sIL-2R at study entry. CONCLUSION: After controlling for the simultaneous effects of other important clinical variables, the level of sIL-2R does not appear to predict the response to methotrexate in patients with refractory RA. Further analysis of cohorts of patients with earlier RA needs to be performed.
Adult↗
The anti-La response of a single MRL/Mp-lpr/lpr mouse: specificity for DNA and VH gene usage.
Autoantibodies to ribonucleoproteins (RNP) occur prominently in human systemic lupus erythematosus and murine lupus models. In previous studies we demonstrated a relationship in MRL/Mp-lpr/lpr (MRL/lpr) mice between antibodies to Sm, an RNP autoantigen, and antibodies to DNA. Thus, many anti-Sm monoclonals bound DNA and expressed the same V region genes as anti-DNA. In addition, many had multiple VHCDR3 Arg residues suggestive of selection by DNA, and some had somatic mutations suggesting selection for mutant B cells by DNA. To determine whether autoantibodies to other RNP antigens are also associated with the anti-DNA response, we have analyzed the response to the La RNP. Six anti-La B cell hybridomas were generated from a single MRL/lpr mouse. Southern blot analysis of Ig V gene rearrangements and V gene sequences indicated two clonally related pairs, suggesting an oligoclonal response. Antibodies from all six hybridomas bound single-stranded DNA, while antibodies from five hybridomas bound double-stranded DNA. Two hybridomas expressed a VH7183 gene which is used by members of two previously reported anti-DNA clones and two anti-Sm/DNA clones of MRL/lpr origin. These data demonstrate an association between the anti-La and anti-DNA responses in MRL/lpr mice, suggesting that cross-reactive anti-RNP and anti-DNA responses are a general feature of autoimmunity in this lupus model.
Amino Acid Sequence↗
The influence of DNA size on the binding of anti-DNA antibodies in the solid and fluid phase.
To elucidate the interaction of anti-DNA antibodies with DNA, the reactivity of lupus sera with single-stranded fragments from calf thymus, Escherichia coli, and salmon testes DNA was investigated. These fragments were generated by digestion with the restriction enzyme HinfI and ranged in size from approximately 100-4000 bases. By ELISA using polystyrene microtiter plates, fragments from all three species were weakly antigenic compared to intact DNA. These fragments, however, were all antigenic when tested as inhibitors in competition-binding assays. The weak antigenicity of fragments could not be explained by poor adherence to the plates since fragments and intact DNA showed similar levels of binding as assessed using biotinylated preparations. Together, these results demonstrate that the antigenicity of DNA fragments is dramatically altered by solid-phase binding and suggest that constraints on topological or conformational rearrangements of DNA in the solid phase limit antibody interaction.
Animals↗
Serial measurement of serum interleukin-2 receptor levels in patients with rheumatoid arthritis: limited evidence for a role of T cell activation in clinical exacerbations.
To investigate the association of T cell activation with clinical exacerbations of RA, we measured serum levels of soluble interleukin-2 receptors (sIL2R), a marker of T cell activation, in serial samples obtained from 23 patients with RA. sIL2R measurements were performed on sera obtained from each patient every 2 weeks for up to 60 weeks, and levels were correlated with swollen joint counts, tender joint counts, physician global assessments, patient global assessments, pain scores, Health Assessment Questionnaire Disability Index scores, and Westergren erythrocyte sedimentation rates measured simultaneously. There were no significant correlations between changes in sIL2R levels and changes in any of the other measures, nor were lead-lag relationships detected, for the group as a whole. Examination of the time courses of individual patients revealed significant positive correlations between changes in sIL2R levels and changes in swollen joint counts in five patients; significant correlations with other measures were present in three or fewer patients. sIL2R levels also varied little over the 2-week time interval of greatest clinical change in each patient. These results suggest either that clinical exacerbations of RA are not associated with changes in T cell activation or that sIL2R levels do not accurately reflect such changes.
Adult↗
Stimulation of murine lymphocyte proliferation by a phosphorothioate oligonucleotide with antisense activity for herpes simplex virus.
To investigate further the immunological properties of nucleic acids, the mitogenicity of a phosphorothioate oligonucleotide (S-oligo 1082) with antisense activity for herpes simplex virus was tested. This compound stimulated proliferation and antibody production by murine lymphocytes in in vitro cultures. Proliferation was dose-dependent and unaffected by T cell depletion. Furthermore, inclusion of a non-mitogenic DNA in the medium did not block stimulation. Since 1082 does not have homology to a known gene involved in lymphocyte activation, these observations suggest that S-oligo antisense compounds may display non-specific activating effects, at least on murine B cells.
Animals↗
Fish oil feeding modulates leukotriene production in murine lupus nephritis.
Diets enriched with fish oil (FO) ameliorate kidney disease in the MRL-lpr/lpr murine model of lupus nephritis. Although the mechanisms of this effect are not known, FO is rich in the polyunsaturated fatty acid eicosapentaenoic acid (EPA) which may have profound effects on eicosanoid metabolism. In MRL-lpr/lpr mice, FO feeding reduces renal production of cyclooxygenase metabolites. However, EPA may also affect the metabolism of arachidonate by the 5-lipoxygenase (5-LO) pathway and enhanced production of 5-LO metabolites has been implicated in the pathogenesis of kidney disease in MRL-lpr/lpr mice. We therefore investigated the effects of FO feeding on production of 5-LO metabolites in 20 week old MRL-lpr/lpr mice. After 8 weeks of dietary supplementation with FO, both renal hemodynamic function and glomerular histology were improved compared to safflower oil (SO) controls. Amelioration of kidney disease was associated with alterations in the pattern of leukotriene production by macrophages and kidneys from FO fed mice. There was a significant decrease in the production of leukotriene B4 (LTB4) and tetraene peptidoleukotrienes by peritoneal macrophages isolated from mice given FO compared to control animals. Similarly, dietary supplementation with FO decreased renal production of LTB4. Reduced production of tetraene leukotrienes was accompanied by a modest increase in the production of pentaene leukotrienes by macrophages from FO fed mice. We speculate that this modulation of leukotriene production by FO feeding may have beneficial effects on renal disease in autoimmune nephritis.
Administration, Oral↗
Characterization of antibodies to bacterial double-stranded DNA in the sera of normal human subjects.
To assess the human antibody response to bacterial double-stranded (ds) DNA, sera from normal human subjects (NHS) were tested by ELISA for binding to highly purified dsDNA from Micrococcus lysodeikticus (MC). Of 38 NHS tested, 19 demonstrated significant activity to MC dsDNA (defined as an OD380 > 1.0 at a 1:100 dilution of serum) without appreciable binding to dsDNA from Escherichia coli or calf thymus. In competition ELISA, antibodies in NHS to MC dsDNA showed equivalent inhibition by MC ssDNA or dsDNA. Antibody specificity was further evaluated by testing the effects of ionic strength on binding. By ELISA, antibodies in NHS had greater binding to MC dsDNA at high ionic strengths than those in systemic lupus erythematosus sera. These results indicate that NHS have antibodies which bind bacterial dsDNA and extend the range of DNA determinants that can be recognized as foreign by the normal immune system.
Adult↗
Molecular characterization of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. Differences from spontaneous anti-DNA in the content and location of VH CDR3 arginines.
Immunization of normal mice with bacterial DNA induces a significant anti-DNA response that includes antibodies resembling some lupus anti-DNA in their binding properties, although lacking specificity for mammalian dsDNA. To determine the structure of these induced antibodies and their relationship to anti-DNA from lupus mice, we have characterized the clonality and selected V-region sequences of a panel of 20 anti-DNA antibodies from 3 BALB/c mice immunized with ssDNA from Escherichia coli. Southern blot analysis of H and L chain rearrangements indicated that two of the animals expressed pairs of clonally related antibodies. Amino acid sequences of 10 of the induced antibodies demonstrated predominant utilization of J558 family VH genes and JH4 in association with various DH, J kappa and V kappa genes. Among the VH CDR3 of these 10 antibodies, 4 displayed arginine residues as a result of N region additions. None of these antibodies, however, had more than one arginine residue in VH CDR3 nor arginines at positions 100 or 100a, characteristic features of lupus antibodies to dsDNA. These results suggest that normal mice immunized with bacterial DNA display certain facets of DNA Ag drive, although lacking the mechanisms for the production of antibodies to mammalian dsDNA.
Amino Acid Sequence↗
V region gene analysis of anti-Sm hybridomas from MRL/Mp-lpr/lpr mice.
Anti-Sm autoantibodies are unique to SLE, but are present in only 25% of patients with this disease. This response also occurs at a similar frequency in mice of the autoimmune MRL strains. Previous analyses of the anti-Sm response in these mice indicate that its occurrence is controlled by stochastic events, and suggest that Sm is the driving Ag. To further elucidate the role of Ag in this response, and to test the hypothesis that the 25% incidence is due to a requirement for particular Ig gene rearrangements or somatic mutations, we have analyzed the specificity and V-region gene sequences of 41 anti-Sm B cell hybridomas derived from nine anti-Sm-positive MRL/Mp-lpr/lpr mice. The majority of hybridomas are specific for the D peptide of the Sm particle. Hybridomas of independent origin express unique VH/V kappa combinations with diverse junctional sequences and are variable in the extent of somatic mutation. Thus, the response does not appear to be dependent upon the occurrence of a rare Ig gene rearrangement or specific somatic mutation. The response exhibits restriction in JH and VH gene use, and in individual mice is oligoclonal, suggestive of Ag selection. In the few B cells for which mutations can be identified, the evidence for selection of mutant B lymphocytes, based on patterns of mutation, is ambiguous. However, there is remarkably little intraclonal diversity, suggesting that the overall mutation rates in these clones are low.
Amino Acid Sequence↗
The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens.
Although DNA is generally considered to be a poor immunogen, recent evidence suggests that DNA from various species differ in their immunological activity and that bacterial DNA can induce the in vitro proliferation of normal murine B cells. To delineate structural features of DNA associated with mitogenic activity, the response of murine lymphocytes to various natural and synthetic polynucleotides was determined. Both ss and dsDNA from two different bacterial strains were equally effective in inducing proliferation. This response was independent of adenosine methylation, since DNA from dam- Escherichia coli stimulated proliferation. Among the synthetic polymers tested, only the duplexes poly(dG).poly(dC), and poly(dG.dC) were mitogenic, while polymers containing dA, dT, or dI alone or in combination with dG and dC were inactive. The mitogenic activity of poly(dG.dC) was eliminated, however, upon substitution of rG for dG or 5medC for dC. The mitogenic activity did not require high molecular weight DNA since active polymers ranged in size from approximately 260 to 800 base pairs. In addition, E. coli DNA fragments of 50-300 and 125-600 bases were mitogenic. Together, these data suggest that the mitogenic activity of DNA is dependent on sequence-specific determinants that can be presented by synthetic DNA duplexes as well as bacterial ss and dsDNA.
Animals↗
Induction of immune-mediated glomerulonephritis in normal mice immunized with bacterial DNA.
Normal mice immunized with bacterial DNA produce high titers of anti-DNA antibodies and represent a new model for autoantibody production in systemic lupus erythematosus. To determine whether DNA immunization can also provoke clinical manifestations of lupus, the occurrence of nephritis in immunized mice was assessed and correlated with levels of anti-DNA as well as antibodies to glomerular antigens. BALB/c mice immunized with Escherichia coli single-stranded DNA in complexes with methylated bovine serum albumin in adjuvant showed increased proteinuria compared to control mice immunized with mBSA alone. Furthermore, DNA immunized mice had significantly greater glomerular proliferative changes and immunoglobulin deposition than control mice. In an in vitro assay, sera from DNA immunized mice exhibited greater binding to glomerular antigens than sera from control mice. Compared to sera, renal eluates from DNA-immunized mice were enriched for anti-DNA and glomerular binding activity. These data indicate that immunization of normal mice with E. coli DNA induces an immune-mediated proliferative glomerulonephritis that is likely secondary to the renal deposition of anti-DNA antibodies.
Animals↗
The fine specificity of monoclonal anti-DNA antibodies induced in normal mice by immunization with bacterial DNA.
To evaluate further bacterial DNA immunization as a model to study antigen drive in the anti-DNA response, the specificity of induced monoclonal anti-DNA antibodies was characterized. A panel of IgM and IgG monoclonal anti-DNA antibodies was produced from spleen cells of BALB/c mice immunized with single-stranded DNA from E. coli complexed to methylated bovine serum albumin in complete Freund's adjuvant. The binding of these antibodies to DNA and non-DNA antigens was tested by ELISA to assess their range of polyspecificity. These monoclonal antibodies were found to bind to nucleic acid as well as non-nucleic acid antigens, such as beta-galactosidase, cardiolipin, Ro, La and Sm. These studies demonstrate that anti-DNA antibodies from normal mice, although induced by bacterial DNA, may display a broad range of antigen recognition and thus resemble lupus anti-DNA antibodies, many of which are polyspecific, in their pattern of cross-reactivity.
Animals↗
Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens.
The Ku autoantigen is a DNA binding factor consisting of 70 and approximately 80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factors in vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of approximately 1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610-705, 8-221, and 1-374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506-541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein.(ABSTRACT TRUNCATED AT 400 WORDS)
3T3 Cells↗