DNA Antibody Production Research Articles

Screening of Environmental Chemicals to Characterize Exposures in Participants With Systemic Lupus Erythematosus.

There is a need to characterize exposures associated with the pathogenesis of systemic lupus erythematosus (SLE). In this pilot study, we explore a hypothesis-free approach that can measure thousands of exogenous chemicals in blood ("exposome") in patients with SLE and unaffected controls. This cross-sectional study analyzed a cohort of patients with prevalent SLE (n=285) and controls (n=106). Plasma was analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). Mass spectrometry features present in at least 25% of all samples were selected for association analysis (n=2,737). Features were matched to potential chemicals using available databases. Association analysis of abundances of features with SLE status was performed, adjusting for age and sex. We also explored features associated with SLE phenotypes, sociodemographic factors, and current medication use. We found 30 features significantly associated with SLE status (Bonferroni P < 0.05). Of these, seven matched chemical names based on databases. These seven features included phthalate metabolites, a formetanate metabolite, and eugenol. The abundance of acid pesticides differed between patients with SLE and controls (Bonferroni P < 0.05). Two unmatched features were associated with a history of lupus nephritis, and one with anti-double-stranded DNA antibody production (Bonferroni P < 0.05). Seventeen features varied by self-reported race and ethnicity, including a polyfluoroalkyl substance (analysis of variance P < 1.69 × 10-5). Eleven features correlated with antimalarials, 6 with mycophenolate mofetil, and 29 with prednisone use. This proof-of-concept study demonstrates that LC-QTOF/MS is a powerful tool that agnostically detects circulating exogenous compounds. These analyses can generate hypotheses of disease-related exposures for future prospective, longitudinal studies.

Interleukin-17 Expression Positively Correlates with Disease Severity of Lupus Nephritis by Increasing Anti-Double-Stranded DNA Antibody Production in a Lupus Model Induced by Activated Lymphocyte Derived DNA

Lupus nephritis is one of the most serious manifestations and one of the strongest predictors of a poor outcome in systemic lupus erythematosus (SLE). Recent evidence implicated a potential role of interlukin-17 (IL-17) in the pathogenesis of lupus nephritis. However, the correlation between IL-17 expression level and the severity of lupus nephritis still remains incompletely understood. In this study, we found that serum IL-17 expression level was associated with the severity of lupus nephritis, which was evaluated by histopathology of kidney sections and urine protein. Of note, we showed that enforced expression of IL-17 using adenovirus construct that expresses IL-17 could enhance the severity of lupus nephritis, while blockade of IL-17 using neutralizing antibody resulted in decreased severity of lupus nephritis. Consistently, we observed an impaired induction of lupus nephritis in IL-17-deficient mice. Further, we revealed that IL-17 expression level was associated with immune complex deposition and complement activation in kidney. Of interest, we found that IL-17 was crucial for increasing anti-double-stranded DNA (dsDNA) antibody production in SLE. Our results suggested that IL-17 expression level positively correlated with the severity of lupus nephritis, at least in part, because of its contribution to anti-dsDNA antibody production. These findings provided a novel mechanism for how IL-17 expression level correlated with disease pathogenesis and suggested that management of IL-17 expression level was a potential and promising approach for treatment of lupus nephritis.

Kidney-infiltrating CD4+ T-cell clones promote nephritis in lupus-prone mice

In systemic lupus erythematosus, CD4+ T cells play key roles in the initiation and promotion of autoantigen-specific humoral immunity, and indirect evidence suggests that T cells are pathogenic effectors in lupus nephritis. The contribution of kidney-infiltrating T cells to nephritis, however, has not been verified because of the difficulty in directly analyzing organ-infiltrating T cells. Here, we examined the pathogenic roles of autoreactive cytokine-expressing CD4+ T cells from the kidneys of early nephritic MRL/lpr mice. Interferon (IFN)-γ-secreting cells were enriched among CD5(high)CD4+ T cells found in the inflamed kidneys. Using single-cell analysis of the T-cell receptor (TCR)(high)CD5(high)CD4+ T cells from the kidneys of early nephritic MRL/lpr mice, two IFN-γ-expressing CD4+ T cell clones, MLK2 and MLK3, were identified. CD4+ T cells transduced with the T-cell receptor genes from each clone responded to splenic dendritic cells in an MHC class II-dependent manner, but not to B cells or macrophages. MLK3-transduced CD4+ T cells proliferated in the spleens of prenephritic mice, promoted nephritis progression upon adoptive transfer, and enhanced the deposition of C3 without promoting anti-double-stranded DNA antibody production. Thus, CD4+ T cells in the inflamed kidneys of MRL/lpr mice contribute to nephritis progression.

Heparin exerts a dual effect on murine lupus nephritis by enhancing enzymatic chromatin degradation and preventing chromatin binding in glomerular membranes

Association of nucleosome-IgG immune complexes with glomerular basement membranes (GBMs) is an important event in the development of lupus nephritis. Preventing this binding and/or increasing nuclease sensitivity of nucleosomes may be viable strategies for the prevention of the disease. Theoretically, heparin may alter nucleosomal structure and increase sensitivity to proteinases and nucleases, and may also inhibit binding of nucleosomes and nucleosome-IgG complexes to basement membrane structures. The aim of this study was to investigate whether and eventually how heparin prevents murine lupus nephritis. Surface plasmon resonance was used to analyze if heparin inhibits binding of nucleosomes to laminin and collagen. The effect of heparin on nuclease- and proteinase-mediated degradation of nucleosomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrophoresis. In vitro results were compared with analyses in vivo in heparin-treated (NZB × NZW)F(1) mice. Anti-double-stranded DNA antibody production, deposition of nucleosome-IgG complexes in GBMs, and development of proteinuria were monitored, and circulating chromatin fragments were quantified using quantitative polymerase chain reaction. In vitro studies demonstrated that heparin increased enzymatic degradation of nucleosomes and almost completely inhibited binding of nucleosomes to laminin and collagen. (NZB × NZW)F(1) mice treated with heparin demonstrated delayed or no antibody production and higher variation of circulating chromatin levels compared with untreated control mice. This effect was accompanied by highly reduced nucleosome-IgG complexes in GBMs and delayed development of nephritis. Increasing the degradation of nucleosomes, reducing their immunogenicity, and preventing binding of nucleosome-IgG complexes in glomeruli together provide an alternative basis for the treatment of lupus nephritis.

The role of DC-STAMP in maintenance of immune tolerance through regulation of dendritic cell function

Regulation of dendritic cell (DC) function is critical for maintaining self-tolerance and preventing autoimmunity. The dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in cell-cell fusion of osteoclasts and foreign body giant cells, but though originally identified in DCs, its specific roles there remain undefined. Here, we report that aged DC-STAMP-deficient mice display several systemic autoimmune symptoms such as spontaneous lymphoproliferation, splenomegaly associated with infiltration of T cells in several organs and increased serum anti-double-stranded DNA antibody production. Although a lack of DC-STAMP did not inhibit DC differentiation or proliferation, antigen presentation activity of DC-STAMP-deficient DCs was significantly up-regulated in both class I and II pathways through increased phagocytotic activity compared with wild-type DCs, an activity likely leading to autoimmunity. Our results indicate that DC-STAMP is required for proper regulation of DC activity and maintenance of immune self-tolerance.

Requirement of toll‐like receptor 7 for pristane‐induced production of autoantibodies and development of murine lupus nephritis

The detection of high titers of antibodies against small nuclear ribonucleoproteins (snRNP) is a diagnostic finding in patients in whom systemic lupus erythematosus (SLE) is suspected. Endogenous RNA molecules within snRNP trigger Toll-like receptor 7 (TLR-7) activation in B cells and dendritic cells, leading to anti-snRNP antibody production, which is associated with the development of immune complex nephritis in SLE. The purpose of this study was to investigate the role of TLR-7 in anti-snRNP antibody production and renal disease in SLE induced by an exogenous factor in the absence of genetic predisposition, using the pristane-induced murine lupus model. Serum autoantibodies, IgG isotypes, and cytokine levels in pristane-treated wild-type and TLR-7-deficient mice were analyzed by enzyme-linked immunosorbent assay. Histopathologic changes in mouse kidneys were determined by light immunofluorescence microscopy. Cell subsets in splenocytes and peritoneal lavage cells from the mice were examined by flow cytometry. We found that anti-snRNP antibody production induced by pristane treatment was entirely dependent on the expression of TLR-7, whereas anti-double-stranded DNA antibody production was not affected by a lack of TLR-7. Impaired anti-snRNP antibody production in TLR-7-deficient mice was paralleled by lower levels of glomerular IgG and complement deposits, as well as less severe glomerulonephritis. TLR-7 is specifically required for the production of RNA-reactive autoantibodies and the development of glomerulonephritis in pristane-induced murine lupus, a model of environmentally triggered SLE in the absence of genetic susceptibility to autoimmunity. Specific interference with TLR-7 activation by endogenous TLR-7 ligands may therefore be a promising novel strategy for the treatment of SLE.

From Vanilla to 28 Flavors: Multiple Varieties of T Regulatory Cells

Numerous T cell subpopulations have now been claimed to exhibit regulatory activity. Shevach discusses the current understanding of the different subsets of T regulatory cells and provides a perspective on the current areas of uncertainly and controversy in the field.

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-γ and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4+CD45RO+ T cells from peripheral blood, the percentage of ICOS+ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [3H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-γ, IL-4 and IL-10 in both SLE and normal T cells. IFN-γ in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.

Genetic dissection of B cell traits in New Zealand black mice. The expanded population of B cells expressing up-regulated costimulatory molecules shows linkage to Nba2.

B cell abnormalities are a prominent feature of the immunologic derangement in NZB and NZB / W mice. We recently demonstrated that these mice have an increased proportion of splenic B cells expressing B7.1 and elevated levels of B7.2 and ICAM-1 that possess the characteristics of marginal zone B cells (CD23(low / -) CD5(-) CD44(hi) CD24(hi) IgD(- / low) IgM(hi)) and are found as early as 4 - 6 weeks of age. These findings suggest that activated B cells in NZB and NZB / W mice could serve a costimulatory function leading to activation of autoreactive T cells. However, it remains unclear whether there is any association between B abnormalities and nephritis in these mice. Here we have used genetic mapping techniques to address this issue. We show that increases in the proportion of B cells expressing costimulatory molecules, serum IgM levels, the number of IgM ELISpots, and IgG anti-single-stranded (ss) DNA antibody production, are significantly associated with a chromosomal region that overlaps with Nba2, a genetic locus previously linked to nephritis. Based on these findings we propose that immune mechanisms leading to polyclonal B cell activation and up-regulation of costimulatory molecules in these mice play a central role in the loss of tolerance that leads to production of pathogenic autoantibodies.

Histone-specific Th0 and Th1 clones derived from systemic lupus erythematosus patients induce double-stranded DNA antibody production.

To investigate whether histone-specific T helper (Th) cells that are able to induce anti-double-stranded DNA (anti-dsDNA) antibodies can be isolated from patients with systemic lupus erythematosus (SLE) and to characterize the cytokine secretion pattern of such Th clones. Peripheral blood mononuclear cells from SLE patients and healthy donors were stimulated with autologous apoptotic cell material or purified histones, expanded with interleukin-2 (IL-2), and cloned by limiting dilution. Histone reactivity of clones was examined by histone-specific proliferation and cytokine release. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Induction of anti-dsDNA antibodies was measured in cocultures of autologous B cells and Th clones by ELISA: Numerous histone-specific T cell receptor (TCR) alpha/beta+ Th clones were established from 2 of 3 patients with active SLE and from 1 of 2 healthy individuals. Most Th clones secreted IL-2, interferon-gamma (IFNgamma), and IL-4, whereas some produced predominantly IL-2 and IFNgamma. Th clones that could stimulate the production of anti-dsDNA antibodies were derived from SLE patients and from a healthy individual. Th cells specific for histones may play an important role in the pathogenesis of SLE by inducing autoantibodies to dsDNA. Both Th1 and Th2 cytokines may be involved in the pathogenesis of SLE. The presence of histone-specific Th cells in a healthy individual indicates the importance of peripheral tolerance for preventing autoimmunity to nuclear antigens.

Genetic control of DNA antibody production: focus on the heavy chain.

Genetic control of DNA antibody production: focus on the heavy chain.

Environmental and other stimuli for DNA antibody production

Environmental and other stimuli for DNA antibody production

Host–microflora interaction in Systemic Lupus Erythematosus (SLE): colonization resistance of the indigenous bacteria of the intestinal tract

Experimental data suggest a role for the microflora in Systemic Lupus Erythematosus (SLE). Anti-ds-DNA antibodies may be pathogenic in SLE by forming immune complexes with DNA. Foreign bacteria in the intestines could constitute the stimulus for anti-ds-DNA antibody production in SLE. Colonization Resistance (CR) is the defence capacity of the indigenous microflora against colonization of the intestines by foreign bacteria. A low CR implies increase of translocation of bacteria and a higher chance of subsequent, possibly DNA-cross-reacting antibacterial antibody production. We measured CR by a comprehensive biotyping technique in healthy individuals and patients with inactive and active SLE. CR tended to be lower in active SLE patients than in healthy individuals (P = 0.09, Wilcoxon one sided, with correction for ties). This could indicate that in SLE more and different bacteria translocate across the gut wall due to a lower CR. Some of these may serve as polyclonal B cell activators or as antigens cross-reacting with DNA.

Comparison of the requirements for cognate T cell help for IgG anti-double-stranded DNA antibody production in vitro: T helper-derived lymphokines replace T cell cloned lines for B cells from NZB.H-2bm12 but not B6.H-2bm12 mice.

We have isolated, from NZB.H-2bm12 mice, several autoreactive cloned T cell lines that provide help for anti-dsDNA IgG antibody production in vitro. The purpose of the work described herein was to examine the requirement for cognate help for the production of anti-DNA antibodies in vitro. Thus, the ability of cloned T cell lines or lymphokines derived from them to provide help for T-depleted spleen cells from both normal B6.H-2bm12 mice and SLE-prone NZB.H-2bm12 mice was examined. Two autoreactive cloned T cell lines were selected for detailed study. 410F T cells respond to APC from both I-Ab and I-Abm12 mice, whereas 410H T cells are restricted to I-Abm12. By using Percoll gradients, B cells from both low density and high density fractions were cultured with autoreactive cloned T cell lines or lymphokines secreted by such cloned T cell lines, and anti-DNA antibody production was determined. Lymphokines elicited IgM anti-ssDNA antibody production from cells in all Percoll fractions from both B6.H-2bm12 and NZB.H-2bm12 mice. Lymphokines did not elicit production of IgG anti-dsDNA antibody production by cells from 2-month-old B6.H-2bm12 mice. In contrast, substantial production of IgG anti-dsDNA antibody was observed for NZB.H-2bm12 cells in response to lymphokines alone. Thus, B cells from NZB.H-2bm12 mice, because of previous activation in vivo, can proceed to IgG anti-dsDNA antibody production in vitro without direct T cell interaction. When we examined direct T cell help for the IgG anti-dsDNA antibody response, we found that we could distinguish the actions of the two cloned T cell lines studied. 410F T cells provided help predominantly for cells from low density Percoll fractions whether the cells were derived from B6.H-2bm12 or NZB.H-2bm12 mice. 410H T cells were capable of providing help for cells from both the low and high density fractions, and this help accounted for more than half of the antibody production in vitro by cells from B6.H-2bm12 mice.

The Effects of Chinese Herbs on Improving Survival and Inhibiting Anti-ds DNA Antibody Production in Lupus Mice

Systemic lupus erythematosus (SLE) is an important autoimmune disease with multiple organ system involvement. From preliminary studies, we have found that six Chinese herbs: Atractylodes ovata, Anqelica sinensis, Cordyceps sinensis, Liqustrum lucidum, Codonopsis pilosula and Homo sapiens can improve defective in vitro interleukin-2 (IL-2) production in patients with SLE. In order to investigate the in vivo effects of these herbs, we used NZB/NZW F1 mice, a typical lupus animal model used to test these herbs. It was found that C. pilosula, H. sapiens and C. sinensis could prolong the life span of female NZB/NZW F1 mice and inhibited anti-ds DNA production. Although A. sinensis could prolong the life span of experimental mice, it did not inhibit the production of anti-ds DNA antibody. These herbs may have great potential for the management of human SLE in the future.

Analysis of immunoglobulin variable region genes from human IgG anti‐DNA hybridomas

The molecular mechanisms leading to anti-double-stranded (ds) DNA antibody production in systemic lupus erythematosus (SLE) are poorly understood. We describe here the immunoglobulin variable region genes of six human hybridomas secreting IgG anti-dsDNA antibodies derived from three SLE patients. The monoclonal IgG anti-dsDNA antibodies have been shown to be of high affinity and no multireactivity was observed (Winkler et al., Clin. Exp. Immunol., 1991. 85: 379). The comparison of the variable region genes expressed in the hybridomas with known germ-line genes as well as with the germ-line counterparts from one patient shows that the VH and VL sequences are somatically mutated. The pattern and extent of the observed somatic mutations are suggestive for an antigen-driven selection of at least four of these B cell clones. Several VH and VL genes used by the hybridomas were found to be expressed in the natural antibody repertoire, in the restricted fetal repertoire and in B cell malignancies expressing the CD5 antigen.

Purification of a novel B cell growth and differentiation factor associated with lupus syndrome

We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mice produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55-kDa protein that we termed nucleobindin (Nuc). Nuc showed not only induction of anti-ssDNA IgG antibody in cultures of B cells from MRL/l mice (greater than 16 weeks old), but also growth activity. Furthermore, antibodies against existing cytokines have so far not been shown to block Nuc activity on these B cells. In view of the fact that Nuc did not boost anti-ssDNA IgG antibody production in cultures of spleen cells of comparable age from MRL/n mice, which develop a mild form of lupus after the age of one year, Nuc may act on pre-activated B cells to help IgG anti-DNA antibody production. Taken together, Nuc is a new kind of growth and differentiation factor associated with lupus syndrome.

Shifts of anti-Sm-specific antibodies in patients with systemic lupus erythematosus: Analysis by counter-immunoelectrophoresis, immunoblotting and RNA-immunoprecipitation

We investigated serial changes in anti-Sm-specific antibodies by counter-immunoelectrophoresis (CIE), immunoblotting (IB) and RNA-immunoprecipitation (RNA-IP) in three patients with SLE. To assess the immuno-regulatory processes underlying anti-Sm antibody production, we compared changes in anti-Sm-specific antibodies with changes in titres of antinuclear and anti-ds DNA antibodies. IB and RNA-IP detected anti-Sm antibodies earlier than did CIE. The induction of the immune responses against the Sm-specific BB′ and D polypeptides occurred separately in two of the three patients. Anti-Sm developed in all three cases after a flare-up of disease, whereas anti-ds DNA antibody levels fluctuated in parallel with disease activity. Thus, anti-Sm and anti-ds DNA antibody production seems to be independently regulated.

Modulation of T-Cell-Mediated Regulation of Antibody Production by Interaction with Specific Immune Complexes

The importance of the phlogogenic effects of immune complexes in disease pathogenesis has been emphasized, but they also have a role in immunoregulation. We examined the effects of DNA: anti-DNA immune complexes, formed at different antigen:antibody ratios, on the ability of T cells to modulate DNA antibody production in vitro. We found that antigen-excess complexes promote IgM anti-DNA synthesis, whereas antibody-excess complexes favor a switch to IgG anti-DNA production. We suggest that interaction of T cells with immune complexes is an important feedback mechanism in control of the immune response.

An established MRL/Mp-lpr/lpr cell line with null cell properties produces a B cell differentiation factor(s) that promotes anti-single-stranded DNA antibody production in MRL spleen cell culture.

The cell line established from the lymph node cells of an MRL/lpr mouse, was found to have null cell properties in that it lacked Thy-1, Lyt-1 and Lyt-2 as well as sIg, and continued to grow in the absence of exogeneously added lymphokines such as IL-2 and IL-3. Interestingly, this cell line (KML1) or a soluble factor(s) produced by it promoted anti-ssDNA antibody production in cultures of MRL/lpr spleen cells. The factor did not induce cell proliferation. Therefore, it is concluded that the cell line KML1 produced at least a B-cell differentiation factor, but not IL-2 or IL-3 as far as detected with the respective lymphokine-dependent cell lines.