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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

The presence of anti–DNA topoisomerase I (anti–topo I) antibody correlates positively with disease severity in patients with systemic sclerosis (SSc). However, the role of induction of anti–topo I antibody production and its potential contribution to the pathogenesis of SSc remain unclear. The aim of this study was to examine the role of anti–topo I antibody in the pathogenesis of SSc.

Methods

To assess the contribution of anti–topo I antibody to the pathogenetic process, dermal sclerosis, pulmonary fibrosis, and cytokine production were examined in mice treated with topo I and either Freund's complete adjuvant (CFA) or Freund's incomplete adjuvant (IFA).

Results

Treatment with topo I and CFA, in contrast to treatment with topo I and IFA, induced skin and lung fibrosis with increased interleukin-6 (IL-6), transforming growth factor β1, and IL-17 production and decreased IL-10 production. Anti–topo I antibody levels were greater in mice treated with topo I and CFA than in mice treated with topo I and IFA. Furthermore, treatment with topo I and CFA increased Th2 and Th17 cell frequencies in bronchoalveolar lavage fluid, whereas treatment with topo I and IFA increased Th1 and Treg cell frequencies. Moreover, loss of IL-6 expression ameliorated skin and lung fibrosis, decreased Th2 and Th17 cell frequencies, and increased Th1 and Treg cell frequencies.

Conclusion

This study is the first to show that treatment with topo I and CFA induces SSc-like skin and lung fibrosis and autoimmune abnormalities. We also suggest that IL-6 plays important roles in the development of fibrosis and autoimmune abnormalities in this novel SSc model.

Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition with an autoimmune background (1). The presence of autoantibodies is a central feature of SSc, since antinuclear antibodies (ANAs), such as anti–DNA topoisomerase I (anti–topo I) antibody, are detected in >90% of patients (2). Furthermore, abnormal activation of several immune cells has been identified in SSc (3). A recent study has shown that skin and lung fibrosis is ameliorated by treatment with cyclophosphamide, an immunosuppressive agent, indicating that immune activation leads to fibrosis through the stimulation of collagen production by fibroblasts (4). Indeed, SSc patients exhibit infiltration of inflammatory cells, especially CD4+ T cells, and elevated serum levels of various cytokines, especially fibrogenic Th2 and Th17 cytokines and transforming growth factor β1 (TGFβ1), a major fibrogenic growth factor, which correlate positively with disease severity (5, 6).

Autoimmune responses with high levels of circulating autoantibodies are commonly detected in patients with rheumatic diseases (7). Furthermore, specific autoantibodies are associated with clinical subsets of a particular autoimmune disease. Anti–topo I antibody is detected more frequently in SSc patients with diffuse cutaneous thickening than in those with limited cutaneous thickening (8). The presence of anti–topo I antibody correlates positively with dermal sclerosis, pulmonary fibrosis, and overproduction of inflammatory cytokines (9–11). These data indicate that serum levels of anti–topo I antibody are associated with disease severity and/or activity in patients with SSc. In addition, anti–topo I antibodies have been detected in mouse models of SSc (12, 13). These studies strongly suggest a close relationship of autoimmune responses to the pathogenesis of SSc. However, the role of induction of anti–topo I antibody production and its potential contribution to the pathogenesis of SSc remain unclear.

A recent study indicated that mice immunized with recombinant human topo I protein emulsified in Freund's complete adjuvant (CFA) and boosted with topo I emulsified in Freund's incomplete adjuvant (IFA) showed anti–topo I antibody production (14). However, these mice did not show dermal and pulmonary fibrosis (14). Previously, in an experimental autoimmune encephalomyelitis (EAE) model, treatment with myelin oligodendrocyte glycoprotein (MOG) and IFA, in contrast to treatment with MOG and CFA, did not induce interleukin-6 (IL-6) production (15). In the absence of IL-6, Th17 cell responses are impaired whereas Treg cell responses are dominant, suggesting that IL-6 is a critical factor that shifts the immune response from Treg cell responses toward pathogenic Th17 cell responses (16, 17). Indeed, in contrast to treatment with MOG and CFA, treatment with MOG and IFA did not trigger antigen-specific production of IL-17 (15). Moreover, treatment with MOG and IFA did not induce EAE symptoms (15, 18). These data suggest that IL-6 induced by CFA plays important roles in the pathogenesis of autoimmune diseases. However, the relative contributions of the SSc-specific antigen, topo I, and IL-6 induced by CFA to the development of SSc remain unknown.

In this study, we investigated the associations of topo I immunization and of IL-6 induced by CFA with the development of SSc, using wild-type (WT) and IL-6–deficient (IL-6−/−) mice. According to our results, treatment with both topo I and CFA increased IL-6 and IL-17 production and Th17 cell frequencies compared to treatment with both topo I and IFA. In contrast, IL-10 production and Treg cell frequencies were greater in WT mice treated with topo I and IFA than in WT mice treated with topo I and CFA. Furthermore, treatment with topo I and CFA induced dermal sclerosis, pulmonary fibrosis, and anti–topo I antibody production, although treatment with topo I and IFA induced only anti–topo I antibody production. In addition, IL-6 deficiency reduced dermal sclerosis, pulmonary fibrosis, and IL-17 and anti–topo I antibody production as well as the increased Th17 cell frequencies induced by treatment with topo I and CFA, while increasing IL-10 production and Treg cell frequencies. These results suggest that subcutaneous treatment with topo I and CFA induces dermal sclerosis, pulmonary fibrosis, and autoimmune abnormalities, which are mainly regulated by IL-6.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Mice.

WT C57BL/6 mice and IL-6−/− mice with a C57BL/6 background were purchased from The Jackson Laboratory. All mice were housed in a specific pathogen–free barrier facility and screened regularly for pathogens. The mice used in these experiments were age 6 weeks. All studies and procedures were approved by the Committee on Animal Experimentation of Nagasaki University Graduate School of Biomedical Sciences.

Topo I and/or adjuvant treatment.

Recombinant human topo I (TopoGEN) was dissolved in saline (500 units/ml). The topo I solution was mixed 1:1 (volume/volume) with CFA H37Ra (Sigma-Aldrich) or IFA (Sigma-Aldrich). These solutions (300 μl) were injected 4 times subcutaneously into a single location on the shaved back of the mice with a 26-gauge needle at an interval of 2 weeks. Human serum albumin (Protea Biosciences) was used as an irrelevant control human protein, as previously described (19–21). Treatment with human serum albumin with or without adjuvant did not affect skin and lung fibrosis, cytokine production, autoantibody production, or Th cell frequencies in bronchoalveolar lavage (BAL) fluid.

Histopathologic assessment of dermal fibrosis.

Morphologic characteristics of skin sections were assessed under a light microscope. All skin sections were obtained from the paramidline, lower back region (the same anatomic site, to minimize regional variations in thickness). Sections were stained with hematoxylin and eosin (H&E). Dermal thickness, defined as the thickness of skin from the top of the granular layer to the junction between the dermis and subcutaneous fat, was examined. Ten random measurements per section were obtained. All of the sections were examined independently by 2 investigators (AY and SS) in a blinded manner.

Histopathologic assessment of lung fibrosis.

Lungs were excised after 4 weeks of treatment and processed as previously described (12, 13). Sections were stained with H&E and with Azan-Mallory stain to identify collagen deposition. The severity of fibrosis was semiquantitatively assessed according to the method described by Ashcroft et al (22). Briefly, lung fibrosis was graded on a scale of 0 to 8 by examining randomly chosen fields of the left middle lobe. The grading criteria were as follows: grade 0 = normal lung; grade 1 = minimal fibrous thickening of alveolar walls; grade 3 = moderate thickening of walls without obvious damage; grade 5 = increased fibrosis with definite damage and formation of fibrous bands; grade 7 = severe distortion of structure and large fibrous areas; and grade 8 = total fibrous obliteration. Grades 2, 4, and 6 were used as intermediate stages between these criteria. In addition, apoptotic cells were examined using the TUNEL assay (Oncor) according to the manufacturer's instructions. Fluorescein isothiocyanate (FITC; green fluorescence)–labeled antidigoxigenin conjugate was applied to detect apoptotic cells. Phycoerythrin (PE; red fluorescence)– conjugated anti–cytokeratin 19 monoclonal antibody (mAb) was used to detect alveolar epithelial cells. These slides were visualized with a fluorescence microscope (Olympus). The percentage of apoptotic epithelial cells was referred to as the apoptosis index, as described previously (23).

Determination of hydroxyproline content in skin and lung tissue.

Hydroxyproline is a modified amino acid uniquely found at a high percentage in collagen. Therefore, the skin and lung tissue hydroxyproline content was determined as a quantitative measure of collagen deposition (24). Punch biopsy samples (6 mm) obtained from the shaved dorsal skin and the harvested right lung of each mouse were analyzed. A hydroxyproline standard solution of 0–6 mg/ml was used to generate a standard curve.

Enzyme-linked immunosorbent assay (ELISA) for serum cytokines, immunoglobulins, and autoantibodies.

Serum levels of IL-4, IL-6, IL-10, IL-17, interferon-γ (IFNγ), TGFβ1, and tumor necrosis factor α (TNFα) were assessed using specific ELISA kits (R&D Systems). Serum Ig concentrations were assessed as described (13), using affinity-purified mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA (SouthernBiotech) to generate standard curves. ANAs were assessed by indirect immunofluorescence staining using HEp-2 substrate cells (Medical & Biological Laboratories) as described (13). The specific ELISA kits were used to measure anti–topo I (Medical & Biological Laboratories), anti–CENP B (Funakoshi), and anti–U1 RNP antibody (Medical & Biological Laboratories). Relative levels of these antibodies were determined for each group of mice, using pooled serum samples. Sera were diluted at log intervals (1:10–1:105) and assessed for relative autoantibody levels as above, except that the results were plotted as optical density (OD) versus dilution (log scale). The dilutions of sera giving half-maximal OD values were determined by linear regression analysis, thus generating arbitrary units per milliliter values for comparison between sets of sera.

RNA isolation and real-time polymerase chain reaction (PCR).

Total RNA was isolated from lower back skin and lung with RNeasy spin columns (Qiagen). Expression of IL-4, IL-6, IL-10, IL-17, IFNγ, TGFβ1, and TNFα was analyzed by TaqMan Assay (Applied Biosystems). GAPDH was used to normalize messenger RNA (mRNA). Relative expression of real-time PCR products was determined using the ΔΔCt method (13).

Preparation of BAL fluid.

BAL fluid cells were prepared as described elsewhere (25). Briefly, both lungs were excised from mice and BAL fluid was collected. T cells were enriched with a mouse CD4+ T cell kit using an AutoMacs isolator (Miltenyi Biotec). More than 99% of these cells were CD4+ when tested with anti-CD4 mAb (Serotec) (data not shown).

Flow cytometry.

Antibodies used in this study included FITC-conjugated anti-mouse mAb to IL-4 (Imgenex), IFNγ (Genetex), IL-17 (Novus Biologicals), and FoxP3 (Lifespan Biosciences) as well as PE-conjugated anti-mouse mAb to CD4 (Serotec). IFNγ, IL-4, IL-17, and FoxP3 production by BAL fluid CD4+ T cells was determined by flow cytometric intracellular cytokine analysis, as previously described (26, 27). All intracellular staining samples were stimulated with phorbol myristate acetate (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) for 5 hours before analysis.

Statistical analysis.

All data are expressed as the mean ± SD. The Mann-Whitney U test was used to determine the level of significance of differences between sample means, and analysis of variance followed by Bonferroni adjustment was used for multiple comparisons.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Subcutaneous injection of topo I and CFA induces dermal sclerosis and pulmonary fibrosis.

Skin and lung fibrosis was histopathologically assessed 2, 4, 6, and 8 weeks after the initiation of topo I treatment. The dermal thickness and lung fibrosis score increased in a time-dependent manner in mice treated with topo I and CFA (Figure 1). Skin fibrosis, lung fibrosis, alveolar epithelial apoptosis, and inflammatory cell infiltration developed during the first 8 weeks of treatment with topo I and CFA, peaked in the eighth week (Figure 1A), and began to resolve 6 weeks after the cessation of treatment (data not shown). After 6 weeks, treatment with topo I and CFA induced significantly greater dermal thickness relative to saline treatment in WT mice (P < 0.01), although there was no significant difference in dermal thickness among mice treated with saline, topo I, and topo I with IFA (Figures 1A and D). In addition, the dermal thickness was similar among untreated mice, saline-treated mice, IFA-treated mice, and CFA-treated mice. Furthermore, there was no significant difference in dermal thickness and inflammatory cell infiltration between the injected site and the other site.

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Figure 1. Skin and lung fibrosis in wild-type (WT) mice treated with saline, DNA topoisomerase I (topo I) alone, topo I and Freund's incomplete adjuvant (IFA), or topo I and Freund's complete adjuvant (CFA). A and B, Skin and lung fibrosis was assessed by quantitatively measuring dermal thickness and the lung fibrosis score 0, 2, 4, 6, and 8 weeks after treatment (A) and skin and lung hydroxyproline content 8 weeks after treatment (B). Each arrow in A indicates a single treatment. C, Shown are representative lung histologic sections obtained after 8 weeks of treatment, stained with hematoxylin and eosin (H&E) (original magnification × 200), Azan-Mallory stain (Azan-M) (original magnification × 40), and TUNEL (red fluorescence) (original magnification × 400). Cytokeratin 19 (CK19; green fluorescence) (original magnification × 400) was used to determine the presence of alveolar epithelial cells. Also shown is the apoptosis index. D, Shown are representative skin histologic sections obtained after 8 weeks of treatment, stained with H&E (original magnification × 40). d indicates dermis; arrow indicates hypodermis beneath the panniculus carnosus. The hypodermal thickness was measured under a light microscope. Each histogram shows the mean ± SD results obtained for 10 mice of each group. † = P < 0.05; †† = P < 0.01 versus WT mice treated with saline or topo I alone. ∗∗ = P < 0.01.

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Similar results were obtained for the lung fibrosis score and apoptosis index (Figures 1A and C). After 8 weeks of topo I administration, WT mice treated with topo I and CFA exhibited extensive inflammatory infiltration, diffuse fibrosis, and alveolar epithelial apoptosis. Topo I treatment with or without IFA did not affect the development of lung fibrosis and epithelial apoptosis. Cutaneous and lung fibrosis was also assessed by quantifying the hydroxyproline content. In mice treated with topo I and CFA, the skin and lung hydroxyproline content was significantly increased compared with that in mice treated with saline, topo I, or topo I with IFA (P < 0.01 for all) (Figure 1B).

Topo I and adjuvant treatment together induce overproduction of cytokines in the serum, skin, and lung.

In the serum (Figure 2A), skin (Figure 2B), and lung (Figure 2C), mice treated with topo I and CFA or topo I and IFA had elevated levels of IL-4, IFNγ, IL-10, TGFβ1, and TNFα compared with mice treated with saline or topo I alone (P < 0.05 for all). Serum, skin, and lung levels of TGFβ1 were higher in mice treated with topo I and CFA than in mice treated with topo I and IFA (P < 0.05 for all), while mice treated with topo I and IFA showed increased levels of IL-10 relative to mice treated with topo I and CFA (P < 0.05 for all). Mice treated with topo I and CFA exhibited elevated levels of IL-6 and IL-17 compared with mice treated with saline, topo I, or topo I with IFA (P < 0.01 for all).

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Figure 2. Levels of interleukin-4 (IL-4), IL-6, interferon-γ (IFNγ), IL-17, IL-10, transforming growth factor β1 (TGFβ1), and tumor necrosis factor α (TNFα) in serum samples (A) and their mRNA expression in the skin (B) and lung (C) from WT mice treated with saline, topo I alone, topo I and IFA, or topo I and CFA. Serum samples were obtained by cardiac puncture 8 weeks after treatment. Serum cytokine levels were assessed using specific enzyme-linked immunosorbent assays. Total RNA from lower back skin and lung was extracted and reverse transcribed to cDNA, and mRNA expression was analyzed using real-time polymerase chain reaction and normalized to the internal control GAPDH. Each histogram shows the mean ± SD results obtained for 10 mice of each group. ∗ = P < 0.05; ∗∗ = P < 0.01 versus WT mice treated with saline or topo I alone. See Figure 1 for other definitions.

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Elevated serum Ig and anti–topo I antibody levels in mice treated with both topo I and adjuvant.

WT mice treated with topo I alone had Ig levels similar to those in saline-treated WT mice. Treatment with both topo I and adjuvant increased serum IgM, IgG1, IgG2a, IgG2b, and IgG3 levels compared with saline treatment (P < 0.05 for all) (Figure 3A), while the levels of IgA were similar between mice treated with both topo I and adjuvant and saline-treated mice. Mice treated with topo I and CFA had increased IgG1 and IgG3 levels compared with mice treated with topo I and IFA (P < 0.05 for both), while there were no significant differences in the levels of other isotypes between mice treated with topo I and IFA and mice treated with topo I and CFA. ANAs were rarely detectable in saline-treated mice and mice treated with topo I alone (5% of mice, or 1 in 20). ANAs with a homogeneous chromosomal staining pattern were detected in 84% of mice (27/32) treated with topo I and CFA, which was similar to the percentage in mice treated with topo I and IFA (78% [25/32]).

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Figure 3. Serum levels of immunoglobulins (A) and autoantibodies (B) in WT mice treated with saline, topo I alone, topo I and IFA, or topo I and CFA. Serum samples were obtained by cardiac puncture 8 weeks after treatment. Serum levels of immunoglobulins and autoantibodies were determined by specific enzyme-linked immunosorbent assays (ELISAs). Horizontal bars represent the mean. Values in parentheses represent the dilutions of pooled sera giving half-maximal optical density (OD) values in anti–topo I, anti–CENP B, and anti–U1 RNP antibody ELISAs, which were determined by linear regression analysis to generate arbitrary units per ml that could be directly compared between each group of mice (n = 6 for each). ∗ = P < 0.05; ∗∗ = P < 0.01 versus WT mice treated with saline or topo I alone. See Figure 1 for other definitions.

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Autoantibody specificities were further assessed by ELISA (Figure 3B). Mice treated with both topo I and adjuvant had increased levels of IgM and IgG autoantibodies to topo I relative to saline-treated mice and mice treated with topo I alone (P < 0.01 for both). Furthermore, IgG anti–topo I antibody production in mice treated with topo I and CFA was greater than that in mice treated with topo I and IFA (P < 0.05). In addition, the levels of anti–topo I antibody increased during the first 8 weeks of treatment with topo I and CFA, peaked in the eighth week, and began to resolve 6 weeks after the cessation of treatment. Treatment with saline, topo I alone, topo I with CFA, or topo I with IFA did not affect levels of autoantibodies to CENP B and U1 RNP.

IL-6 loss attenuates the development of skin and lung fibrosis induced by treatment with topo I and CFA.

In IL-6−/− mice treated with topo I and CFA, skin fibrosis, lung fibrosis, and epithelial apoptosis similar to that in WT mice treated with topo I and CFA developed during the first 8 weeks of treatment with topo I and CFA and peaked in the eighth week (Figure 4). However, after 6 and 8 weeks, IL-6−/− mice treated with topo I and CFA showed moderate thickening of dermal tissue that was significantly less (31% and 32%, respectively) than that found in WT mice treated with topo I and CFA (P < 0.01), but still greater than that in saline-treated WT mice (P < 0.01) (Figures 4A and C). Saline-treated WT and IL-6−/− mice showed similar dermal thickness (data not shown). Similar results were obtained for the lung fibrosis score and apoptosis index (Figures 4A and D). After 8 weeks, WT mice treated with topo I and CFA exhibited extensive inflammatory cell infiltration, fibrosis, and alveolar epithelial apoptosis. IL-6 deficiency reduced such histologic changes. Skin and lung fibrosis was also assessed by quantifying hydroxyproline content. The skin and lung hydroxyproline content in IL-6−/− mice treated with topo I and CFA was significantly lower than that in WT mice treated with topo I and CFA (P < 0.01), but the content remained higher than that in saline-treated WT mice (P < 0.05) (Figure 4B).

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Figure 4. Skin and lung fibrosis in WT mice treated with saline or with topo I and CFA, and in interleukin-6–deficient (IL-6−/−) mice treated with topo I and CFA. A and B, Skin and lung fibrosis was assessed by quantitatively measuring dermal thickness and the lung fibrosis score 0, 2, 4, 6, and 8 weeks after treatment (A) and skin and lung hydroxyproline content 8 weeks after treatment (B). Each arrow in A indicates a single treatment. C, Shown are representative skin histologic sections obtained after 8 weeks of treatment, stained with H&E (original magnification × 40). d indicates dermis; arrow indicates hypodermis beneath the panniculus carnosus. D, Shown are representative lung histologic sections obtained after 8 weeks of treatment, stained with H&E (original magnification × 200), Azan-Mallory stain (original magnification × 40), and TUNEL (red fluorescence) (original magnification × 400). Cytokeratin 19 (green fluorescence) (original magnification × 400) was used to determine the presence of alveolar epithelial cells. Also shown is the apoptosis index. Each histogram shows the mean ± SD results obtained for 10 mice of each group. † = P < 0.05; †† = P < 0.01 versus WT mice treated with topo I and CFA. ∗ = P < 0.05; ∗∗ = P < 0.01 versus saline-treated WT mice. See Figure 1 for other definitions.

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IL-6 deficiency suppresses overproduction of cytokines, Ig, and autoantibodies induced by treatment with topo I and CFA.

Serum levels of IL-6 were not detected in IL-6−/− mice (Figure 5A). Furthermore, there was no difference in serum levels of IL-4, IFNγ, IL-17, IL-10, TGFβ1, and TNFα between saline-treated IL-6−/− mice and saline-treated WT mice. In contrast, 8 weeks after treatment with topo I and CFA, serum levels of all cytokines examined were increased in WT mice (P < 0.01). However, IL-6−/− mice showed a significant decrease in serum IL-17 levels relative to WT mice (P < 0.01). In contrast, IL-6−/− mice showed significantly increased production of IL-10 relative to WT littermates (P < 0.01). Similar results were obtained for skin and lung mRNA expression (data not shown). Furthermore, IL-6−/− mice treated with topo I and CFA showed significantly lower levels of IgM, IgG1, IgG2a, IgG2b, and IgG3 compared with WT mice treated with topo I and CFA (P < 0.05), but these levels remained higher than those in saline-treated WT mice (Figure 5B). Serum levels of autoantibodies were also examined (Figure 5C). IL-6−/− mice treated with topo I and CFA had decreased levels of IgG autoantibodies to topo I compared with WT mice treated with topo I and CFA (P < 0.05).

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Figure 5. Serum levels of cytokines (A), immunoglobulins (B), and anti–topo I antibodies (C) in WT mice treated with saline or with topo I and CFA, and in interleukin-6–deficient (IL-6−/−) mice treated with topo I and CFA. Serum samples were obtained by cardiac puncture 8 weeks after treatment. Serum levels of cytokines, immunoglobulins, and anti–topo I antibodies were determined by specific enzyme-linked immunosorbent assays (ELISAs). In A, each histogram shows the mean ± SD results obtained for 10 mice of each group. In B and C, horizontal bars represent the mean. In C, values in parentheses represent the dilutions of pooled sera giving half-maximal optical density (OD) values in anti–topo I antibody ELISAs, which were determined by linear regression analysis to generate arbitrary units per ml that could be directly compared between each group of mice (n = 6 for each). † = P < 0.05; †† = P < 0.01 versus WT mice treated with topo I and CFA. ∗ = P < 0.05; ∗∗ = P < 0.01 versus saline-treated WT mice. IFNγ = interferon-γ; TGFβ1 = transforming growth factor β1; TNFα = tumor necrosis factor α (see Figure 1 for other definitions).

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Th cell balance in BAL fluid from mice treated with saline, topo I alone, or both topo I and adjuvant.

We investigated Th1, Th2, Th17, and Treg cell frequencies in BAL fluid from WT mice treated with saline, topo I alone, topo I and IFA, or topo I and CFA, and from IL-6−/− mice treated with topo I and CFA (Figure 6). The percentages of CD4+ BAL fluid cells were 15.2%, 14.9%, 16.7%, 15.8%, and 15.4% in saline-treated WT mice, WT mice treated with topo I alone, WT mice treated with topo I and IFA, WT mice treated with topo I and CFA, and IL-6−/− mice treated with topo I and CFA, respectively. The populations expressing IFNγ, IL-4, IL-17, or FoxP3 did not overlap (data not shown), which is consistent with previous studies (26).

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Figure 6. Th1, Th2, Th17, and Treg cell frequencies in bronchoalveolar lavage (BAL) fluid from WT mice treated with saline, topo I alone, topo I and IFA, or topo I and CFA, and from interleukin-6–deficient (IL-6−/−) mice treated with topo I and CFA. A, Th1, Th2, Th17, and Treg cell frequencies were determined by surface CD4 expression and intracellular expression of interferon-γ (IFNγ), IL-4, IL-17, and FoxP3, respectively, as previously described (26). BAL fluid was analyzed by flow cytometry after 8 weeks of topo I or saline treatment. Data are representative of 3 independent experiments. Percentages of Th1, Th2, Th17, and Treg cells are shown in each upper right quadrant. B, Shown are summaries of Th1, Th2, Th17, and Treg cell frequencies in each group. Each histogram shows the mean ± SD results obtained for 10 mice of each group. ∗ = P < 0.05; ∗∗ = P < 0.01 versus WT mice treated with saline or topo I alone. See Figure 1 for other definitions.

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There were no significant differences in Th1, Th2, Th17, and Treg cell frequencies between saline-treated WT mice and WT mice treated with topo I alone. WT mice treated with topo I and IFA exhibited significantly increased Th1, Th2, and Treg cell frequencies compared with saline-treated WT mice (P < 0.01 for all). Similarly, WT mice treated with topo I and CFA exhibited significantly increased frequencies of Th1 cells (P < 0.01), Th2 cells (P < 0.01), Th17 cells (P < 0.01), and Treg cells (P < 0.05) relative to saline-treated WT mice. In addition, Th1 and Treg cell frequencies were significantly lower in WT mice treated with topo I and CFA than in WT mice treated with topo I and IFA (P < 0.05 for both). Moreover, WT mice treated with topo I and CFA displayed higher frequencies of Th2 and Th17 cells compared with WT mice treated with topo I and IFA (P < 0.05 for both). IL-6−/− mice treated with topo I and CFA exhibited significantly increased frequencies of Th1, Th2, and Treg cells compared with saline-treated WT mice. Furthermore, Th1 and Treg cell frequencies were significantly increased in IL-6−/− mice treated with topo I and CFA compared with WT mice treated with topo I and CFA (P < 0.05 and P < 0.01, respectively). In contrast, IL-6−/− mice treated with topo I and CFA exhibited significantly reduced Th2 and Th17 cell frequencies compared with WT mice treated with topo I and CFA (P < 0.05 and P < 0.01, respectively).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The precise mechanisms involved in the pathogenesis of SSc remain unknown, although autoimmunity is considered to be involved (3). The presence of anti–topo I antibody has been clinically associated with a more severe form of SSc that exhibits diffuse cutaneous and lung involvement (3, 9, 10, 28). In addition, a recent study showed that 20% of anti–topo I antibody–positive patients lost anti–topo I antibodies during the disease course and had a favorable outcome, suggesting the clinical importance of anti–topo I antibody levels in patients with SSc (29). The topo I protein is a ubiquitous and indispensable enzyme involved in DNA replication and protein transcription (30). Human topo I has >93% sequence identity to the 766 amino acid residues of mouse topo I (GenBank accession no. L20632). It has been demonstrated that immunization using a mutated self antigen is capable of inducing an autoreactive response that is more potent than that using the bona fide autoantigen in mice (31). Therefore, we immunized mice with recombinant human topo I as described previously (14, 32). The present study is the first to demonstrate that subcutaneous injection of topo I with CFA induces skin and lung fibrosis, hypergammaglobulinemia, and anti–topo I antibody production in WT mice (Figures 1 and 3), generating many characteristics of human SSc. Furthermore, treatment with topo I and CFA increased the production of various fibrogenic cytokines (Figure 2). Collectively, these results indicate that mice treated with topo I and CFA show SSc-like fibrosis and might be a novel animal model of human SSc.

In this study, treatment with topo I and IFA, in contrast to treatment with topo I and CFA, did not induce skin and lung fibrosis (Figure 1). Previous studies have demonstrated that CFA is essential for development of autoimmune responses, such as autoimmune encephalomyelitis, in each of the induction protocols (18). In contrast, IFA injection can prevent induction by CFA (33). This may be explained by the fact that CFA treatment induces IL-6 production, whereas IFA injection cannot affect IL-6 production (15, 34, 35). Indeed, in our present study, WT mice treated with topo I and CFA exhibited significantly higher levels of IL-6 relative to mice treated with topo I and IFA (Figure 2). Moreover, a recent study has shown that both IL-6 induced by CFA and exogenous IL-6 injection augment autoantibody production in immunized mice (36). In the present study, the levels of IgG anti–topo I antibody, IgG1, and IgG3 in WT mice treated with topo I and CFA were greater than those in WT mice treated with topo I and IFA (Figure 3). These results suggest that IL-6 induced by CFA contributes to development of fibrosis and augments autoantibody production. Furthermore, a recent case report has shown that the anti–IL-6 receptor antibody tocilizumab decreased skin sclerosis in SSc patients (37). In fact, IL-6 deficiency inhibited the development of fibrosis with decreased autoantibody production in mice treated with topo I and CFA (Figures 4 and 5). Thus, immunization with topo I can induce dermal and pulmonary fibrosis and hypergammaglobulinemia, which requires IL-6 induced by CFA.

Previous studies have demonstrated a fibrogenic effect of Th2 cytokines, such as IL-4 and IL-6 (13, 24, 38). Th17 cytokines, such as IL-17, also have a fibrogenic effect on dermal, pulmonary, and cardiac fibroblasts (39, 40). Indeed, SSc patients exhibit elevated serum levels of these cytokines, which promote collagen synthesis (5, 6, 27, 39, 41, 42). Some studies have also shown that IFNγ, a Th1 cytokine, has an antifibrotic effect (26, 27, 43, 44). In addition, IL-10 produced by Treg cells has antifibrotic and antiinflammatory effects on fibrotic diseases (45). Previously, we and others confirmed that IL-4 and/or IL-17 stimulation increased proliferation and collagen production of dermal fibroblasts, while these processes were inhibited by IFNγ and IL-10 (27, 45). The results of the present study indicate differential expression levels of these cytokines in mice treated with both topo I and adjuvant (Figure 2). Treatment with topo I and CFA induced significantly higher production of IL-6, IL-17, and TGFβ1 in parallel with increased dermal and pulmonary fibrosis. Treatment with topo I and IFA enhanced IL-10 production, which was accompanied by inhibited fibrosis. Thus, the differential expression levels of these cytokines that were induced by topo I and adjuvant treatment might contribute to the development of skin and lung fibrosis.

It is possible that treatment with topo I and adjuvant alters the frequencies of fibrogenic Th2 and Th17 cells and antifibrogenic Th1 and Treg cells. This may result in differential production of cytokines, which may then directly or indirectly influence the development of dermal sclerosis, pulmonary fibrosis, and autoimmune abnormalities. A recent study indicated that CD4+ cells play an important role in fibrosis, although these cells are a minority population (27). Moreover, previous studies have demonstrated that IL-6 together with TGFβ1 is capable of inducing Th17 cells (15). Furthermore, in the absence of IL-6, Th17 cell responses are impaired, whereas Treg cell responses are dominant, suggesting that IL-6 is a critical factor that shifts the immune response from a Treg cell response toward a pathogenic Th17 cell response (16). Consistent with these findings, the results of the present study showed that treatment with topo I and CFA increased Th17 cell frequencies and decreased Treg cell frequencies in parallel with increased IL-6 and TGFβ1 overproduction (Figures 2 and 6). In contrast, treatment with topo I and IFA increased Treg cell frequencies and decreased Th17 cell frequencies. Furthermore, lack of IL-6 expression inhibited accumulation of Th17 cells and increased numbers of Treg cells in mice treated with topo I and CFA (Figure 6).

Recently, other studies suggested that IL-6 also promotes Th2 cell differentiation and simultaneously inhibits Th1 cell polarization (46, 47). In the absence of any polarizing cytokine, IL-6 directs the differentiation of the CD4+ cells to a Th2 phenotype but not to a Th1 phenotype, since cells differentiated in the presence of IL-6 produce high amounts of IL-4 but not IFNγ (46, 47). IL-4 promotes Th2 differentiation but inhibits Th1 differentiation, although IFNγ stimulates Th1 differentiation but suppresses Th2 differentiation (48–50). Indeed, in the present study, treatment with topo I and CFA increased Th2 cell frequencies and decreased Th1 cell frequencies in WT mice (Figure 6). Moreover, IL-6 deficiency inhibited augmentation of Th2 cell frequencies and increased Th1 cell frequencies. Collectively, treatment with topo I and CFA may lead to increased Th2 and Th17 cell frequencies in parallel with deteriorated skin and lung fibrosis, whereas topo I and IFA treatment leads to increased Th1 and Treg cell frequencies that are accompanied by inhibited fibrosis of skin and lung.

To date, few studies have addressed the role of induction of anti–topo I antibody production and its potential association with the pathogenesis of SSc. This is the first systematic study to reveal that treatment with topo I and CFA induces SSc-like dermal sclerosis, pulmonary fibrosis, and autoimmune abnormalities in mice. However, the exact role of anti–topo I antibody in fibrogenesis and overproduction of cytokines remains unclear. Future studies, in which anti–topo I antibodies are isolated from mice treated with topo I and adjuvant and transferred into recipient mice, will be needed to clarify the exact role of anti–topo I antibody in this pathogenesis. We also suggest that IL-6 plays important roles in the development of fibrosis and autoimmune abnormalities in this novel model of SSc induced by treatment with topo I and CFA. These results provide additional clues to understanding the complexity of the pathogenesis of SSc.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Sato had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Yoshizaki, Sato.

Acquisition of data. Yoshizaki, Yanaba, Ogawa.

Analysis and interpretation of data. Yoshizaki, Ogawa, Asano, Kadono, Sato.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Ms M. Yozaki, Ms A. Usui, and Ms K. Shimoda for technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES