Article Text

Extended report
Antifibroblast antibodies from systemic sclerosis patients bind to α-enolase and are associated with interstitial lung disease
  1. B Terrier1,
  2. M C Tamby1,
  3. L Camoin3,4,
  4. P Guilpain1,2,
  5. A Bérezné2,
  6. N Tamas1,
  7. C Broussard3,4,
  8. F Hotellier3,4,
  9. M Humbert5,
  10. G Simonneau5,
  11. L Guillevin2,
  12. L Mouthon1,2
  1. 1
    Paris Descartes University, Faculty of Medicine, UPRES EA 4058, Paris, France
  2. 2
    Paris Descartes University, Faculty of Medicine, Department of Internal Medicine and Reference Center for Necrotizing Vasculitis and Systemic Sclerosis, Cochin Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France
  3. 3
    Cochin Institute, Paris Descartes University, CNRS (UMR 8104), Plate-Forme Protéomique Paris 5, Paris, France
  4. 4
    Inserm U567, Paris, France
  5. 5
    Paris Sud University, Faculty of Medicine, Department of Pneumology and French Reference Center for Pulmonary Arterial Hypertension, Antoine-Béclère Hospital, AP-HP, Clamart, France
  1. Correspondence to Dr L Mouthon, Laboratoire d’Immunologie, UPRES EA 4058, Pavillon Gustave Roussy, 4e étage, Université Paris Descartes, 8 rue Méchain, 75014 Paris, France; luc.mouthon{at}cch.aphp.fr

Abstract

Objective: To identify target antigens of antifibroblast antibodies (AFA) in systemic sclerosis (SSc) patients.

Patients and Methods: In the first part, sera from 24 SSc patients (12 with pulmonary arterial hypertension (PAH) and 12 without) and 36 idiopathic PAH patients, tested in pooled sera for groups of three, were compared with a sera pool from 14 healthy controls (HC). Serum IgG reactivity was analysed by the use of a two-dimensional electrophoresis and immunoblotting technique with normal human fibroblasts antigens. In the second part, serum IgG reactivity for two groups: 158 SSc, 67 idiopathic PAH and 100 HC; and 35 SSc and 50 HC was tested against α-enolase from Saccharomyces cerevisiae and recombinant human (rHu) α-enolase, respectively, on ELISA.

Results: In the first part, α-enolase was identified as a main target antigen of AFA from SSc patients. In the second part, 37/158 (23%) SSc patients, 6/67 (9%) idiopathic PAH patients and 4/100 (4%) HC (p<0.001) had anti-S cerevisiae α-enolase antibodies; 12/35 (34%) SSc patients and 3/50 (6%) HC had anti-rHu α-enolase antibodies (p = 0.001). In SSc, the presence of anti-S cerevisiae α-enolase antibodies was associated with interstitial lung disease (ILD), decreased total lung capacity (73.2% vs 89.7%; p<0.001) and diffusion capacity for carbon monoxide (47.4% vs 62.3%; p<0.001), and antitopoisomerase 1 antibodies (46% vs 21%; p = 0.005) but not anticentromere antibodies (11% vs 34%; p = 0.006). Results were similar with rHu α-enolase testing.

Conclusion: In SSc, AFA recognise α-enolase and are associated with ILD and antitopoisomerase antibodies.

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Systemic sclerosis (SSc) is a connective tissue disorder characterised by excessive collagen deposition in the dermis and internal organs, together with vascular hyperreactivity and obliteration phenomena.1 2 Antinuclear antibodies are present in 80–90% of cases.3 Three autoantibodies are specific for SSc and are mutually exclusive: anticentromere antibodies, associated with limited cutaneous SSc,4 antitopoisomerase 1 antibodies, associated with interstitial lung disease (ILD)5 and anti-RNA-polymerase III antibodies, associated with scleroderma renal crisis.6 In addition, the serum of SSc patients has been found to contain non-specific autoantibodies, including antifibrillin 1,7 anti-endothelial cells8 9 10 11 and antifibroblast antibodies (AFA).12 13 14 15 16 17 In SSc patients, AFA can activate fibroblasts and induce extracellular matrix protein synthesis.13 15 Antibodies directed against platelet-derived growth factor receptor were recently identified in SSc patients. These antibodies were reported to induce tyrosine phosphorylation and reactive oxygen species accumulation and stimulate type I collagen gene expression and myofibroblast phenotype conversion in normal human primary fibroblasts.15 However, the presence of stimulatory anti-platelet-derived growth factor receptor antibodies in SSc patients remains controversial.18 19

In a previous study, using a proteomic approach combining two-dimensional electrophoresis (2-DE) and immunoblotting, with normal human fibroblasts as a source of self-antigens, we identified in patients with idiopathic pulmonary arterial hypertension (PAH) and SSc-related PAH, specific target antigens of AFA that play key roles in cell biology and the maintenance of homeostasis.20 Therefore, we decided to use the same approach to identify the target antigens of AFA in SSc patients.

Patients and methods

Serum samples

In the first part of the study, in order to identify the target antigens of AFA, serum samples were obtained from 24 patients with SSc (12 with PAH confirmed by right heart catheterisation (RHC) and 12 without) and 36 patients with PAH confirmed by RHC (24 with idiopathic PAH, six with familial PAH and six with dexfenfluramine-related PAH) and 14 healthy controls (HC) who did not differ significantly from patients in terms of age or sex. In the second part of the study, on the basis of validation of the identified target antigen by ELISA and phenotypical correlations, we included 158 patients with SSc, 67 patients with PAH confirmed by RHC and 100 HC. Sera from all patients and HC were aliquoted and stored at −80°C for testing. All patients gave their written informed consent according to the ethics committee of the La Pitié-Salpêtrière Hospital Group.

SSc patients fulfilled the criteria for SSc of the American Rheumatism Association21 and/or the Leroy and Medsger criteria.22 Limited SSc was defined by Raynaud’s phenomenon and abnormalities seen on capillaroscopy and/or SSc-specific autoantibodies in the absence of skin involvement; limited cutaneous SSc was defined, in addition to the previous criteria, by skin thickening in areas distal to the elbows and knees;23 and diffuse cutaneous SSc was defined by skin thickening proximal, as well as distal, to the elbows and knees.23 None of the patients had another connective tissue disease or cancer.

For 2-DE and immunoblotting, patients were separated by distinct phenotypes: (1) idiopathic PAH; (2) familial PAH; (3) dexfenfluramine-related PAH; (4) SSc–PAH and (5) SSc patients without PAH. Sera from groups of three patients with an identical PAH and/or SSc phenotype were pooled. Sera from the 14 HC were also pooled. We thus tested eight pools of sera for patients with idiopathic PAH, two pools for patients with familial PAH, two pools for patients with dexfenfluramine-related PAH, four pools for SSc patients with PAH, four pools for SSc patients without PAH and one pool for HC. For ELISA experiments, serum samples for SSc patients, idiopathic PAH patients and HC were tested individually.

Clinical, biological and immunological parameters were recorded by the use of a standardised form as detailed (see supplemental data available online only).

Cell culture

Normal human dermal fibroblasts were obtained from skin biopsies performed in HC. Biopsy specimens were cut and seeded into Petri dishes and cultured as previously described.20

Detection of antibody reactivity with cell antigens using 2-DE and immunoblotting

Antibody reactivity was analysed by the use of a 2-DE and immunoblotting technique with normal human dermal fibroblasts as previously reported.20 Cell culture, protein extraction, protein quantification, two-dimensional gel electrophoresis, two-dimensional blots and protein identification by mass spectrometry on 2-DE gels were performed as detailed (see supplemental data available online only).20 24 25 26 Identified proteins are given in table 1.

Table 1

Characteristics of cellular proteins identified by sera of patients with SSc

Detection and validation of antibody reactivity against Saccharomyces cerevisiae and human recombinant α-enolase by ELISA

Serum IgG reactivity from 158 SSc patients, 67 PAH patients and 100 HC was tested by ELISA against Saccharomyces cerevisiae α-enolase (Sigma, St Louis, Missouri, USA), and serum IgG reactivity from 35 SSc patients and 50 HC was tested against recombinant human (rHu) α-enolase (Abnova, Taipei, Taiwan). ELISA was performed as detailed in the supplemental data (available online only). Samples were considered positive for anti-S cerevisiae or anti-rHu α-enolase antibodies when optical density was greater than or equal to the mean plus 2 SD the value for HC.

Statistical analysis

Data are presented as medians and percentages. Fisher’s exact test or the χ2 test was used as appropriate to compare qualitative values, and the Mann–Whitney non-parametric test was used to compare quantitative values; p<0.05 was considered significant. Statistical analyses involved the use of InStat (version 3.00). For 2-DE and protein identification by mass spectrometry, analyses were performed as previously described.20

Results

Characteristics of patients assessed for AFA by the proteomic approach

The clinical and immunological characteristics of PAH patients and SSc patients are summarised in table 2. Ten patients had limited SSc. Patients with idiopathic PAH were significantly younger than those with SSc with or without PAH (38.9 (SD 15.1) vs 54.8 (SD 16.6) years; p = 0.001). The sex ratio was similar between the two groups. The disease duration of PAH was similar for patients with idiopathic PAH and those with SSc (27.4 (SD 41.4) vs 25.3 (SD 19.4) months; p = 0.57).

Table 2

Clinical and immunological characteristics of PAH patients and SSc patients assessed for AFA by 2-DE

Serum IgG reactivity in HC and in patients with PAH and/or SSc with fibroblast antigens

From proteins extracted from normal human fibroblasts, 859 protein spots were detected on silver nitrate staining, and 436 (SD 118) spots were successfully transferred onto polyvinylidene difluoride membranes. Serum IgG from the sera pools for SSc patients recognised more protein spots than serum IgG from HC (84 (SD 29) vs 43 (SD 22); p = 0.03). Serum IgG from the 12 sera pools for PAH patients and eight sera pools for SSc patients recognised 92 (SD 30) and 72 (SD 24) spots, respectively. Serum IgG from sera for PAH patients recognised a significantly greater number of protein spots than those for HC (p<0.05), with no significant difference in recognition between SSc patients and HC.

Serum IgG from the four sera pools for SSc patients with PAH and from the four sera pools for SSc patients without PAH recognised 81 (SD 12) and 63 (SD 32) protein spots, respectively (not significant).

Comparison of the binding of serum IgG antibodies between SSc patients and HC and identification of target antigens of AFA

We used computer analysis to assess the binding of serum IgG antibodies to target antigens in pools of sera for SSc patients and HC. We selected protein spots shared by SSc patients and HC, but recognised more intensely in SSc patients than in HC; and protein spots recognised in more than 75% of the pools of sera of SSc patients and not recognised by the pool of HC. We thus selected 14 protein spots recognised by serum IgG in sera from SSc patients (fig 1).

Figure 1

Two-dimensional silver-stained protein pattern of total protein extracted from healthy donor fibroblasts. Identification of the positions of the 13 reactive spots was arbitrarily assigned by a computer program. GCP, glutamate carboxy-peptidase; G6PD, glucose-6-phosphate-deshydrogenase; HSP27, heat-shock protein 27; Kelch-like ECH, Kelch-like ECH-associated protein 1; PHF15, protein Jade-2; PI3 kinase, phosphatidyl inositol 3 kinase; UGT1A, UDP-glucuronosyltransferase 1–2; ZFP51, zinc finger protein 51.

Thirteen of the 14 spots were successfully identified on mass spectrometry. The target antigens of AFA recognised in sera of SSc patients and not HC are listed in table 1. The target antigens of AFA recognised more intensely by sera of SSc patients than those of HC were caldesmon, UDP-glucuronosyltransferase 1–2, p22Dokdel and α-enolase (fig 2). The proteins identified and indications regarding the reliability of these assignments are in table 1.

Figure 2

Serum IgG reactivity in pulmonary arterial hypertension (PAH) patients, systemic sclerosis (SSc) patients and healthy controls with fibroblast antigens by two-dimensional electrophoresis and immunoblotting focusing on the area corresponding to reactivity to α-enolase. Protein spots presented in a selected area are from 45 to 50 kDa and isoelectric point from 6.50 to 7.50. IPAH, idiopathic pulmonary arterial hypertension; MW, molecular weight.

Detection of anti-α-enolase antibodies by ELISA

Sera for patients with SSc (n  =  158), idiopathic PAH (n  =  67) and HC (n  =  100) were assessed for anti-S cerevisiae α-enolase antibodies by ELISA. SSc patients more frequently than HC showed anti-S cerevisiae α-enolase antibodies (37/158 (23%) vs 4/100 (4%); p<0.001). No significant difference was found between idiopathic PAH patients and HC (6/67 (9%) vs 4/100 (4%); fig 3). On ELISA, SSc patients (n  =  35) more frequently than HC (n  =  50) showed anti-rHu α-enolase antibodies (12/35 (34%) vs 3/50 (6%); p = 0.001; data not shown).

Figure 3

Detection of anti-S cerevisiae α-enolase antibodies by ELISA in systemic sclerosis (SSc) patients, pulmonary arterial hypertension (PAH) patients and healthy controls. The line represents the positivity threshold for anti-S cerevisiae α-enolase antibodies, defined by optical density (OD) greater than or equal to the mean plus 2 SD of the value for healthy controls.

Clinical and immunological characteristics of SSc patients with anti-α-enolase antibodies

The clinical and immunological characteristics of SSc patients assessed for anti-α-enolase antibodies are depicted in table 3. Patients with anti-S cerevisiae α-enolase antibodies (n  =  37) more frequently than those without antibodies (n  =  121) showed decreased total lung capacity (73.2% vs 89.7%; p<0.001), decreased forced vital capacity (73.0% vs 87.1%; p = 0.016), restrictive syndrome with total lung capacity less than 80% (76% vs 32%; p<0.001), pulmonary fibrosis (27% vs 12%; p = 0.03), decreased diffusion capacity for carbon monoxide (47.4% vs 62.3%; p<0.001) and antitopoisomerase 1 antibodies (46% vs 21%; p = 0.005), and less frequently showed anticentromere antibodies (11% vs 34%; p = 0.006). Moreover, patients with anti-S cerevisiae α-enolase antibodies more frequently claimed black ethnicity (26% vs 12%; p = 0.06) and were younger (44.2 (SD 16.7) vs 48.2 (SD 14.6); p = 0.08), but not significantly, than those without antibodies (table 3). Among the 32 patients with SSc–PAH, nine had pulmonary fibrosis and 23 had no pulmonary fibrosis on computed tomography scan. Four out of the nine (44%) patients with pulmonary fibrosis and eight out of 23 (35%) of those without pulmonary fibrosis had anti-α-enolase antibodies (p = 0.70). Also, capillaroscopic abnormalities (megacapillaries and/or avascular areas) were found in 17 of 27 (63%) patients with anti-α-enolase antibodies and in 63 of 97 (65%) of those without (p = 1.00).

Table 3

Clinical and immunological associations in SSc patients by anti-S cerevisiae α-enolase antibody profile

Among patients assessed for anti-rHu α-enolase antibodies, 12 (34%) were positive for the antibodies and 23 (66%) were negative. Patients with anti-rHu α-enolase antibodies more frequently than those without antibodies exhibited ILD with decreased total lung capacity (69.3% vs 89.4%; p = 0.006), total lung capacity less than 80% (73% vs 29%; p = 0.05), decreased diffusion capacity for carbon monoxide (43.9 vs 64.5; p = 0.005; data not shown) and decreased forced vital capacity (67.6% vs 86.7%; p = 0.08) and pulmonary fibrosis (42% vs 13%; p = 0.09), although not significantly. Patients with anti-rHu α-enolase antibodies more frequently than those without antibodies showed antitopoisomerase 1 antibodies (67% vs 22%; p = 0.02; data not shown). As observed with anti-S cerevisiae α-enolase antibodies, patients with anti-rHu α-enolase antibodies more frequently claimed black ethnicity (42% vs 13%; p = 0.09) and were younger (42.1 (SD 13.8) vs 49.2 (SD 16.9); p = 0.08), but not significantly, than those without antibodies (data not shown).

Discussion

We used a 2-DE and immunoblotting technique and identified 13 target antigens of AFA in SSc patients. These target antigens include glucose-6-phospate deshydrogenase, glutamate carboxypeptidase, heat-shock protein 27, zinc finger protein 51, PHF15, phosphatidyl-inositol-3 kinase, Kelch-like ECH-associated protein 1, caldesmon, UDP-glucuronosyltransferase 1–2, p22Dokdel and isoforms of α-enolase. These proteins are involved in cytoskeletal regulation, cell contraction, oxidative stress, cell energy metabolism and in different pathways, playing key roles in cell biology and the maintenance of homeostasis.

Among these protein antigens, we selected α-enolase to validate our approach and establish phenotypic associations because anti-α-enolase antibodies have been found in a large variety of autoimmune and inflammatory diseases including SSc,27 and because IgG reactivity directed against α-enolase identified in SSc patients was particularly higher than that in HC.

Alpha-enolase, also called non-neuronal enolase, belongs to a family of glycolytic enzymes.28 In addition to its glycolytic function, α-enolase has many other functions related to its subcellular location.28 Alpha-enolase is expressed at the surface of many eukaryotic cells, such as stimulated haematopoietic cells (neutrophils, B and T lymphocytes, monocytes), epithelial, neuronal and endothelial cells,28 29 30 31 32 and may serve as a strong receptor and activator of plasminogen.28 30 However, upregulation of α-enolase did not contribute to increased plasminogen activation in c-jun transformed rat fibroblasts.33 When located in the nucleus, α-enolase is a Myc-binding protein (MBP-1), playing a crucial role in the regulation of cell growth and differentiation. In addition, α-enolase is upregulated in endothelial cells in the context of hypoxic stress and may also play a direct or indirect role in the site-specific organisation of tubules.34

Anti-α-enolase antibodies have been found in a large variety of autoimmune and inflammatory diseases including patients who reacted with centrosomes in systemic rheumatic diseases,34 antineutrophil cytoplasm antibody-associated systemic vasculitides,35 ulcerative colitis and Crohn’s disease,36 primary sclerosing cholangitis,37 systemic lupus erythematosus,38 mixed cryoglobulnaemia,38 SSc,38 rheumatoid arthritis (RA),39 Behçet’s disease,40 multiple sclerosis,41 Hashimoto’s encephalopathy42 and paraneoplastic retinopathy such as cancer-associated retinopathy.43 In HC, antibodies against α-enolase have been found in 0–6%,36 38 43 44 45 and recently we reported that α-enolase was one of the main targets of natural antiendothelial cell antibodies.24

Taking into account the wide spectrum of diseases associated with anti-α-enolase antibodies, these antibodies do not seem to help in the diagnosis of a specific autoimmune disease. However, a recent study showed the presence of anticitrullinated α-enolase antibodies in patients with RA, and that these antibodies were specific for RA.46 Besides its possible diagnostic relevance, anti-α-enolase antibodies could be used as prognostic markers in systemic lupus erythematosus and cryoglobulinaemia as the presence of anti-α-enolase antibodies was associated with renal involvement,35 38 47 but these findings remain controversial.48

In previous work in SSc, the number of patients tested was small and no correlation was clearly made between the detection of anti-α-enolase antibodies and clinical manifestations, except for severe organ involvement.38 In the present work, we found anti-S cerevisiae α-enolase and anti-rHu α-enolase antibodies in 23% and 33% of SSc patients, respectively, significantly more frequently than in HC. Moreover, anti-α-enolase antibodies were associated with ILD and antitopoisomerase 1 antibodies. These findings further support that anti-α-enolase antibodies could contribute to the pathophysiology of SSc by inducing fibroblast proliferation or activation, as it has previously been reported that anti-α-enolase antibodies could induce cell proliferation, endothelial cell injury through the generation of immune complexes and activation of the complement cascade, and inhibit the binding of plasminogen to α-enolase with perturbations of the intravascular and pericellular fibrinolytic system.27 Also, the presence of both anti-S cerevisiae α-enolase and anti-rHu α-enolase antibodies may indicate a role for microbial enolase in the initiation of autoimmunity in SSc patients.

In previous work, we detected AFA in 30% of patients with SSc-associated PAH and 40% of patients with idiopathic PAH.17 In SSc patients, AFA have been shown to induce fibroblast activation in vitro and a pro-adhesive and pro-inflammatory phenotype of these cells.13 Target antigens of AFA in PAH patients were recently identified by our group.20 Patients with PAH and those with SSc share most of these target antigens: glucose-6-phosphate dehydrogenase, glutamate carboxypeptidase, heat-shock protein 27, zinc finger protein 51, PHF15, phosphatidylinositol 3-kinase, Kelch-like ECH-associated protein 1 and α-enolase. This finding supports the concept that common features take part in the pathophysiology of idiopathic PAH and SSc.

We also found new target antigens of AFA in SSc patients: caldesmon, UDP-glucuronosyltransferase 1–2 and p22Dokdel. These proteins play key roles in cell biology: caldesmon is an actin and myosin-binding protein implicated in the regulation of actomyosin interactions in smooth muscle cells and fibroblasts, and p22Dokdel, a truncated p21Ras GTPase activating protein-associated 62-kDa protein isoform, is an early substrate of various tyrosine phosphorylation pathways.49

In conclusion, our results confirm the presence of AFA in SSc patients and provide evidence that these antibodies recognise cellular targets, in particular α-enolase, playing key roles in cell biology and maintenance of homeostasis. We validated our findings by using S cerevisiae α-enolase and rHu α-enolase. Anti-α-enolase antibodies are associated with ILD and antitopoisomerase antibodies. Finally, the usefulness of these antibodies for SSc screening, diagnosis or follow-up needs to be further confirmed by extensive laboratory screening of large and geographically different groups of SSc patients and in other patient groups with connective tissue disease having ILD.

Acknowledgments

The authors would like to thank Nicolas Dupin for providing normal human fibroblasts. MCT, MH, LG and LM are members of the Groupe Français de Recherche sur la Sclérodermie.

REFERENCES

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Supplementary materials

Footnotes

  • ▸ Additional supplemental data are published online only at http://ard.bmj.com/content/vol69/issue2

  • Funding This work was supported by grants from the Association des Sclérodermiques de France (ASF), the Legs Poix, Chancellerie des Universités, Académie de Paris, France, Actelion Pharmaceuticals France and a “Contrat d’Investigation et de Recherche Clinique” (CIRC 05066) from the Assistance Publique-Hôpitaux de Paris. BT received financial support from the Direction Régionale de l’Action Sanitaire et Sociale (DRASS) d’Ile de France. MCT is a recipient of grants from Avenir Mutualiste des Professions Libérales and Indépendantes, ASF and Actelion Pharmaceuticals.

  • Competing interests None.

  • Ethics approval Ethics approval was obtained from the ethics committee of the La Pitié-Salpêtrière Hospital Group.

  • Patient consent Obtained.

  • Provenance and Peer review Not commissioned; externally peer reviewed.

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