Autoimmunity to Vimentin Is Associated with Outcomes of Patients with Idiopathic Pulmonary Fibrosis

Abstract

Autoimmunity has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF); however, the repertoire of autoantigens involved in this disease and the clinical relevance of these autoimmune responses are still being explored. Our initial discovery assays demonstrated that circulating and intrapulmonary vimentin levels are increased in IPF patients. Subsequent studies showed native vimentin induced HLA-DR-dependent in vitro proliferation of CD4 T cells from IPF patients and enhanced the production of IL-4, IL-17, and TGF-β1 by these lymphocytes in contrast to normal control specimens. Vimentin supplementation of IPF PBMC cultures also resulted in HLA-DR-dependent production of IgG with anti-vimentin specificities. Circulating anti-vimentin IgG autoantibody levels were much greater in IPF subjects from the University of Alabama at Birmingham (n = 102) and the University of Pittsburgh (U. Pitt., n = 70) than in normal controls. Anti-vimentin autoantibody levels in IPF patients were HLA biased and inversely correlated with physiological measurements of lung function (i.e., forced expiratory volumes and diffusing capacities). Despite considerable intergroup differences in transplant-free survival between these two independent IPF cohorts, serious adverse outcomes were most frequent among the patients within each population that had the highest anti-vimentin autoantibody levels (University of Alabama at Birmingham: hazard ratio 2.5, 95% confidence interval 1.2-5.3, p = 0.012; University of Pittsburgh: hazard ratio 2.7, 95% confidence interval 1.3-5.5, p = 0.006). These data show that anti-vimentin autoreactivity is prevalent in IPF patients and is strongly associated with disease manifestations. These findings have implications with regard to the pathogenesis of this enigmatic disease and raise the possibility that therapies specifically directed at these autoimmune processes could have therapeutic efficacy.

FIGURE 1.

Vimentin expression is increased in IPF. (A) Plasma vimentin levels are greater in IPF patients than in controls (Con). Each dot represents an individual subject, and horizontal lines and numbers indicate the median values. (B) IHC shows that vimentin is expressed more in IPF lungs, along with α-SMA and collagen types 1 and 3 (per Picro Sirius Red stains) compared with normal specimens. Images are representative of studies in eight specimens. Scale bars, 50 μm. (C) Staining density was quantified, and the average positive area density was calculated from 10 random areas. Densities are denoted in arbitrary units. (D) Vimentin was measured in the supernatants of HMEC-1 cell cultures after incubation with TGF-β1 for various durations (left panel) and various TGF-β1 concentrations (right panel). *p < 0.05, **p < 0.01.

FIGURE 2.

Supplementation of PBMC cultures with native vimentin induced proliferation of CD4 T cells from IPF patients. (A) Representative line graphs showing CFSE dilution in gated CD4 T cells from controls (Con.) and IPF patients. Dose dependence was evident in increasing vimentin concentrations from top to bottom. (B) Quantitation of aggregate CFSE assays in Con. (n = 10) and IPF (n = 15). Representative line graphs showing downregulation of CD4 T cell FOXP3 (C) and aggregate compilations of the same (D). Cultures in these studies were stimulated or not with vimentin (1 μg/ml). **p < 0.01.

FIGURE 3.

Vimentin induces production of IL-4, TGF-β1, and IL-17 among CD4 T cells from IPF patients. (A) Representative line graphs of gated CD4 T cells from Control (Con.) and IPF PBMC cultures, with and without supplementation by native vimentin (1 μg/ml). (B) Analyses of results in 10 Con and 15 IPF preparations. Results of other cytokine assays are depicted in Supplement Fig. 1. **p < 0.01.

FIGURE 4.

IPF CD4 T cell proliferative responses to vimentin are HLA class II dependent. (A) Representative line graphs depicting that addition of F(ab′)₂ anti-human HLA-DR (2 μg/ml) inhibits vimentin-induced (1 μg/ml) CD4 T cell proliferation. (B) Aggregate results of CFSE proliferation assays among Control (Con., n = 10) and IPF specimens (n = 15). *p < 0.05, **p < 0.01.

FIGURE 5.

Anti-vimentin autoantibodies, HLA-DRβ1*15 biases, and discordance between two autoantibody responses in IPF patients. (A) Incubation of IPF PBMC cultures with native vimentin (Vim; 1 μg/ml) and without (Medium) increased levels of anti-vimentin autoantibodies; these responses were blunted by prior addition of F(ab′)₂ anti-human HLA-DR (2 μg/ml). (B) Plasma levels of anti-vimentin IgG autoantibodies were greatest among IPF patients in each cohort. Horizontal lines and numbers denote median values. (C) Anti-vimentin autoantibody concentrations were lower among the IPF patients who were positive for HLA-DRβ1*15 at UAB and U. Pitt. (see also Supplemental Table I). (D) There were no apparent correlations between autoantibody responses to vimentin and HSP70 among the IPF subjects (U. Pitt.) who had received equal measures of each. *p < 0.01, **p < 0.001.

FIGURE 6.

Correlations of anti-vimentin autoantibody responses and clinical manifestations of IPF. (A) Plasma anti-vimentin autoantibody concentrations were inversely correlated with FVC and DLCO (as percentages of predicted values) in both IPF patient cohorts. (B) Transplant-free survival during the next 2 y was similarly reduced in both cohorts among the IPF patients in the tertiles with the highest circulating anti-vimentin levels. (C) Measures of anti-vimentin and anti-HSP70 autoantibodies were available in a limited number of IPF patients. Those who had greater than average levels of both autoantibodies (Both High, n = 10) had lesser FVC percentage predicted than did the other patients (Other) who had only a single or no increased autoantibody concentration. There was no significant difference between FVC percentage predicted of patients with a single increased autoantibody (anti-vimentin or anti-HSP70) (n = 24) and those in whom both autoantibodies were below mean levels (n = 8) (64 ± 18% versus 69 ± 12%, respectively). (D) Transplant–free survival was also much worse in this IPF subpopulation who had increased levels of both autoantibodies compared with the other subjects. *p = 0.017.