Patients with idiopathic pulmonary fibrosis with antibodies to heat shock protein 70 have poor prognoses

Abstract

Rationale: Diverse autoantibodies are present in most patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that specific autoantibodies may associate with IPF manifestations.

Objectives: To identify clinically relevant, antigen-specific immune responses in patients with IPF.

Methods: Autoantibodies were detected by immunoblots and ELISA. Intrapulmonary immune processes were evaluated by immunohistochemistry. Anti-heat shock protein 70 (HSP70) IgG was isolated from plasma by immunoaffinity. Flow cytometry was used for leukocyte functional studies.

Measurements and main results: HSP70 was identified as a potential IPF autoantigen in discovery assays. Anti-HSP70 IgG autoantibodies were detected by immunoblots in 3% of 60 control subjects versus 25% of a cross-sectional IPF cohort (n = 122) (P = 0.0004), one-half the patients with IPF who died (P = 0.008), and 70% of those with acute exacerbations (P = 0.0005). Anti-HSP70 autoantibodies in patients with IPF were significantly associated with HLA allele biases, greater subsequent FVC reductions (P = 0.0004), and lesser 1-year survival (40 ± 10% vs. 80 ± 5%; hazard ratio = 4.2; 95% confidence interval, 2.0-8.6; P < 0.0001). HSP70 protein, antigen-antibody complexes, and complement were prevalent in IPF lungs. HSP70 protein was an autoantigen for IPF CD4 T cells, inducing lymphocyte proliferation (P = 0.004) and IL-4 production (P = 0.01). IPF anti-HSP70 autoantibodies activated monocytes (P = 0.009) and increased monocyte IL-8 production (P = 0.049). ELISA confirmed the association between anti-HSP70 autoreactivity and IPF outcome. Anti-HSP70 autoantibodies were also found in patients with other interstitial lung diseases but were not associated with their clinical progression.

Conclusions: Patients with IPF with anti-HSP70 autoantibodies have more near-term lung function deterioration and mortality. These findings suggest antigen-specific immunoassays could provide useful clinical information in individual patients with IPF and may have implications for understanding IPF progression.

Figures

Figure 1.

Antigens of idiopathic pulmonary fibrosis (IPF). (A) Antigenicity of IPF lung explant extracts was greatest among the most acidic protein fractions (lowest pI). Proliferation was determined by bromodeoxyuridine (BrdU) incorporation among autologous hilar lymph node CD4 T cells (6). (B) Further fractionations of the acidic protein preparations by size filtration indicated the most immunogenetic antigens among the low pI proteins were greater than 50 kD. In these cases, proliferation was measured by 3H-thymidine because the preparations were limiting. (C) Prevalences of circulating anti–heat shock protein 70 (HSP70) IgG detected by immunoblots (IB) are depicted in healthy normal control subjects and patients with IPF. Respective subject numbers are denoted within columns. (D) HLA Class II DRβ1*11 alleles were underrepresented, whereas DRβ1*15 was overrepresented among the patients with IPF with anti-HSP70 autoantibodies. Prevalence is defined as the proportion of subjects with at least one allele copy. (E) Addition of rHSP70 to peripheral blood mononuclear cell cultures resulted in greater proliferation of CD4 T cells from patients with IPF, ascertained by BrdU incorporation, than in concurrent control cultures with no added antigens (Cnt), or cultures supplemented instead with glucose-regulated protein 78 (GRP78) (n = 24). Proliferation within GRP78-supplemented cultures did not significantly differ from those of control subjects. (F) IPF CD4 T cell IL-4 production was also more augmented by HSP70 than by the GRP78 (n = 15). The latter did not significantly differ from controls. CI = confidence interval; OR = odds ratio; SI = specific index.

Figure 2.

Lung immunohistochemistry. Columns from left to right depict, respectively, expression of heat shock protein 70 (HSP70), IgG immune complexes (IgG), complement deposits (C3), and isotype controls. Rows, from top to bottom respectively, depict end-stage idiopathic pulmonary fibrosis (IPF) lungs explanted during therapeutic pulmonary transplantations and normal lungs harvested during multiorgan retrievals but not used in therapeutic transplantations (n = 6 each). IPF specimens were depicted at 20× to better illustrate anatomical localizations of the HSP70, IgG, and C3. Normal lung sections are shown at 10× to optimally depict the overall paucity of the expressions/depositions in these specimens.

Figure 3.

Monocytes functional studies. (A) In comparison to concurrent, autologous control preparations treated with normal IgG, incubations with the idiopathic pulmonary fibrosis (IPF) autoantibody resulted in increased expression of CD69 in all but one specimen (n = 12). Aggregate mean increments induced by these single anti–heat shock protein 70 (HSP70) IgG treatments for 18 hours were 6 ± 1%. (B) Specific index (SI) (6) of CD69 expression, defined as percentages of CD69+ monocytes in IPF HSP70 IgG cultures minus that of the normal IgG cultures. (C) IPF anti-HSP70 increased IL-8 production of monocytes relative to normal IgG treatment.

Figure 4.

Clinical correlates of heat shock protein 70 (HSP70) autoreactivity determined by immunoblot (IB). (A) Surviving subjects with idiopathic pulmonary fibrosis (IPF) with anti-HSP70 autoantibodies detected on IB testing (anti-HSP70+) (n = 14) had greater subsequent decrements of FVC, as % predicted values (FVC%p), and an insignificant tendency for greater decrements of percent predicted diffusing capacity for carbon monoxide (DlCO%p), compared with the subjects without this autoantibody (anti-HSP70−, n = 59). Pulmonary function determinations were made approximately 6 months after the plasma sample acquisitions (see text). (B) Patients with IPF with anti-HSP70 autoantibodies detected by IB (n = 30) had worse prognoses than those who were autoantibody negative (n = 92). Major (adverse) events are defined as either deaths or lung transplantations. Cross hatches denote censored events, and numbers in parentheses denote subjects censored at the end of observation. (C) The proportion of subjects with IPF who died during the next year was threefold greater among those with anti-HSP70 autoantibodies (anti-HSP70+) compared with the anti-HSP70−. Tx = transplanted. (D) Among patients with IPF who did not have lung transplantations during the observation period, those with anti-HSP70 autoantibodies (n = 25) had worse prognoses than the autoantibody-negative subpopulation (n = 77). CI = confidence interval.

Figure 5.

Heat shock protein 70 (HSP70) autoreactivity determined by ELISA. (A) Outcomes were near identical among subjects with idiopathic pulmonary fibrosis (IPF) (n = 121) and non-IPF interstitial lung disease (ILD) (n = 51). Post hoc analyses of absolute 1-year survival limited to those subjects who did not have transplantations during the observation period were also similar among these experimental cohorts (74 ± 4% vs. 78 ± 7% for IPF and ILD, respectively; P = 0.62). (B) Anti-HSP70 IgG ELISA optical density (OD) values were near identical in subjects with IPF and subjects with non-IPF ILD, and both were greater than among healthy control subjects (n = 59). The P value here is per Kruskal-Wallis (three-group) comparison. Values for both IPF and ILD were significantly greater than normal subjects in post hoc two-group analyses using Mann-Whitney tests. The dashed line denotes the mean plus SD of the values in normal subjects here. Thick horizontal lines denote the mean of each subpopulation. (C) Subjects with IPF with anti-HSP70 ELISA OD in the highest quartile (Highest ELISA) had nonsignificant trends for greater subsequent (∼ 6-mo) decrements of FVC % predicted (FVC%p) and percent predicted diffusing capacity for carbon monoxide (DlCO%p). (D) Subjects with IPF in the highest quartile of anti-HSP70 ELISA OD values (Highest ELISA) more frequently had major adverse events (deaths or lung transplantations) during the year after their specimen acquisitions. In contrast, anti-HSP70 autoantibody measures had no associations with clinical manifestations in the non-IPF ILD disease control cohort (see online supplement E3). CI = confidence interval.