Autoreactivity to glucose regulated protein 78 links emphysema and osteoporosis in smokers

Abstract

Rationale: Emphysema and osteoporosis are epidemiologically associated diseases of cigarette smokers. The causal mechanism(s) linking these illnesses is unknown. We hypothesized autoimmune responses may be involved in both disorders.

Objectives: To discover an antigen-specific autoimmune response associated with both emphysema and osteoporosis among smokers.

Methods: Replicate nonbiased discovery assays indicated that autoimmunity to glucose regulated protein 78 (GRP78), an endoplasmic reticulum chaperone and cell surface signaling receptor, is present in many smokers. Subject assessments included spirometry, chest CT scans, dual x-ray absorptiometry, and immunoblots for anti-GRP78 IgG. Anti-GRP78 autoantibodies were isolated from patient plasma by affinity chromatography, leukocyte functions assessed by flow cytometry, and soluble metabolites and mediators measured by immunoassays.

Measurements and main results: Circulating anti-GRP78 IgG autoantibodies were detected in plasma specimens from 86 (32%) of the 265 smoking subjects. Anti-GRP78 autoantibodies were singularly prevalent among subjects with radiographic emphysema (OR 3.1, 95%CI 1.7-5.7, p = 0.003). Anti-GRP78 autoantibodies were also associated with osteoporosis (OR 4.7, 95%CI 1.7-13.3, p = 0.002), and increased circulating bone metabolites (p = 0.006). Among emphysematous subjects, GRP78 protein was an autoantigen of CD4 T-cells, stimulating lymphocyte proliferation (p = 0.0002) and IFN-gamma production (p = 0.03). Patient-derived anti-GRP78 autoantibodies had avidities for osteoclasts and macrophages, and increased macrophage NFkB phosphorylation (p = 0.005) and productions of IL-8, CCL-2, and MMP9 (p = 0.005, 0.007, 0.03, respectively).

Conclusions: Humoral and cellular GRP78 autoimmune responses in smokers have numerous biologically-relevant pro-inflammatory and other deleterious actions, and are associated with emphysema and osteoporosis. These findings may have relevance for the pathogenesis of smoking-associated diseases, and development of biomarker immunoassays and/or novel treatments for these disorders.

Figure 1. Anti-GRP78 autoantibodies, airflow obstruction, and emphysema.

A.) Anti-GRP78 autoantibody prevalence was not significantly associated with COPD severity (GOLD stages) . Numbers within columns denote subject n. B.) Anti-GRP78 autoantibodies are significantly associated with emphysema prevalence (%) in the smoking subjects. OR  =  odds ratio; CI  =  confidence interval. C.) Emphysema scores per CT were also significantly greater among those subjects with anti-GRP78 autoantibodies.

Figure 2. Anti-GRP78 autoantibody association with osteoporosis, bone metabolism markers, and concurrent low bone density and emphysema.

A.) T scores (left panel) and osteoporosis prevalence (at either/both hip or spine) (right panel) among the smoking cohort. B.) Serum levels of bone turnover metabolite collagen type 1 cross-linked C-telopeptide (CTX) were greatest among smokers with anti-GRP78 autoantibodies. The lowest, second lowest, middle, second highest, and highest lines represent 10^th, 25^th, median, 75^th, and 90^th percentiles, respectively. Means are denoted by solid squares. C.) Serum levels of bone turnover metabolite type 1 (N-terminal) procollagen (P1NP) were greatest among smokers with anti-GRP78 autoantibodies. D.) The relationship between GRP78 autoantibody positivity and the concurrent co-existences of low BMD and emphysema in smokers is significant in both genders, but greatest in males.

Figure 3. GRP78 expression in lung, bronchoalveolar lavage fluid, alveolar macrophages, and bone marrow derived osteoclasts.

A.) Compared to normal lung (Left panel), emphysematous lungs (middle and right panels) demonstrated increased immunostaining in macrophages (yellow arrows) and alveolar epithelial cells (blue arrows). Magnification x100 left and middle panels, and magnification x400 in the right panel, n = 3. B.) GRP78 was also greater in bronchoalveolar lavage fluid (BALF) from emphysematous lungs compared to normal preparations. Lanes 1 and 8 are rGRP78 standards. Lanes 2–7 are BALF from individual emphysematous lung explants; lanes 9–14 are BALF from normal lung explants. All specimen lanes were loaded with equal amounts of BALF proteins. C.) Indirect immunofluorescent assays showed anti-GRP78 IgG isolated from patients bind to alveolar macrophages from normal lung explants (panel a), and osteoclasts derived from bone marrow (panel b). Normal human IgG control is illustrated in panel c.

Figure 4. Cellular effects of autoantibodies to GRP78 on macrophages.

A.) Mean fluorescence intensity (MFI) for phosphorylated NFkB among paired, concurrent autologous CD14⁺ derived macrophages was increased in all 10 normal specimens after incubation with patient-derived autoantibodies to GRP78 (α-GRP78), relative to control cells treated with normal human IgG. Patient derived anti-GRP78 autoantibodies also increased macrophage production of IL-8 (B.), CCL-2 (C.) and MMP9 (C.). Population means are denoted with a horizontal line.

Figure 5. CD4 T-cell autoreactivity to GRP78.

A.) Addition of GRP78 to PBMNC cultures increased CD4 T-cell proliferation (BrdU uptake) relative to media controls (no added protein) (n = 47), unlike addition of elastin split products (ESP). P_c denotes alpha level corrected for multiple comparisons. B.) GRP78-induced CD4 T-cell proliferation was greatest among cultures from smokers with emphysema (n = 34). Specific indices (SI) of proliferation were calculated as %CD4 T-cells incorporating BrdU in GRP78-supplemented cultures minus incorporation in concurrent media controls. C.) GRP78 also increased percentages of CD4 T-cells that produced IFN-gamma again, unlike effects of ESP. D.) IFN-gamma production was also greatest in the CD4 T-cells from the emphysematous smokers. Specific indices (SI) were calculated as %CD4 T-cells producing IFN-gamma production in GRP78-supplemented cultures minus that of concurrent controls.