Plasma B lymphocyte stimulator and B cell differentiation in idiopathic pulmonary fibrosis patients

Abstract

We hypothesized B cells are involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive, restrictive lung disease that is refractory to glucocorticoids and other nonspecific therapies, and almost invariably lethal. Accordingly, we sought to identify clinically associated B cell-related abnormalities in these patients. Phenotypes of circulating B cells were characterized by flow cytometry. Intrapulmonary processes were evaluated by immunohistochemistry. Plasma B lymphocyte stimulating factor (BLyS) was assayed by ELISA. Circulating B cells of IPF subjects were more Ag differentiated, with greater plasmablast proportions (3.1 ± 0.8%) than in normal controls (1.3 ± 0.3%) (p < 0.03), and the extent of this differentiation correlated with IPF patient lung volumes (r = 0.44, p < 0.03). CD20(+) B cell aggregates, diffuse parenchymal and perivascular immune complexes, and complement depositions were all prevalent in IPF lungs, but much less prominent or absent in normal lungs. Plasma concentrations of BLyS, an obligate factor for B cell survival and differentiation, were significantly greater (p < 0.0001) in 110 IPF (2.05 ± 0.05 ng/ml) than among 53 normal (1.40 ± 0.04 ng/ml) and 90 chronic obstructive pulmonary disease subjects (1.59 ± 0.05 ng/ml). BLyS levels were uniquely correlated among IPF patients with pulmonary artery pressures (r = 0.58, p < 0.0001). The 25% of IPF subjects with the greatest BLyS values also had diminished 1-y survival (46 ± 11%), compared with those with lesser BLyS concentrations (81 ± 5%) (hazard ratio = 4.0, 95% confidence interval = 1.8-8.7, p = 0.0002). Abnormalities of B cells and BLyS are common in IPF patients, and highly associated with disease manifestations and patient outcomes. These findings have implications regarding IPF pathogenesis and illuminate the potential for novel treatment regimens that specifically target B cells in patients with this lung disease.

Figures

Figure 1. Phenotypes of circulating B-cells

A.) Cells were stained with panels of mAb and gated on CD19+, CD20−, CD3−, and IgD− B-cells. Plasmablasts among these (CD27 hi, CD38hi) are denoted in the upper right quadrant of this example. B.) Selected phenotypic characteristics of circulating B-cells among COPD (n=29), IPF (n=25) and normal controls (n=20). The lowest, second lowest, middle, second highest, and highest box points represent 10th, 25th, median, 75th, and 90th percentiles, respectively. Means are represented by squares. Trans = transitional (recent bone marrow emigrant B-cell), and successively differentiated developmental stages: non-isotype switched (IgA−, IgG−) memory (nsMem), isotype (IgG) switched memory (sMEM) and efficient antibody-producing plasmablasts (PB). Absolute sMEM values are 10x those depicted in this figure. P values denote Kruskal-Wallis comparisons of the three cohorts; *denotes the group that is significantly different from the other two. Note increased B-cell differentiation among the disease cohorts (COPD and IPF) relative to healthy controls, and the general similarities between those two lung disease subpopulations. C.) The percentage of plasmablasts (PB) among circulating B-cells was inversely correlated with FVC, as a percentage of predicted values (FVC%p) in IPF subjects. D.) The percentage of plasmablasts (PB) among circulating B-cells was inversely correlated with DLCO%p in COPD subjects.

Figure 2. Intrapulmonary CD20+B-cells

A.) Representative images show sections from normal lungs (left panel) and IPF lungs (middle panel) immunostained for CD20. These B-cells are only infrequently positive for Ki-67 (right panel) (×100). B.) Direct counting confirmed CD20+ cells are more numerous in IPF compared to normal lung specimens.

Figure 3. BLyS levels

A.) Plasma BLyS concentrations were significantly greater in IPF subjects (n=110) than in either COPD (n=90) or normals (n=53). B.) Among the IPF cohort, BLyS levels were highest among those who died during the next year (n=25), and in those who had pulmonary artery hypertension (PAH) (n=17), compared to those with lesser pulmonary artery (PA) mean pressures (Nl) (n=28). PAH was defined as PA mean pressures >25 mmHg with PA wedge (PAW) pressures <15 mmHg. Subjects with PAW >15 were not tabulated here (n=5). These five excluded subjects had PA mean pressures >25, and their BLyS concentrations were 1.92±0.32 ng/ml). C.) BLyS concentrations were correlated with pulmonary artery pressures among IPF subjects. D.) The quartile (25%) of IPF subjects with greatest BLyS levels (High) had worse outcomes than those subjects with lesser BLyS levels (Low). Cross-hatches and numbers in parentheses denote censored events (end of observation). E.) Absolute mortality was also greater among IPF patients with the highest quartile plasma BLyS concentrations (High) after omission of the subpopulation that had lung transplantations during the observation interval.

Figure 4. Intrapulmonary IgG and C4d

In contrast to normal lungs, the IPF sections are characterized by more intense IgG and C4d staining throughout the parenchyma, and particularly intense staining around blood vessels (black arrows). Staining was also present in lymphoid aggregates (red arrows) and intraluminal cells and debris (green arrows). Inset images show the lack of staining in non-immune controls (magnification ×100).