B cell antigen presentation promotes Th2 responses and immunopathology during chronic allergic lung disease

Abstract

Background: The role of B cells in allergic asthma remains undefined. One mechanism by which B cells clearly contribute to allergic disease is via the production of specific immunoglobulin, and especially IgE. Cognate interactions with specific T cells result in T cell help for B cells, resulting in differentiation and immunoglobulin secretion. Proximal to (and required for) T cell-dependent immunoglobulin production, however, is antigen presentation by B cells. While interaction with T cells clearly has implications for B cell function and differentiation, this study investigated the role that B cells have in shaping the T cell response during chronic allergic lung disease.

Methodology/principal findings: In these studies, we used a clinically relevant mouse model of chronic allergic lung disease to study the role of B cells and B cell antigen presentation in this disease. In these studies we present several novel findings: 1) Lung B cells from chronically allergen challenged mice up-regulated MHC II and costimulatory molecules CD40, CD80 and CD86. 2) Using in vitro studies, B cells from the lungs of allergen challenged mice could present antigen to T cells, as assessed by T cell proliferation and the preferential production of Th2 cytokines. 3) Following chronic allergen challenge, the levels of Th2 cytokines IL-4 and IL-5 in the lungs and airways were significantly attenuated in B cell -/- mice, relative to controls. 4) B cell driven Th2 responses and mucus hyper secretion in the lungs were dependent upon MHC II expression by B cells.

Conclusions/significance: Collectively, these results provide evidence for antigen presentation as a novel mechanism by which B cells contribute to chronic allergic disease. These findings give new insight into the mechanisms by which B cells promote asthma and other chronic diseases.

Figure 1. Accumulation of B cells in the lungs during chronic allergic lung disease.

(A) Chronic model of cockroach antigen-induced allergic lung disease. Mice were immunized intraperitoneally (i.p.) and subcutaneously (s.c.), followed by 4 intranasal (i.n.) challenges, and two intratracheal (i.t.) inoculations. (B) Kinetics of B and T cell accumulation in the lungs of allergen challenged mice. The numbers of CD4⁺, CD8⁺, and CD19⁺ cells were determined by flow cytometric analysis of enzymatically digested lungs. Each time point represents the mean of 5 mice per group ±SEM * = p<0.05 vs. naïve lung . Similar results were observed in two independent experiments. (C) Cockroach antigen-specific B cells in the lungs, lymph nodes, spleen, and bone-marrow of naïve and allergen challenged (day 37) mice. The frequency of specific B cells was determined by a combination of ELISPOT and flow cytometry, as described in Materials and Methods. Bars represent the mean of 5 mice per group ±SEM * = p<0.05 vs. naïve. Similar results were observed in two independent experiments.

Figure 2. Expression of MHC II and costimulatory molecules by B cells from the lungs of chronic allergen challenged mice (day 37).

Lung leukocytes were recovered from the lungs via enzymatic digests and the expression of MHC II, CD40, CD80, and CD86 on CD19⁺ cells were determined by flow cytometry. Representative histograms (from a group of n = 5) are shown. Shaded histograms represent isotype controls, with overlays representing MHC II, CD40, etc.

Figure 3. Th2 cytokine production in pulmonary B cell/T cell co-cultures.

Lung lymphocytes were isolated from chronic allergen challenged mice (day 37) by enzymatic digest and enriched by density gradient centrifugation. Lung CD4⁺ T cells and CD19⁺ B cells were isolated via fluorescence activated cell sorting (FACS), and co-cultured at a ratio of 4∶1 (T∶B), with and without cockroach antigen. CD4⁺T and CD19⁺B cells were >99% pure, and contained <0.05% CD11c⁺ cells, as determined by post-sort analysis. (A) Cytokine levels in culture supernatants were determined via Bioplex-based multiplex assay after 48 hours in culture. Bars represent the mean of triplicate wells ±SEM. * = p<0.05 vs. T cell/B cell co-cultures without cockroach antigen. Similar results were observed in three independent experiments. (B) In a parallel experiment, cytokine mRNA levels were determined after 24 hour in culture via quantitative PCR. Bars represent the mean of triplicate wells ±SEM. * = p<0.05 vs. cells without antigen. Similar results were observed in two independent experiments.

Figure 4. In vitro proliferative responses of T cells co-cultured with pulmonary B cells.

Lymphocytes were isolated from chronic allergen challenged mice via enzymatic digest and density gradient centrifugation. CD4⁺ T cells and pulmonary CD19⁺ B cells were isolated via magnetic separation (MACS). Purity of isolated cell populations was >96%. CD4⁺ T cells were stained with CFSE and co-cultured with B cells with and without cockroach antigen. Cells were harvested at 72 hours post-culture, and analysed via flow cytometry. (A) Representative histogram: shaded line –T cells+CRA (250 PNU/ml), solid line – T cells+B cells+CRA (PNU/ml) (B) Percentage of dividing cells and division index were calculated using Flowjo™ software. Bars represent the mean of triplicate wells ±SEM. * = p<0.05 vs. T cells alone † = p<0.05 versus all other groups NS = not significant. Similar results were observed in two independent experiments.

Figure 5. Lung pathophysiology in control and B cell −/− mice.

(A) Airway hyperreactivity in control and B cell −/− mice. AHR was assessed by whole body plethysmography. The Y-axis corresponds to the increase in airway resistance observed following methacholine challenge. Bars represent the mean of 8–9 mice per group ±SEM. * = p<0.05 (B) Representative histology of lung sections from control allergen challenged and B cell−/− mice. H&E: Hemotoxylin and eosin, PAS: periodic acid Schiff's stain. (C) Histological scoring of PAS-stained lung sections. Bars represent the mean of 5 mice per group ±SEM.

Figure 6. Lung leukocyte and lymphocyte recruitment to the lungs of chronic allergen challenged mice.

(A) B cell −/− and control mice were chronically challenged with allergen. The numbers of CD45⁺, CD4⁺, CD8⁺, and CD19⁺ cells in the lung parenchyma were determined via flow cytometry of enzymatic digests of whole lungs. * = p<0.05 vs. control allergic mice. (B and C) Lung eosinophilia in the lungs of chronic allergen challenged mice. The frequency (B) and absolute number (C) of eosinophils in control and B cell −/− mice were determined from cytospins of enzymatically digested lungs. Bars represent the mean of 5 mice per group ±SEM. Similar results were obtained in three independent experiments.

Figure 7. Th1/Th2 associated cytokines in the lungs of allergen challenged mice.

B cell −/− and control mice were chronically challenged with allergen, and cytokine levels were assesed in bronchoalveolar lavage (B) and lung homogenates (A) via Bioplex multiplex assay. Bars represent the mean of 6 mice per group ±SEM, except naive, for which n = 2. (C) Intracellular cytokine staining of CD4 T cells from the lungs of control and B cell−/− allergen challenged mice. Bars represent the mean of 6 mice per group ±SEM. * = p<0.05 versus control allergen challenged. Similar results were obtained in two independent experiments.

Figure 8. Role of B cell expression of MHC II in allergic disease.

(A) Lung cytokines and (B) mucus-associated gene expression in the lungs of µMT/I-A^b−/− mice and µMT/WT mice. Mice with MHC II deficient (µMT/I-A^b−/− mice) or normal (µMT/WT ) B cells were generated using mixed bone marrow chimeras, and chronically challenged with allergen. The levels of cytokines in the lungs were determined from lung homogenate samples via Bioplex multiplex assay. The expression of mucus associated genes was assessed by real-time PCR using whole lung RNA. Bars represent the mean ±SEM of 4 mice per group. * = p<0.05 Similar results were obtained in two independent experiments.