An obligatory role for lung infiltrating B cells in the immunopathogenesis of obliterative airway disease induced by antibodies to MHC class I molecules

Abstract

Using a murine model, we demonstrated that endobronchial administration of antibodies (Abs) to major histocompatibility complex (MHC) class I results in cellular infiltration, epithelial metaplasia, fibrosis and obstruction of the small airways (obliterative airway disease [OAD]) mediated predominantly by Th17 responses to self-antigens. This resembles bronchiolitis obliterans syndrome developed following human lung transplantation. Since B cells play a crucial role in induction of autoimmune responses, we defined the role of B cells and its antigen presenting properties in induction of OAD in this study. Anti-MHC class I was administered endobronchially in B(-/-) and wild-type mice. In contrast to wild type, B(-/-) animals did not demonstrate cellular infiltration, epithelial metaplasia and obstruction of airways following anti-MHC. Frequency of K-α1 tubulin and CollagenV-specific IL-17 cells was significantly decreased in B(-/-) mice. As expected, Abs against self-antigens and germinal center formation were not developed in B(-/-) mice. Thus, we conclude that B cells and its antigen presenting capacity play an important role in induction of immune responses to self-antigens and immunopathogenesis of OAD following the administration of anti-MHC. Therefore, strategies to block B-cell and its antigen presenting functions should be considered for preventing the development of chronic rejection.

Figure 1. Increased infiltration of B cells in lungs of mice administered anti-MHC Abs endobronchially

Anti-H2k^b or control (C1.18.4) Ab was administered endobronchially in C57BL/6 mice on days 1, 2, 3. The lungs were harvested on day 4 and analyzed for the number of infiltrating cells by flow cytometry using anti-CD19 Abs. There was a significant increase in the number of B cells in the lung following anti-MHC Ab administration. Represented as mean number of B cells with 5 mice in each group. *Denotes p<0.05

Figure 2. B cell depletion results in decreased IL-17 secreting cells when stimulated with low concentrations of self-antigens

Splenocytes (3×10⁵ per well) from B6.129S2-Igh-6^tm1Cgn/J (B−/−) or control C57BL/6 mice (Wild type – WT) were stimulated with 1μg, 250ng and 100 ng of K-α1T or ColV (cells cultured in triplicate for each concentration of antigen) and the frequency of IL-17 secreting cells analyzed using ELISPOT. In another set of experiment B cells from wild type mice were added to the T cells from B6.129S2-Igh-6^tm1Cgn/J or B cells were depleted from the splenocytes of wild type mice and stimulated with self-antigens to determine the frequency of IL-17 secreting T cells. The frequency of IL-17 secreting T cells were lower in splenocytes from B6.129S2-Igh-6^tm1Cgn/J and wild type mice depleted of B cells (at 100ng antigen concentration) when compared to wild type mice as well as B cells added splenocytes from B6.129S2-Igh-6^tm1Cgn/J mice. Frequency of IL-17 secreting cells following stimulation at higher antigen concentration (250ng and 1μg) did not differ between the groups. Results are expressed as mean ± SD spots per million cells observed in 5 mice per group. * Denotes P<0.05

Figure 3. Significant reduction in the pro-inflammatory cytokines and chemokines in B cell deficient mice following anti-MHC Ab administration

B6.129S2-Igh-6^tm1Cgn/J (B−/−) or control C57BL/6 mice (Wild type) were treated with anti-MHC class I Abs day 1, 2 and 3. We analyzed the expression levels of cytokines and chemokines in the lungs by quantitative real-time PCR on Day 4. There was a significant reduction in the expression of TNF-α (4.6 folds), IL-6 (6.8 folds), IL-2 (2.7 folds), IFN-γ (8.3 folds) and CXCL12 (14.4 folds) in the lungs of B cell deficient mice when compared to the wild type controls. Data representative of mean ± SD fold change observed, with 5 mice per group.

Figure 4. B cell deficiency results in a significant reduction in the frequency of IFN-γ and IL-17 secreting T cells against K-α1T and ColV in the lung following anti-MHC Ab administration

B6.129S2-Igh-6^tm1Cgn/J (B cell KO) or control C57BL/6 mice were treated with anti-MHC class I Abs on day 1, 2, 3, 6 and weekly thereafter. T cells infiltrating the lungs were harvested by collagenase digestion and the frequency of T cells secreting IFN-γ, IL-4 and IL-17 on stimulation with K-α1T and ColV (3×10⁵ cells/well cultured in triplicate for each antigen) were analyzed by ELISPOT. A significant reduction in the frequency of T cells against K-α1T and ColV was observed in B cell deficient mice when compared to controls. IFN-g secreting cells to LPS in vitro did not differ among the groups. Data represented as mean ± SD spots per million cells (spm) of 5 mice per group.

Figure 5. Alloimmune responses are preserved in B cell KO mice albeit of a lower magnitude compared to wild type mice

B6.129S2-Igh-6^tm1Cgn/J (B cell KO) or control C57BL/6 (Wild type) mice were immunized twice with 1 million BALB/c splenocytes intraperitoneally at weekly intervals. Frequency of IFN-γ secreting cells that respond to the BALB/c splenocytes were analyzed by ELISPOT. B cell deficient mice immunized with BALB/c splenocytes mounted an IFN-γ response against the mismatched MHC antigens albeit of a lower magnitude when compared to their wild type controls. Represented as mean spots per million cells of 5 mice per group.

Figure 6

A: Decreased auto-Ab production in B cell deficient mice following anti-MHC Ab administration. B6.129S2-Igh-6^tm1Cgn/J (B cell KO) or control C57BL/6 (wild type) mice were treated with anti-MHC class I Abs day1, 2, 3, 6 and weekly thereafter. Serum collected at day 30 was analyzed for auto-Abs against K-α1T and ColV by ELISA. Abs to K-α1T and ColV were significantly reduced in B cell deficient mice when compared to wild type following administration of anti-MHC Abs. Represented as mean ± SD μg/mL of 5 mice per group. * Denotes p<0.05 B: Formation of germinal centers is significantly reduced in the B cell deficient mice following administration of anti-MHC Abs when compared to wild type controls. B6.129S2-Igh-6^tm1Cgn/J (B cell KO) or control C57BL/6 (wild type) mice were treated with anti-MHC class I Abs day 1, 2, 3, 6 and weekly thereafter. Spleens from the mice were harvested on day 30 and analyzed for the germinal center formation by staining with anti-PNA Abs. Immunohistochemical analysis of the spleen from the wild type mice demonstrated significantly increased number of germinal centers in comparison to B cell deficient mice.

Figure 7. B cell deficiency results in a significant reduction in cellular infiltration around vessel and bronchiole, hyperplasia of the bronchial epithelium and fibrosis

B6.129S2-Igh-6^tm1Cgn/J (B cell KO), control C57BL/6 (wild type), and B cell KO mice with passive transfer of 2.5×10⁶ B cells were treated with either anti-MHC class I (anti-H2kb), isotype (C1.184) or nonspecific anti-MHC class I (anti H2kd) Abs on day 1, 2, 3, 6 and weekly thereafter (5 mice in each group). The lungs were harvested on day 30. Formalin preserved tissues were sectioned randomly and analyzed by H&E (a – e) and Masson's trichrome staining (d – i). Images were captured and representative area depicted in the figure. At least 4 out of 5 mice from each group demonstrated similar lesions in various sections of the lung. None of the B cell KO mice treated with anti-H2kb (b, g) demonstrated any significant fibrosis or airway obliteration, with minimal cellular infiltration and epithelial hyperplasia compared to wild type mice treated with anti-H2kd (c, h). B cell KO mice with passive transfer of B cells (e, j) demonstrated lesions similar to wild type mice. Wild type mice treated with non-specific anti-MHC class I (anti H2kd) (d, i) demonstrated no lesions. a,f: B cell KO with isotype control Abs; b,g: B cell KO with anti H2kb; c,h: wild type with anti-H2kb; d,i: wild type with anti-H2kd (non specific anti-MHC); e,j: B cell KO with passive transfer of B cells. B: bronchiole and V: vessels. Blue staining in trichrome stains denotes collagen deposition in the lungs.

Figure 8. Morphometric analysis showing reduced cellular infiltration, epithelial hyperplasia, fibrosis and luminal occlusion in B cell KO mice and in B cell KO mice with passive transfer of auto-Abs

A: B6.129S2-Igh-6^tm1Cgn/J (B cell KO), control C57BL/6 (wild type), B cell KO mice with passive transfer of 2.5×10⁶ B cells (purified from splenocytes using B cell enrichment kit, Miltenyi Biotech) and B cell KO mice with passive transfer of 250ng each of Abs to ColV and K-α1T (on day 1) were treated with anti-MHC class I (anti-H2kb Abs on day 1, 2, 3, 6 and weekly thereafter (5 mice in each group). The lungs were harvested on day 30. Formalin preserved tissues were sectioned randomly and analyzed by H&E and Masson's trichrome staining. Images were captured and sections morphometric analysis performed using Optimas software (Media cybernetics). B-cell KO mice demonstrated significantly lesser bronchioles and vessels with cellular infiltration and epithelial hyperplasia. On passively transferring auto-Abs, only some bronchioles and vessels had epithelial hyperplasia or cellular infiltration with absence of any fibrosis when compared to wild type mice or B cell KO mice with passively transferred B cells. % occurrence refers to the percentage of bronchioles/vessels with cellular infiltration, epithelial hyperplasia, fibrosis or luminal occlusion per total number of bronchioles/vessels visualized in the lung sections. Data is mean ± SD of that observed in 5 mice per group. B: H&E and trichrome staining of the lungs. C: ELISPOT analysis of the lung infiltrating T cells specific for K-α1T and ColV in wild type mice, B−/− mice and B−/− mice passively transferred B cells from wild type mice.

Figure 8. Morphometric analysis showing reduced cellular infiltration, epithelial hyperplasia, fibrosis and luminal occlusion in B cell KO mice and in B cell KO mice with passive transfer of auto-Abs

A: B6.129S2-Igh-6^tm1Cgn/J (B cell KO), control C57BL/6 (wild type), B cell KO mice with passive transfer of 2.5×10⁶ B cells (purified from splenocytes using B cell enrichment kit, Miltenyi Biotech) and B cell KO mice with passive transfer of 250ng each of Abs to ColV and K-α1T (on day 1) were treated with anti-MHC class I (anti-H2kb Abs on day 1, 2, 3, 6 and weekly thereafter (5 mice in each group). The lungs were harvested on day 30. Formalin preserved tissues were sectioned randomly and analyzed by H&E and Masson's trichrome staining. Images were captured and sections morphometric analysis performed using Optimas software (Media cybernetics). B-cell KO mice demonstrated significantly lesser bronchioles and vessels with cellular infiltration and epithelial hyperplasia. On passively transferring auto-Abs, only some bronchioles and vessels had epithelial hyperplasia or cellular infiltration with absence of any fibrosis when compared to wild type mice or B cell KO mice with passively transferred B cells. % occurrence refers to the percentage of bronchioles/vessels with cellular infiltration, epithelial hyperplasia, fibrosis or luminal occlusion per total number of bronchioles/vessels visualized in the lung sections. Data is mean ± SD of that observed in 5 mice per group. B: H&E and trichrome staining of the lungs. C: ELISPOT analysis of the lung infiltrating T cells specific for K-α1T and ColV in wild type mice, B−/− mice and B−/− mice passively transferred B cells from wild type mice.

Figure 8. Morphometric analysis showing reduced cellular infiltration, epithelial hyperplasia, fibrosis and luminal occlusion in B cell KO mice and in B cell KO mice with passive transfer of auto-Abs

A: B6.129S2-Igh-6^tm1Cgn/J (B cell KO), control C57BL/6 (wild type), B cell KO mice with passive transfer of 2.5×10⁶ B cells (purified from splenocytes using B cell enrichment kit, Miltenyi Biotech) and B cell KO mice with passive transfer of 250ng each of Abs to ColV and K-α1T (on day 1) were treated with anti-MHC class I (anti-H2kb Abs on day 1, 2, 3, 6 and weekly thereafter (5 mice in each group). The lungs were harvested on day 30. Formalin preserved tissues were sectioned randomly and analyzed by H&E and Masson's trichrome staining. Images were captured and sections morphometric analysis performed using Optimas software (Media cybernetics). B-cell KO mice demonstrated significantly lesser bronchioles and vessels with cellular infiltration and epithelial hyperplasia. On passively transferring auto-Abs, only some bronchioles and vessels had epithelial hyperplasia or cellular infiltration with absence of any fibrosis when compared to wild type mice or B cell KO mice with passively transferred B cells. % occurrence refers to the percentage of bronchioles/vessels with cellular infiltration, epithelial hyperplasia, fibrosis or luminal occlusion per total number of bronchioles/vessels visualized in the lung sections. Data is mean ± SD of that observed in 5 mice per group. B: H&E and trichrome staining of the lungs. C: ELISPOT analysis of the lung infiltrating T cells specific for K-α1T and ColV in wild type mice, B−/− mice and B−/− mice passively transferred B cells from wild type mice.