Antibodies to MHC class I induce autoimmunity: role in the pathogenesis of chronic rejection

Abstract

Alloimmunity to mismatched donor HLA-Ags and autoimmunity to self-Ags have been hypothesized to play an important role in immunopathogenesis of chronic rejection of transplanted organs. However, it is not known what role, if any, alloimmune response plays in inducing autoimmunity. To test whether Ab-developed posttransplantation to mismatched donor MHC induces autoimmunity and chronic rejection, we developed a murine model wherein anti-MHC class I Abs or control (C1.18.4/anti-keratin) were administered intrabronchially into native lungs. Animals receiving anti-MHC class I, but not control Abs, developed marked cellular infiltration around vessels and bronchiole of lung by day 15, followed by epithelial hyperplasia, fibrosis, and occlusion of the distal airways similar to chronic rejection following human lung transplantation. Lungs of mice receiving anti-MHC class I showed increased expression of chemokines, their receptors, and growth factors, and induced IL-17 as well as de novo Abs to self-Ags, K-alpha1 tubulin, and collagen V. IL-17 neutralization by anti-IL-17 resulted in reduction of autoantibody and lesions induced by anti-MHC class I Abs. Thus, our results indicate that Abs to donor MHC can induce autoimmunity, mediated by IL-17, which plays a pivotal role in chronic rejection postlung transplantation. Therefore, approaches to prevent autoimmunity should be considered for the treatment of chronic rejection postlung transplantation.

Figures

Figure 1. Administration of anti-MHC class I Abs developed significant cellular infiltration around vessel and bronchiole as well as hyperplasia of the bronchial epithelium (H&E stain)

Anti-H2kd or control (C1.18.4) Ab or anti-keratin antibody was administered endobronchially in BALB/c mice on days 1, 2, 3, 6 and weekly thereafter. The lungs were harvested on day 15 or 30 and analyzed by H&E and trichrome staining. A representative picture from data obtained from 5 mice is presented in the figure. Original magnification: × 100, insets: ×400. (A, B, C, G, H and I are sections stained with H& E; D, E, F, J, K, and L are trichrome staining. A, and D: anti-H2Kd treated mice on day 15; G and J : anti-H2Kd treated mice on day 30; B, E: C1.18.4 Ab treated mice on day 15; H, K: C1.18.4 Ab treated mice on day 30; C,F: anti-keratin Ab treated mice on day 15; I, L: anti-keratin Ab treated mice on day 30. Lungs from the anti-H2kd Ab treated mice showed significant cellular infiltration around bronchia and vessel (A) and hyperplasia of the bronchial epithelium (G) are marked by the arrows. A significant increase in the fibroproliferation, collagen deposition and luminal occlusion was observed in the lungs of the anti-H2kd Ab treated mice (J). Mice treated with isotype control Ab or anti-keratin Ab showed no significant morphological changes compared to the anti-H2Kd Ab treated mice (B, C, E, F, H, I, K and L).

Figure 2. Administration of anti-MHC class I Abs induces significant increase in the CD4+ and CD11b+ cells in the lungs

Anti-H2kd (A, D, G, J, M) or control (C1.18.4) Ab (B, E, H, K, N) or anti-keratin antibody (C, F, I, L, O) was administered endobronchially in BALB/c mice on days 1, 2, 3, 6 and weekly thereafter. The lungs were harvested on day 15 or 30 and analyzed for infiltration of CD4+ T cells, CD11b and C4d deposition by immunohistochemistry using mouse anti-CD4, anti-mouse CD11b Abs anti-mouse C4d antibodies. CD4 + cells infiltrated around the bronchiole (arrow head) and vessel at both 15 and 30 days (A, G) along with significant infiltration of CD11b + cells around the bronchiole (arrow head) and vessel at 15 and 30 days (D, J) in anti-H2kd administered mice whereas no cellular infiltration around the bronchiole and vessel was observed in isotype control (B,E, H and K) or anti-keratin antibody administered mice (C, F, I and L). A significant increase in the C4d deposition was observed in the lungs of anti-H2kd administered mice (M) when compared to control antibody administered mice (N, O). A significant increase in the levels of CD4+ T cells and CD11b+ cells was observed in the lungs of mice treated with anti-H2Kd Ab compared to mice treated with isotype control Ab.

Figure 3. Administration of anti-MHC class I Ab increases the frequency of IFN-γ, IL-4 and IL-17 secreting T cells against K-α 1 tubulin and ColV in the lung

(A) Frequency of the IFN-γ secreting T cells against K-α 1 tubulin and ColV. (B) Frequency of the IL-4 secreting T cells against K-α 1 tubulin and ColV and C: Frequency of the IL-17 secreting T cells against K-α 1 tubulin and ColV. Anti-H2Kd or control Ab was administered endobronchially in BALB/c mice on days 1, 2, 3, 6 and weekly thereafter. The lungs were harvested on day 15 and 30. 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-α 1 tubulin and collagen were analyzed by ELISPOT. The values are represented as means ± SD using data obtained from 5 ELISPOT assays. The grey bars represent the isotype control, the solid bars represent the T cells isolated from lungs on day 15 and the black bars represent the T cells isolated on day 30. A significant increase in the frequency of IFN-γ, IL-4 and IL-17 secreting T cells against K-α 1 tubulin and ColV was observed in mice administered anti-H2kd Ab when compared to controls (*=P

Figure 4. Administration of anti-MHC class I Abs increases the expression of chemokines, cytokines and growth factor in the lungs

(A) Expression levels of chemokines and their receptors. (B) Expression levels of the cytokines. (C) Expression levels of the growth factor. Anti-H2Kd or control Ab was administered endobronchially in BALB/c mice on days 1, 2, 3, 6 and weekly thereafter. The lungs were harvested on day 4 (chemokines) and 15 (cytokines and growth factor) and analyzed using a quantitative real-time PCR array. RNA was extracted from the lung tissue and 1μg of the RNA was reverse transcribed and used as template for the quantitative real time PCR array (chemokines and receptor PCR array, common cytokines PCR array and growth factor PCR array, Superarray Inc, Frederick, MD). CT values obtained from the quantitative real-time PCR were analyzed by using the PCR array analysis software from Superarray Inc. The samples were normalized using the expression levels of the house keeping genes GAPDH, and HPRT1. The data represents the average values obtained from 3 different experiments. A significant (p>0.05) increase in the expression of chemokines (CCL9: 9.2, CCR2: 10.6, CXCL1: 6.35 and CXCL12: 8.0), cytokines (IL-10: 4.0, IL-12b: 5.66, IL-17f: 12.17, Inhba: 13.0, Tnfsf11: 6.5, Tnfsf15: 8.0, Tnfsf 4: 21.11, CD40 lg: 5.28) and growth factors (Bmp6: 4.30, Bmp8a: 10.88, Bmp8b: 5.28, Fgf6: 5.07, Fgf2: 37.46 and IL-4: 290.82) was observed in mice administered anti-H2Kd compared to controls.

Figure 5. Administration of Anti-MHC class I Abs leads to development of autoAbs against self antigens K-α1 tubulin and ColV

Anti-H2Kd or control Ab was administered endobronchially in BALB/c mice (n=5 each) on days 1, 2, 3, 6 and weekly thereafter. Sera were collected from the mice on day 15, 30 and 60. ELISA was performed for Abs to self antigens in the serum of mice treated with anti-MHC Abs or control Abs using purified K-α1 tubulin or ColV and as antigen. Administration of anti-H2Kd Ab results in the increased concentrations of anti-K-α 1 tubulin (A) and ColV (B) specific Abs on days 15, 30 and 60 compared to control mice treated with C1.18.4 Abs.

Figure 6. Neutralization of IL-17 blocks the induction of autoimmune responses and development of bronchial occlusion

Anti-H2Kd or control Ab was administered endobronchially in BALB/c mice on days 1, 2, 3, 6 and weekly thereafter. 50 μg of neutralizing anti-IL17 or control Abs were administered intravenously on day 1, 3, 5 and weekly thereafter (n=5 each). Serum and lungs of these mice were harvested on day 30 and analyzed for the development of auto-Abs and histopathological analysis. (A) Quantitation of the anti-tubulin Abs by ELISA showed a 3 fold reduction in the concentrations of anti-tubulin Abs in IL-17 neutralized mice compared to controls. (B) H& E staining of the lungs showed a significant reduction in T cell infiltration and bronchial occlusion similar to that observed in control mice treated with isotype control Ab.