Figure 1.
Histology from lungs harvested on day 0, 30 and 60 following intrabronchial administration of M5/114 (MHC class II Ab) or LTF-2 (isotype control) to C57BL/6 mice on day 1,2,3,6 and weekly.
(A–F) represent lungs analyzed by H&E staining; (G–L) represent lungs analyzed by trichrome staining; and (M) represents morphometric analysis performed using NIS-Elements BR software to quantitate the epithelial hyperplasia, cellular infiltration, fibrosis, luminal occlusion and neutrophil infiltration (assayed by myeloperoxidase activity). The data is represented as a mean ± SEM over a 5 different animals in each group, the significance (p-value <0.05) was determined by student-t-test and represented with an asterisk (*).
Figure 2.
Immunohistochemistry from lungs harvested on day 30 and 60 following intrabronchial administration of M5/114 (MHC class II Ab) or LTF-2 (isotype control) to C57BL/6 mice.
(A–D) represent staining for CD4+ T cells; (E–H) represent staining for CD8+ T cells; (I and J) represent morphometric analysis to quantitate the total cellular infiltration, CD8 infiltration, CD4 infiltration on day 15, 30 and 60 around bronchioles (I) and blood vessels (J). The data is represented as a mean ± SEM over a 5 different animals in each group, the significance (p-value <0.05) was determined by student-t-test and represented with an asterisk (*).
Figure 3.
Analysis of the humoral immune response on serum collected from day 7, 15, 30 and 60 following administration of MHC class II Abs or isotype C57BL/6 animals.
(A,B) represent the serum concentration of Abs to self-Ags ColV (A) and Kα1T (B) analyzed by ELISA. The data is represented as a mean ± SEM over a 5 different animals in each group, the significance (p-value <0.05) was determined by student-t-test and represented with an asterisk (*).
Figure 4.
Analysis of the cellular immune response on lung infiltrating lymphocytes collected on day 7, 15, 30 and 60 following administration of MHC class II Abs or isotype C57BL/6 animals, analyzed by ELISpot.
(A,B) represent the ColV (A) and Kα1T (B) specific IL-10 responses; (C,D) represent the ColV (C) and Kα1T (D) specific IL-17 responses; (E, F) represent the ColV (E) and Kα1T (F) specific IFN-γ responses. The data is represented as a mean ± SEM over a 5 different animals in each group, the significance (p-value <0.05) was determined by student-t-test and represented with an asterisk (*).
Figure 5.
Analysis of the MHC class II expression and macrophage phenotype prior to and following the administration of MHC class II Abs.
(A) represent MHC class II (I-Akα) expression in the lung analyzed by PCR on day 0, 15, 30 and 60 following the administration of M5/114; (B–D) represent the immunohistochemistry on harvested lungs with MHC class II staining prior to administration of MHC class II Ab (B), day 60 of isotype administration (C) and day 60 of MHC class II Ab administration; (E) represent the morphometric quantitation of the MHC class II expression in the lung on day 0, 30 and 60 following the administration of MHC class II Ab or isotype Ab; (F, G) represent the phenotype of the lung infiltration macrophages following administration of MHC class II Ab analyzed by flow cytometry, green represent F4/80 staining (MΦ), orange represent CD11c staining (MΦ1), and red represent CD206 staining (MΦ2); and (G) represent the ratio of MΦ2 to MΦ1 macrophage phenotype on day 0, 7, 15, 30 and 60. The data is represented as a mean ± SEM over a 5 different animals in each group, the significance (p-value <0.05) was determined by student-t-test and represented with an asterisk (*).
Figure 6.
Gene expression analysis of the chemokines, their receptors and growth factors on the on the mRNA collected from the lungs harvested on day 7.
The data is represented as a mean ± SEM over a 5 different animals in each group.
Figure 7.
Histology from lungs harvested on day 30 following adoptive transfer of day 60 macrophages isolated at day 60 from MHC class II Ab administered mice.
0.1, and 1×105 CD11b+ macrophages intrabronchially, and single intrabronchial administration of MHC class II Ab was done for adoptive transfer experiments. (A, B) H&E staining of the lungs collected from day 30 without (A) and with (B) adoptive transfer of 1×105 CD11b+ macrophages following single administration of MHC class II Ab. (C, D) Trichrome staining of the lungs collected from day 30 without (C) and with (D) adoptive transfer of 1×105 CD11b+ macrophages following single administration of MHC class II Ab; and (E) serum concentration of Abs to self-Ags ColV and Kα1T analyzed by ELISA. The data is represented as a mean ± SEM over a 5 different animals in each group, the significance (p-value <0.05) was determined by student-t-test and represented with an asterisk (*).
Figure 8.
Induction of Th17 cell from naïve T cells by lung infiltrating macrophages.
(A) Flow cytometry analysis for the expression of IL-1β, TGF-β, IL-6 and IL-23 cytokines on CD4+gated population; (B) gene expression of IL-1β, TGF-β, IL-6 and IL-23 in macrophages collected from lungs; (lane1) and peritoneum (lane 3) day 60 MHC class II Ab administered mice, lane 2 and 4 represent macrophages collected from lung and peritoneum of isotype administered animals. (B) represent IL-17 concentration from the media collected by co-culture of 1∶1 ratio of naïve T cells collected from splenocytes of wild type C57BL/6 mice and cultured with macrophages collected from lung (1) and peritoneum (3) of day 60 MHC class II Ab administerd mice, 2 and 4 represent co culture with macrophages collected from lung (2) and peritoneum (4) of day 60 Isotype Ab administered mice, (5 in panel B) represent lung infiltrating macrophages of day 60 MHC class II Ab administered mice in the absence of naïve T cells. The data is represented as a mean ± SEM over a 5 different experiments, the cohort with significant expression of IL-17 (p-value <0.005) compared to isotype Ab administered group (5) were represented with an asterisk (*).