Article Figures & Data
Figures
Figure 1. Representative photomicrographs of histologic features of renal injury in normal C57BL/6 (WT), IL-12p40−/−, and IFN-γ−/− mice with experimental autoimmune glomerulonephritis (EAG). Photomicrographs at low and high power demonstrate relatively mild renal injury in WT mice with EAG (A and B), less severe injury in IL-12p40−/− mice (C and D), more significant but relatively mild injury in the majority of IFN-γ−/− mice (E and F), and severe injury in some IFN-γ−/− mice with diffuse interstitial disease and more severe glomerular injury (G and H), including a significant degree of glomerular crescent formation (all periodic acid Schiff stain; A, C, E, and G low power; B, D, F, and H high power).
Figure 2. Glomerular pathology and leukocytes in EAG. (A and B) IL-12p40−/− mice have a lower proportion of glomeruli affected than WT mice and a trend to decreased glomerular cell numbers, whereas IFN-γ−/− mice have increased numbers of abnormal glomeruli compared with WT mice and increased cell nuclei compared with IL-12p40−/− mice. Analysis of leukocytes in glomeruli (C through F) show increased numbers of CD4+ cells, M1/70+ cells, and Gr-1+ leukocytes in IFN-γ−/− mice compared with IL-12p40−/− mice. *P < 0.05 versus IL-12p40−/− mice; **P < 0.01 versus IL-12p40−/− mice; ***P < 0.01 versus WT (ANOVA).
Figure 3. Analysis of interstitial leukocytic infiltrates in mice with EAG, showing fewer interstitial neutrophils in IL-12p40−/− mice and a trend to increased CD4+ cells in IFN-γ−/− mice. *P < 0.05 versus both C57BL/6 and IFN-γ−/− mice (ANOVA).
Figure 4. Immunohistochemical detection of leukocytes in EAG showing a predominant CD4+ cell and macrophage infiltrate in representative sections of more severely affected IFN-γ−/− mice. Positive cells are shown as a black reaction product. CD4+ cells in WT mice (A), IL-12p40−/− mice (B), and IFN-γ−/− mice (C). (D) A more significantly affected IFN-γ−/− mouse. CD8+ cells WT (E), IL-12p40−/− mice (F), IFN-γ−/− mice (G), and a more significantly affected IFN-γ−/− mouse (H). In general, CD8+ cells were less evident in IFN-γ−/− mice, including the dense infiltrates, than CD4+ cells. M1/70+ cells in WT mice (I), IL-12p40−/− mice (J), and IFN-γ−/− mice (K and L). Gr-1+ neutrophils in WT mice (M), IL-12p40−/− mice (N), and IFN-γ−/− mice (O and P). M1/70 detects both macrophages and neutrophils. Most of the M1/70+ cells were not Gr-1+, indicating a predominance of macrophages. Medium-power views using DAB (black reaction product) and nuclear fast red counterstain.
Figure 5. Semiquantitative analysis of adhesion molecule expression in kidneys of mice with EAG. (A and B) Upregulated P-selectin expression in the absence of IFN-γ, the difference reaching statistical significance (compared with both WT mice and IL-12p40−/− mice) in glomerular P-selectin. (C) No significant alteration in the expression of intracellular adhesion molecule 1 (ICAM-1) in glomeruli of the three groups of mice with EAG, but D shows significantly increased tubulointerstitial ICAM-1 expression in IFN-γ−/− mice compared with both WT and IL-12p40−/− mice. *P < 0.05 versus both WT and IL-12p40−/− mice; **P < 0.05 versus IL-12p40−/− mice and P < 0.01 versus WT mice (ANOVA).
Figure 6. Immunofluorescent detection of adhesion molecule expression (P-selectin and ICAM-1) in EAG, showing in WT mice expression of P-selectin (A) and ICAM-1 (E) that was little different in the absence of IL-12p40 (P-selectin [B], ICAM-1 [F]). In IFN-γ−/− mice, increases in both P-selectin (C and D) and ICAM-1 (G and H; within the interstitium) were evident. The increased adhesion molecule expression observed was a feature of EAG in IFN-γ−/− mice regardless of the relative degree of injury (C and G show a less affected mouse; D and H show a more severely affected IFN-γ−/− mouse). Medium-power views using FITC (P-selectin) and PE (ICAM-1) as fluorochromes.
Figure 7. Autoantibodies in EAG. Linear staining of Ig was observed to a similar degree in C57BL/6 WT, IL-12p40−/−, and IFN-γ−/− mice (A through C); semiquantitative analysis in D. There was no significant difference in serum anti α3-α5(IV)NC1 antibody titers (E) between the three groups of mice.
Figure 8. Dermal delayed-type hypersensitivity (DTH) responses in mice that were sensitized to α3-α5(IV)NC1. WT mice (n = 4) received an injection of vehicle alone (□), IFN-γ−/− mice (n = 5) received an injection of vehicle alone (▥), and IFN-γ−/− mice (n = 5) were given daily recombinant murine IFN-γ (rmIFN-γ;
) from sensitization. Responses to collagenase solubilized murine renal basement membrane 48 h after challenge (A) showed an increase in IFN-γ−/− mice that was reversed by daily rmIFN-γ intraperitoneally. Responses to bovine α3-α5(IV)NC1 at both 24 and 48 h showed a similar pattern with suppression of DTH responses by rmIFN-γ, although results in IFN-γ−/− mice that were given vehicle alone did not reach statistical significance compared with WT mice. *P < 0.05 versus IFN-γ−/− mice; **P < 0.001 versus both other groups at the same time point (ANOVA).
Tables
Table 1. Serum and glomerular IgG subclasses in C57BL/6 WT, IL-12p40−/− and IFN-γ−/− mice with EAGa
C57BL/6 WT IL-12p40−/− IFN-γ−/− a Sera were assessed by ELISA, and glomerular deposition was assessed by direct immunofluorescence (see Materials and Methods for details). Results are expressed as the OD405 mean ± SEM and the mean ± SEM of a semiquantitative score for each animal. WT, wild type mice; IL-12p40−/−, interleukin-12p40 deficient mice; IFN-γ−/−, IFN-γ deficient mice. b Values for normal mouse sera were 0.098 ± 0.069 for IgG1 and 0.004 ± 0.002 for IgG2a. c P < 0.05 versus C57BL/6. d P < 0.01 versus IL-12p40−/− mice. IgG1 serum (OD405) 0.343 ± 0.081b 0.469 ± 0.092 0.503 ± 0.112 glomerular (0–3+) 2.2 ± 0.2 2.4 ± 0.2 1.5 ± 0.2c IgG2a serum (OD405) 0.600 ± 0.183b 1.060 ± 0.030 0.221 ± 0.010d glomerular (0–3+) 0.62 ± 0.12 0.61 ± 0.27 0.10 ± 0.10 Table 2. Renal function and urinary protein excretion in C57BL/6 WT, IL-12p40−/−, and IFN-γ−/− mice with EAGa
Serum Creatinine (μmol/L) Urinary Protein (mg/24 h) a Results are expressed as the mean ± SEM. EAG, experimental autoimmune glomerulonephritis; GN, glomerulonephritis. b P < 0.01 versus other three groups (ANOVA). c Results in this group exclude two oligoanuric mice. Normal mice no GN 10.9 ± 1.0 1.2 ± 0.3 C57BL/6 WT GN 12.9 ± 1.0 2.6 ± 0.3b IL-12p40−/− GN 10.9 ± 1.2 1.4 ± 0.1 IFN-γ−/− GN 27.3 ± 11.2 1.4 ± 0.4c Table 3. CD4+ cells, CD8+ cells, proliferation, and apoptosis in draining lymph nodes four weeks after immunization of WT and IFN-γ−/− micea
WT Mice IFN-γ−/− Mice a Results are expressed in % of live cells, as the mean ± SEM. b P = 0.038 versus WT (t test). CD4+ cells 20.8 ± 1.6 25.1 ± 0.8b CD8+ cells 21.0 ± 1.4 21.0 ± 0.7 CD4+/BrdU+ cells 1.16 ± 0.06 1.53 ± 0.14b CD8+/BrdU+ cells 0.43 ± 0.08 0.51 ± 0.16 Apoptotic cells 9.5 ± 0.7 10.2 ± 0.7
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