Fig 1.
Experimental design for intranasal cSiO2 exposure.
Beginning at 9 wk of age, NZBWF1 and C57Bl/6 mice were dosed intranasally with 25 μl PBS containing 0, 0.25 mg or 1.0 mg cSiO2 once per wk, for 4 wk. Proteinuria was monitored over the course of the experiment and all animals sacrificed at 24 wk of age.
Fig 2.
cSiO2 exposure accelerates development of proteinuria in NZBWF1 mice.
Proteinuria was monitored weekly until sacrifice 12 wk after the final cSiO2 exposure when most mice exposed to 1.0 mg cSiO2 were over threshold (≥ 300 mg/dl). Proteinuria was not detected in NZBWF1 mice dosed with 0.25 mg cSiO2 or vehicle. C57Bl/6 mice exposed to cSiO2 or vehicle did not develop detectable proteinuria over the course of the experiment.
Fig 3.
cSiO2 exposure increases severity of lupus nephritis in kidneys of NZBWF1 mice.
Representative light photomicrographs of PASH stained kidney sections in NZBWF1 mice at 24 wks of age exposed to vehicle (A) and 1.0mg cSiO2 (B). NZBWF1 mice exposed to 1.0mg cSiO2 developed extensive glomerulonephritis (black arrow) and tubular proteinosis (asterisk). NZBWF1 mice were individually graded for lupus nephritis following the modified ISN/RPS classification system as described in the Materials and Methods (C). Animals exposed to cSiO2 developed more severe lesions characteristic of lupus nephritis than vehicle-exposed mice. cSiO2 dose significantly correlated with lupus nephritis (Spearman rank-order coefficient = 0.64, p < 0.05).
Fig 4.
cSiO2 exposure in C57Bl/6 mice induced kidney lesions resembling lupus nephritis.
Representative light photomicrographs of PASH stained kidney sections in C57Bl/6 mice at 24 wks of age exposed to vehicle (A) and 1.0mg cSiO2 (B). Some renal histopathological lesions were observed in cSiO2-exposed C57Bl/6 mice (B) with notable renal tubular proteinosis (asterisk) and several glomeruli with global mesangial hypercellularity (white arrows). C57Bl/6 mice were individually graded for lupus nephritis as described in the Materials and Methods (C). Animals exposed to cSiO2 developed more severe lesions characteristic of lupus nephritis than vehicle exposed mice.
Fig 5.
cSiO2 elicits perivascular and peribronchiolar lymphocytic infiltration in lungs of NZBWF1 and C57Bl/6 mice.
Representative light photomicrographs of H&E stained lung sections from NZBWF1 mice exposed to vehicle (A), 0.25 mg cSiO2 (B), or 1.0 mg cSiO2 (C) and C57Bl/6 mice given vehicle (D), or 1.0mg cSiO2 (E). Br = bronchiole, bv = bronchial vasculature, ap = alveolar parenchyma. Black arrows indicate lymphocytic infiltration in perivascular and peribronchial regions. Lymphocytic infiltration was semi-quantitatively graded as described in the Materials and Methods (Table 1).
Table 1.
cSiO2 exposure increases severity of lymphocytic cell infiltration in lungs of NZBWF1 mice relative to C57Bl/6 mice.
Fig 6.
Marked accumulation of IgG producing plasma cells occurs in lungs of cSiO2-exposed NZBWF1 mice.
Representative immunohistochemical photomicrographs of IgG in lungs of NZBWF1 mice. Photomicrographs taken at low magnification are shown in A and B whereas images at high magnification are shown in C and D. Vehicle-exposed mice did not indicate positive IgG staining (A,C). cSiO2 induced marked infiltration of IgG-laden lymphocytes peripheral to both blood vessels (bv) and bronchiole airways (BA) (black arrows in B, white arrows in D). IgG was also detected extracellularly within alveolar parenchyma (ap) (stippled arrows in B, black arrows in D).
Fig 7.
Intranasal cSiO2 exposure induces infiltration of CD45R+ and CD3+ lymphocytes in lungs that resemble ectopic lymphoid tissue.
Representative light photomicrographs of lung tissue sections from mice treated with 0.0 mg cSiO2 (vehicle controls; A, D, E, H, I and L) or 1.0 mg cSiO2 (B, C, F, G, J and K). Some lung sections were immunohistochemically stained to identify B lymphocytes (CD45R+) (A, B, E, F, I, and J), while others (C, D, G, H, K, and L) were immunohistochemically stained to identify T lymphocytes (CD3+). Photomicrographs taken at high magnifications of blood vessels (v) and bronchioles (b) are illustrated in E, F, G, H and I, J, K, L, respectively. In cSiO2-treated mice, both bronchiolar airways and blood vessels were fully or partially circumscribed by thick interstitial infiltrates of mononuclear cells (arrows in B and C). These infiltrates were primarily comprised of B lymphocytes (solid arrows in F and J) and T lymphocytes (stippled arrows in G and K). B cells tended to form distinct focal aggregates (solid arrows in F and J) and T cells (stippled arrows in G and K) were more diffusely distributed throughout the peribronchiolar and perivascular lymphoid infiltrates. Control mice had only a few widely scattered B (solid arrow in E, I) and T (stippled arrow in H, L) cells present in the alveolar parenchyma (a), but no distinct lymphoid cell cuffing around the bronchioles or blood vessels. All tissues were counterstained with hematoxylin. e, airway epithelium.
Fig 8.
Intranasal cSiO2 exposure induces infiltration of lymphocytes and PMN leukocytes in BALF of NZBWF1 and C57Bl/6 mice.
Cytometric slides prepared from BALF were stained with Diff-Quick and 200 cells per slide identified as monocytes/macrophages (Mo), lymphocytes (Lymph) or polymorphonuclear (PMN) cells. In NZBWF1 mice, group mean ± SEM at 0.0 mg cSiO2 were 97.2 ± 0.4%, 2.4 ± 0.3%, and 0.5 ± 0.1% for Mo, Lymph and PMN, respectively. Group mean ± SEM for 0.25 mg cSiO2 group were 50.8 ± 2.9%, 32.8 ± 3.5%, and 14.0 ± 1.8%, respectively and for the 1.0 mg cSiO2 group were 39.3 ± 1.7%, 43.0 ± 2.5%, and 18.7 ± 1.8%, respectively. In C57Bl/6 mice, group mean ± SEM at 0.0 mg cSiO2 were 95.9 ± 0.9%, 3.1 ± 0.7% and 1.0 ± 0.2% for Mo, Lymph, and PMN, respectively. Group mean ± SEM for 1.0mg cSiO2 were 59.9 ± 3.7%, 22.6 ± 3.7, and 17.3 ± 2.1% for Mo, Lymph, and PMN, respectively.
Fig 9.
cSiO2 exposure elevates IgG (A), IgA (B), and IgM (C) concentrations in BALF of NZBWF1 and C57Bl/6 mice.
Total immunoglobulins in BALF were measured by ELISA. Data are group mean ± SEM (n = 7–8/gp) and were analyzed by one-way ANOVA on Ranks with Dunn’s method (NZBWF1) or Mann-Whitney Rank Sum Test (C57Bl/6). Asterisk indicates a statistically significant difference between cSiO2 treatment and vehicle control (p < 0.05). In BALF of NZBWF1 mice, cSiO2 dose correlated significantly (p < 0.05) with IgG (Spearman rank-order correlation coefficient = 0.80), IgA (Spearman rank-order correlation coefficient = 0.72), and IgM (Spearman rank-order correlation coefficient = 0.85).
Fig 10.
cSiO2 exposure increases proinflammatory cytokines MCP-1 (A, D), TNF-α (B, E), and IL-6 I (C, F) in BALF and plasma of NZBWF1 mice.
Proinflammatory cytokine concentrations in BALF and plasma were determined by cytometric bead array. Data are group mean ± SEM (n = 5–8/gp) and were analyzed by one-way ANOVA on Ranks with Dunn’s method. Asterisk indicates a statistically significant difference in analyte between cSiO2 treatment and vehicle control (p < 0.05). cSiO2 dose correlated significantly (p < 0.05) with BALF MCP-1 (Spearman rank-order correlation coefficient = 0.90), BALF TNF-α (Spearman rank-order correlation coefficient = 0.89), and BALF IL-6 (Spearman rank-order correlation coefficient = 0.82). cSiO2 dose also correlated significantly (p < 0.05) with plasma TNF-α (Spearman rank-order correlation coefficient = 0.60), and IL-6 (Spearman rank-order correlation coefficient = 0.49). n.d. indicates not detected.
Fig 11.
cSiO2 exposure increases anti-dsDNA antibodies (A) and anti-nuclear antibodies (B) in plasma of NZBWF1 mice.
Autoreactive antibodies in plasma at sacrifice were measured by ELISA. Data are group mean ± SEM (n = 7–8/gp) and were analyzed by One-Way ANOVA on Ranks with Dunn’s method. Asterisk indicates statistically significant difference in antibody concentration between cSiO2 treatment and vehicle control (p < 0.05). cSiO2 dose significantly correlated (p < 0.05) with plasma anti-dsDNA Ab’s (Spearman rank-order correlation coefficient = 0.62) and anti-nuclear Ab’s (Spearman rank-order correlation coefficient = 0.58).