BALB/c-Fcgr2bPdcd1 mouse expressing anti-urothelial antibody is a novel model of autoimmune cystitis

Abstract

We report the impact of anti-urothelial autoantibody (AUAb) on urinary bladder phenotype in BALB/c mice deficient of the FcγRIIb and PD-1. AUAb was present in serum samples from approximately half of the double-knockout (DKO) mice, as detected by immunofluorescence and immunoblots for urothelial proteins including uroplakin IIIa. The AUAb-positive DKO mice showed degeneration of urothelial plaque and umbrella cells, along with infiltration of inflammatory cells in the suburothelial layer. TNFα and IL-1β were upregulated in the bladder and the urine of AUAb-positive DKO mice. Voiding behavior of mice was analyzed by the Voided Stain on Paper method. 10-week-old and older AUAb-positive DKO mice voided significantly less urine per void than did wild type (WT) mice. Furthermore, administration of the AUAb-containing serum to WT mice significantly reduced their urine volume per void. In summary, this report presents a novel comprehensive mouse model of autoimmune cystitis.

Figure 1. DKO mice expressing AUAb have degenerated urothelium.

Histological analyses of bladder tissue of WT (A, D, G), AUAb-positive DKO (B, E, H), and AUAb-negative DKO (C, F, I) mice are displayed. (A–C) Hematoxylin and eosin (H&E) stain showed smaller epithelial cells including umbrella cells in the bladder of AUAb-positive DKO mice (B) than WT (A) and AUAb-negative DKO mice (C). (D–F) Scanning electron microscopy (SEM) revealed epithelial plaque in the bladder of WT (D) and AUAb-negative DKO mice (F); however, the size of plaques was decreased in AUAb-positive DKO mice (E). (G–I) The intensity of staining for UPKIIIa was reduced in the urothelial layer of AUAb-positive DKO mice (H) compared to WT (G) and AUAb-negative DKO mice (I). Bars indicate 30 μm in A–C, G–I, 10 μm in D–F.

Figure 2. C-Kit expressing cells are significantly elevated in the suburothelial layer of AUAb-positive DKO mice.

(A–F) H&E staining (A–C) and immunohistochemistry for c-Kit (D–F) are shown. There were many inflammatory cells beneath the urothelium of AUAb-positive DKO mice and c-Kit-positive cells were observed in the lesion (B, E), though there were no infiltration of inflammatory cells nor c-Kit-positive cells in the specimen of WT (A, D) and AUAb-negative DKO (C, F) mice. (G) There was a significant increase in the number of c-Kit-positive cells in the bladder of AUAb-positive DKO mice (: n = 5) compared to WT (: n = 5) and AUAb-negative DKO mice (: n = 5) under microscopic observation (**p<0.01). (H–O) Immunohistochemistry for the other important inflammatory cells is shown for bladder of WT (H, J, L, N) and AUAb-positive DKO mice (I, K, M, O). CD4 (T-cell marker: H, I), CD11c (dendritic cell marker: J, K) and B220 (activated B-cell marker: L, M) were prominent in the bladder of AUAb-positive DKO mice (I, K, M) compared to the WT bladder that had no infiltration of inflammatory cells (H, J, L) (positive cells are indicated by white arrows), while there were no remarkable staining of F4/80 (macrophage marker: N, O) in AUAb-positive DKO bladder (O) and there were no infiltration of inflammatory cells in WT bladder (N). Bars indicate 100 μm in A–C, 30 μm in D–F, H–O.

Figure 3. Higher level of TNFα is detected in the bladder and the urine of AUAb-positive DKO mice.

(A) TLDA indicated that mRNA expression level of six inflammatory molecules was significantly upregulated in the bladder of AUAb-positive mice compared to WT mice (†† p<0.01). Urine protein assay also revealed that seven inflammatory proteins were significantly upregulated in the urine of AUAb-positive DKO mice compared to WT mice († p<0.05). IL-1β and TNFα were expressed higher in both. (B) TNFα mRNA was elevated in the stomach and bladder of AUAb-positive DKO mice (*p<0.05). (C) ELISA assay for mouse TNFα has revealed that AUAb-positive DKO mice urine samples contain significantly higher level of TNFα than WT mice (*p<0.05).

Figure 4. AUAb-containing serum is pathogenic to bladder storage function.

(A) Urine volume per void of female AUAb-positive DKO mice (: n = 7), female AUAb-negative DKO mice (: n = 6), and female WT mice (: WT, n = 10) was recorded from 4-week-old to 16-week-old by the VSOP method. From 10 to 16-week-old, there were significant differences (**p<0.01) in urine volume per void between AUAb-positive DKO mice and WT mice, although there were no significant differences between AUAb-negative DKO mice and WT mice (upper panel). The data adjusted by body weight are shown in the middle panel and the data shown as urine volume per void index (adjusted at 4-week's urine volume per void) are in the lower panel. There were also significant differences between AUAb-positive DKO () and WT () from 10 to 16-week-old (*p<0.05), although there were no significant differences between AUAb-negative DKO () and WT () except the urine volume adjusted by body weight at 10-week-old (#p<0.05). (B) Serum samples from AUAb-positive DKO mice (, n = 5) and serum samples from WT mice (, n = 5) were injected into WT female mice intraperitoneally (300 μl/body at 4-, 7-, 10-week-old; arrow). The urine volume per void recorded by VSOP method revealed a significant decrease of urine volume per void by administration of the serum from AUAb-positive DKO mice, compared with the serum from WT mice (shown as urine volume per void index). Data are expressed as means ± SE for each group. (C) H&E staining of the mouse bladder after injection of AUAb-containing serum (upper right) showed the disarrangement of urothelial plaque, which was not observed in the bladder after injection of control serum (upper left). The intensity of staining for UPKIIIa was reduced in the urothelial layer of the bladder of AUAb-containing serum injected mice (lower left) compared to the bladder of control serum injected mice (lower right). Bars indicate 100 μm.