Fas/CD95 is required for gastric mucosal damage in autoimmune gastritis  Aiden C.J. Marshall, Frank Alderuccio, Ban-Hock Toh  Gastroenterology  Volume 123, Issue 3, Pages 780-789 (September 2002) DOI: 10.1053/gast.2002.35383 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Screening strategy for lpr (Fas mutant) mice. (A) The mutation of the Fas gene involves insertion of a retrotransposon (ETn) between exons 2 and 3. A combination of 3 primers (numbered arrows) was used in a PCR to differentiate wild-type and mutant Fas genes. (B) PCR products were separated by agarose gel electrophoresis. The wild-type Fas allele produced a product of 180 base pairs and the mutant gene a product of 560 base pairs. Gastroenterology 2002 123, 780-789DOI: (10.1053/gast.2002.35383) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Fas expression in parietal cells of mice with EAG. Five-micrometer cryostat stomach sections of (D–F) 10-week-old normal and (A–C) gastritic BALB/cCrSlc mice were stained with (A and D) biotinylated monoclonal antibody reactive with Fas or (B and E) mouse monoclonal antibody reactive with the gastric H+/K+ ATPase β subunit in parietal cells. Bound biotinylated antibody was detected with streptavidin Texas Red (red) and H+/K+ ATPase reactivity with anti-mouse Ig fluorescein isothiocyanate (green). Images were captured by confocal microscopy, and C and F represent staining overlay with yellow staining indicating colocalization of Fas expression on parietal cells. Gastroenterology 2002 123, 780-789DOI: (10.1053/gast.2002.35383) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Incidence of autoimmune gastritis in neonatally thymectomized BALB/cCrSlc lpr/lpr mice. C3H/HeJ lpr mice were backcrossed 8 times to BALB/cCrSlc and intercrossed to generate wild-type (wt/wt), heterozygous (lpr/wt), and Fas-deficient lpr/lpr mice. Mice were thymectomized at day 3 and assessed at 10 weeks for H+/K+ ATPase antibodies by ELISA, parietal cell autoantibodies by indirect immunofluorescence (IF), and gastritis by H&E staining of paraffin-embedded stomach sections. ELISA reactivity is indicated by absorbance at 490 nm. Controls included positive (+) and negative (−) sera. ■, Parietal cell reactivity; □, lack of parietal cell reactivity or gastritis. Gastritis was scored as destructive (■) or nondestructive (▨). Gastroenterology 2002 123, 780-789DOI: (10.1053/gast.2002.35383) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Parietal cell autoantibodies and gastritis in neonatally thymectomized mice. H&E-stained stomach sections of (A) 10-week-old neonatally thymectomized lpr/lpr mouse without gastritis, (B) lpr/lpr mouse with nondestructive gastritis, and (C) wild-type littermate. Lpr/lpr mice without gastritis or with nondestructive gastritis (arrow) retained intact zymogenic (Z) and parietal cell (P) regions. In contrast, thymectomized wild-type mice developed a prominent mononuclear cell infiltrate within the gastric mucosa (arrow), accompanied by cellular destruction (arrowhead) and tissue hypertrophy. Sera from thymectomized lpr/lpr without gastritis or with nondestructive gastritis did not develop parietal cell autoantibodies (D and E) in contrast to thymectomized heterozygous lpr/wt (not shown) or wild-type littermates (F). Bar = 100 μm. Gastroenterology 2002 123, 780-789DOI: (10.1053/gast.2002.35383) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Transfer of lymphocytes from thymectomized lpr/lpr mice to nu/nu mice. A total of 2 × 107 nondepleted or CD25+-depleted pooled whole splenocytes and lymph node cells from neonatally thymectomized (nTx) BALB/cCrSlc lpr/lpr or wild-type (wt) mice were transferred to T-cell–deficient BALB/c nu/nu recipients. Recipients were killed 10 weeks after transfer and examined for autoantibody production and gastric pathology. (A) Gastric H+/K+ ATPase and parietal cell reactive antibodies were determined by ELISA and indirect immunofluorescence (IF), respectively, and indicated by absorbance at 490 nm (■). ■, Destructive gastritis assessed histologically by conventional H&E-stained stomach sections; ▨, nondestructive gastritis. ■, Cellular destruction assessed using a modified Maxwell's stain. Modified Maxwell's stained sections of (B) normal mouse stomach or stomach of nu/nu mouse transferred with cells from thymectomized lpr/lpr mice (C) or from wild-type mice (D). Note extensive areas of yellow staining in C and D compared with normal stomach (B). Bar = 100 μm. Gastroenterology 2002 123, 780-789DOI: (10.1053/gast.2002.35383) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Transfer of autoimmune gastritis from gastritic mice to nu/nu lpr/lpr mice. A total of 4 × 107 splenocytes from neonatally thymectomized BALB/cCrSlc mice with EAG including parietal cell reactivity and gastric destruction were transferred to wild-type nu/nu, heterozygous lpr/wt nu/nu, and lpr/lpr nu/nu mice (A). Gastric H+/K+ ATPase reactivity was measured by ELISA, parietal cell reactivity by indirect immunofluorescence (IF), and gastritis by histology on H&E-stained stomach sections. ■, Parietal cell reactivity or destructive gastritis; ▨, nondestructive gastritis. Control antibody included monoclonal antibodies 1H9 and 2B6 reactive with H+/K+ ATPase and negative control ET1. (B) Nu/nu lpr/lpr mice deficient in Fas expression did not develop EAG, but a nondestructive gastritis was evident (arrow). (C) EAG was induced in wild-type nu/nu mice, including a prominent mononuclear cell infiltrate within the gastric mucosa (arrow) and parietal (P) and zymogenic (Z) cell destruction (arrowhead). Bar = 100 μm. Gastroenterology 2002 123, 780-789DOI: (10.1053/gast.2002.35383) Copyright © 2002 American Gastroenterological Association Terms and Conditions