Involvement of OX40-OX40L interactions in the intestinal manifestations of the murine acute graft-versus-host disease  Eckhard Stüber, Alexander von Freier, Dana Marinescu, Ulrich R. Fölsch  Gastroenterology  Volume 115, Issue 5, Pages 1205-1215 (November 1998) DOI: 10.1016/S0016-5085(98)70092-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. Animals were killed at day 6, and the jejunum was removed and fixed in formalin. After embedding in paraffin, 6–8-μm sections were cut and the slides were stained with H&E according to standard procedures. (C and D) Villus length and crypt depth were measured by using a graded ocular in at least 15–20 complete, straight crypt-villus axes per slide of 7 syngeneic control (■) and 15 semiallogeneic (2) animals. (C) The average (±SD) of these measurements; (D) calculated ratio of crypt depth to villus length. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. Animals were killed at day 6, and the jejunum was removed and fixed in formalin. After embedding in paraffin, 6–8-μm sections were cut and the slides were stained with H&E according to standard procedures. (C and D) Villus length and crypt depth were measured by using a graded ocular in at least 15–20 complete, straight crypt-villus axes per slide of 7 syngeneic control (■) and 15 semiallogeneic (2) animals. (C) The average (±SD) of these measurements; (D) calculated ratio of crypt depth to villus length. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. Animals were killed at day 6, and the jejunum was removed and fixed in formalin. After embedding in paraffin, 6–8-μm sections were cut and the slides were stained with H&E according to standard procedures. (C and D) Villus length and crypt depth were measured by using a graded ocular in at least 15–20 complete, straight crypt-villus axes per slide of 7 syngeneic control (■) and 15 semiallogeneic (2) animals. (C) The average (±SD) of these measurements; (D) calculated ratio of crypt depth to villus length. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. Animals were killed at day 6, and the jejunum was removed and fixed in formalin. After embedding in paraffin, 6–8-μm sections were cut and the slides were stained with H&E according to standard procedures. (C and D) Villus length and crypt depth were measured by using a graded ocular in at least 15–20 complete, straight crypt-villus axes per slide of 7 syngeneic control (■) and 15 semiallogeneic (2) animals. (C) The average (±SD) of these measurements; (D) calculated ratio of crypt depth to villus length. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. Animals were killed at day 3, and the jejunum was removed and snap-frozen. Cryosections of 6–8 μm were prepared, and the slides were stained with Oncor ApopTag Direct Apoptosis in situ detection kit and counterstained with propidium iodide (orange). Green (FITC) fluorescent nuclei indicate TUNEL-positive cells undergoing apoptosis. (C) TdT-negative control slide derived from the same animal as in A. (D) Jejunal cryosections of a semiallogeneic GVH animal were first incubated with anti-CD4 and anti-CD8 antibodies followed by a staining procedure with the ApopTag Direct Apoptosis in situ detection kit. Sections were not counterstained with propidium iodide. Brown-stained CD4- and CD8-positive cells are distinct from the green/yellow fluorescent nuclei of the crypt epithelium, indicating ongoing apoptosis. (E) FITC-positive cells were counted in at least 15–20 high-power fields per slide. Data represent averages (■) + SD (2) of 6 syngeneic control and 12 semiallogeneic animals. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. Animals were killed at day 3, and the jejunum was removed and snap-frozen. Cryosections of 6–8 μm were prepared, and the slides were stained with Oncor ApopTag Direct Apoptosis in situ detection kit and counterstained with propidium iodide (orange). Green (FITC) fluorescent nuclei indicate TUNEL-positive cells undergoing apoptosis. (C) TdT-negative control slide derived from the same animal as in A. (D) Jejunal cryosections of a semiallogeneic GVH animal were first incubated with anti-CD4 and anti-CD8 antibodies followed by a staining procedure with the ApopTag Direct Apoptosis in situ detection kit. Sections were not counterstained with propidium iodide. Brown-stained CD4- and CD8-positive cells are distinct from the green/yellow fluorescent nuclei of the crypt epithelium, indicating ongoing apoptosis. (E) FITC-positive cells were counted in at least 15–20 high-power fields per slide. Data represent averages (■) + SD (2) of 6 syngeneic control and 12 semiallogeneic animals. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. At day 6, the animals were injected with BrdU (5 mg/kg body wt) 1 hour before they were killed, and the jejunum was removed and fixed in formalin. After paraffin embedding, 6–8-μm sections were prepared and the slides were stained for BrdU-positive cells (i.e., proliferating cells) with a Calbiochem BrdU detection kit. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Irradiated B6D2F1 mice received either (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) or (B) 80 × 106 B6D2F1 lymphocytes (syngeneic control) by IP injection. At day 6, the animals were injected with BrdU (5 mg/kg body wt) 1 hour before they were killed, and the jejunum was removed and fixed in formalin. After paraffin embedding, 6–8-μm sections were prepared and the slides were stained for BrdU-positive cells (i.e., proliferating cells) with a Calbiochem BrdU detection kit. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Irradiated B6D2F1 mice received (A–D) 80 × 106 B6D2F1 lymphocytes (syngeneic), (E–I) C57/BL6 lymphocytes (semiallogeneic) and human IgG1, or (J–M) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. Cryosections were stained with (A, E, F, and J) antitenascin, (B, G, and K) antifibronectin, (C, H, and L) antilaminin, or (D, I, and M) anti–collagen type IV. Antibody binding was detected as described in Materials and Methods. (Because of the very long exposition time at this magnification [100×] and the low level of tenascin expression, the background color also changed to a slightly darker green [F].) Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Irradiated B6D2F1 mice received in 2 separate experiments 80 × 106 B6D2F1 lymphocytes (syngeneic) (lanes 1 and 2), C57/BL6 lymphocytes (semiallogeneic) and human IgG1 (lanes 3 and 4), or OX40-Ig (200 μg daily on days 0–5, lanes 5 and 6) by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. RNA was prepared, and reverse-transcription PCR specific for tenascin, fibronectin, laminin, and β-actin mRNA was performed as described in Materials and Methods. PCR products were separated by polyacrylamide gel electrophoresis, and bands were visualized using ethidium bromide. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Irradiated B6D2F1 mice received 80 × 106 C57/BL6 lymphocytes (semiallogeneic) by IP injection. Animals were killed 60 hours later, and the spleens were removed and snap-frozen. Cryosections of 6–8 μm were prepared, and the slides were stained with either (A) anti-OX40 or (B) OX40-Ig to detect OX40L (see Materials and Methods). Controls were performed using polyclonal rabbit IgG or human IgG1, respectively. No unspecific staining could be observed in these control slides (data not shown). (C) Irradiated B6D2F1 mice were treated as described earlier. Animals were killed 60 hours later, and splenic lymphocytes were prepared. Cells were incubated with the polyclonal anti-OX40 antibody (or rabbit IgG as control) and counterstained with anti-rabbit IgG-FITC, and FITC-positive cells were counted by flow cytometry. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Irradiated B6D2F1 mice received 80 × 106 C57/BL6 lymphocytes (semiallogeneic) by IP injection. Animals were killed 60 hours later, and the spleens were removed and snap-frozen. Cryosections of 6–8 μm were prepared, and the slides were stained with either (A) anti-OX40 or (B) OX40-Ig to detect OX40L (see Materials and Methods). Controls were performed using polyclonal rabbit IgG or human IgG1, respectively. No unspecific staining could be observed in these control slides (data not shown). (C) Irradiated B6D2F1 mice were treated as described earlier. Animals were killed 60 hours later, and splenic lymphocytes were prepared. Cells were incubated with the polyclonal anti-OX40 antibody (or rabbit IgG as control) and counterstained with anti-rabbit IgG-FITC, and FITC-positive cells were counted by flow cytometry. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Irradiated B6D2F1 mice received 80 × 106 C57/BL6 lymphocytes (semiallogeneic) by IP injection. Animals were killed 60 hours later, and the spleens were removed and snap-frozen. Cryosections of 6–8 μm were prepared, and the slides were stained with either (A) anti-OX40 or (B) OX40-Ig to detect OX40L (see Materials and Methods). Controls were performed using polyclonal rabbit IgG or human IgG1, respectively. No unspecific staining could be observed in these control slides (data not shown). (C) Irradiated B6D2F1 mice were treated as described earlier. Animals were killed 60 hours later, and splenic lymphocytes were prepared. Cells were incubated with the polyclonal anti-OX40 antibody (or rabbit IgG as control) and counterstained with anti-rabbit IgG-FITC, and FITC-positive cells were counted by flow cytometry. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Irradiated B6D2F1 mice received (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) and human IgG1 or (B) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and fixed in formalin. After paraffin embedding, 6–8-μm sections were cut and the slides were stained with H&E according to standard procedures. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Irradiated B6D2F1 mice received (A) 80 × 106 C57/BL6 lymphocytes (semiallogeneic) and human IgG1 or (B) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and fixed in formalin. After paraffin embedding, 6–8-μm sections were cut and the slides were stained with H&E according to standard procedures. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Irradiated B6D2F1 mice received (A and B) 80 × 106 B6D2F1 lymphocytes (syngeneic), (C and D) C57/BL6 lymphocytes (semiallogeneic) and human IgG1, or (E and F) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. Cryosections were stained afterwards with (A, C, and E) anti-CD4 or (B, D, and F) anti-CD8. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Irradiated B6D2F1 mice received (A and B) 80 × 106 B6D2F1 lymphocytes (syngeneic), (C and D) C57/BL6 lymphocytes (semiallogeneic) and human IgG1, or (E and F) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. Cryosections were stained afterwards with (A, C, and E) anti-CD4 or (B, D, and F) anti-CD8. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Irradiated B6D2F1 mice received (A and B) 80 × 106 B6D2F1 lymphocytes (syngeneic), (C and D) C57/BL6 lymphocytes (semiallogeneic) and human IgG1, or (E and F) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. Cryosections were stained afterwards with (A, C, and E) anti-CD4 or (B, D, and F) anti-CD8. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Irradiated B6D2F1 mice received (A and B) 80 × 106 B6D2F1 lymphocytes (syngeneic), (C and D) C57/BL6 lymphocytes (semiallogeneic) and human IgG1, or (E and F) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. Cryosections were stained afterwards with (A, C, and E) anti-CD4 or (B, D, and F) anti-CD8. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Irradiated B6D2F1 mice received (A and B) 80 × 106 B6D2F1 lymphocytes (syngeneic), (C and D) C57/BL6 lymphocytes (semiallogeneic) and human IgG1, or (E and F) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. Cryosections were stained afterwards with (A, C, and E) anti-CD4 or (B, D, and F) anti-CD8. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Irradiated B6D2F1 mice received (A and B) 80 × 106 B6D2F1 lymphocytes (syngeneic), (C and D) C57/BL6 lymphocytes (semiallogeneic) and human IgG1, or (E and F) OX40-Ig, 200 μg daily on days 0–5, by IP injection. Animals were killed at day 6, and the jejunum was removed and snap-frozen. Cryosections were stained afterwards with (A, C, and E) anti-CD4 or (B, D, and F) anti-CD8. Gastroenterology 1998 115, 1205-1215DOI: (10.1016/S0016-5085(98)70092-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions