Volume 125, Issue 5, Pages 1341-1354 (November 2003) Reduced migration of fibroblasts in inflammatory bowel disease: role of inflammatory mediators and focal adhesion kinase1   Sandra Nicole Leeb, Daniela Vogl, Manuela Gunckel, Stephan Kiessling, Werner Falk, Michael Göke, Jürgen Schölmerich, Cornelia Maria Gelbmann, Gerhard Rogler  Gastroenterology  Volume 125, Issue 5, Pages 1341-1354 (November 2003) DOI: 10.1016/j.gastro.2003.07.004

Figure 1 Immunocytochemical characterization of CLPF. The first column shows stainings of control-CLPF, the second column shows stainings of CD-CLPF, and the third column shows stainings of UC-CLPF. All cultures showed a positive and homogeneous red staining with anti-fibroblast antibody ASO2 (row A), vimentin (row B), and vinculin (row C). Smooth muscle actin (row D), myosin (row E), and desmin (F) were also found in the investigated CLPF cultures but not ubiquitously. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 2 Reduced migration of CD- and UC-CLPF. The migration was induced with 24 hours (A) or 48 hours (B) conditioned media of control-, CD-, or UC-CLPF. CD-CLPF (shaded bars) and UC-CLPF (solid bars) showed a reduced migratory response toward conditioned media when compared with control-CLPF (open bars). Statistics: (A) ∗∗Mann-Whitney rank sum test, P < 0.0001; ∗t test, P = 0.0005; #t test, P = 0.006; §t test, P = 0.002. (B) ∗∗Mann-Whitney rank sum test, P < 0.0001; ∗t test, P = 0.0004; #Mann-Whitney rank sum test, P = 0.05; §Mann-Whitney rank sum test, P = 0.02. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 3 Inhibition of the migration of control-CLPF by IFN-γ and TNF. Twenty-four-hour-conditioned medium of CLPF with additional IFN-γ (A) or TNF (B) in the indicated concentrations was used for migration assays with CLPF in the modified Boyden chamber. Migration induced with conditioned medium without IFN-γ or TNF addition was used as control. The migration is given as the percentage of the control: (A) 100% = 62 CLPF/hpf; (B) 100% = 50 CLPF/hpf. Statistics: (A) 20 ng/mL IFN-γ Mann-Whitney rank sum test, P = 0.0341; 30 ng/mL IFN-γ t test, P = 0.0018; 40 ng/mL IFN-γ t test, P = 0.0006; 50 ng/mL IFN-γ Mann-Whitney rank sum test, P < 0.0001. (B): 10 ng/mL TNF t test, P = 0.0455; 20 ng/mL TNF t test, P = 0.0137; 30 ng/mL TNF t test, P = 0.0088. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 4 Reduced migration of control-CLPF after preincubation with IFN-γ and TNF. CLPF were incubated for 3 days with IFN-γ (10 ng/mL) or TNF (20 ng/mL) or with both cytokines simultaneously (A and B). Furthermore, CLPF were incubated for 3 days with IFN-γ (10 ng/mL) or TNF (20 ng/mL) or with both cytokines simultaneously and cultured for another 7 days without any cytokine (C and D). Subsequently, it was investigated whether 24 hours (solid bars) and 48 hours (open bars) conditioned media of control-CLPF (A and C) or 24 hours (solid bars) and 48 hours (open bars) conditioned media of CD-CLPF (B and D) induce migration in these cells. Statistics: (A) ∗Wilcoxon signed rank test, P = 0.0005; ∗∗paired t test, P < 0.0001. (B): #Mann-Whitney rank sum test, P = 0.06; §Wilcoxon signed rank test, P = 0.009; ∗∗paired t test, P < 0.0001; ∗paired t test, P < 0.0006. (C): #Wilcoxon signed rank test, P = 0.05; ∗Wilcoxon signed rank test, P = 0.004; §paired t test, P = 0.013; ∗∗paired t test, P < 0.0001. (D) #paired t test, P = 0.002; §paired t test, P = 0.001; ∗paired t test, P = 0.003; ##paired t test, P = 0.0003; ∗∗paired t test, P = 0.0002. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 5 Cell viability analyses. XTT-based colorimetric analyses for quantification of cell viability of control-CLPF incubated for 3 days with IFN-γ (10 ng/mL) or TNF (20 ng/mL) or with both cytokines simultaneously and of CD-CLPF and UC-CLPF cultured in parallel (A) and of cytokine-treated control-CLPF cultured for another 7 days without any cytokine and of CD-CLPF and UC-CLPF cultured in parallel (B). IFN-γ/TNF-incubated control-CLPF, CD-, and UC-CLPF showed no decreased cell viability. CLPF of 3 different control, CD and UC patients were used in the experiments. Statistics: (A): #Paired t test, P = 0.055; ∗Wilcoxon signed rank test, P = 0.02. (B) ∗Paired t test, P = 0.03. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 6 Cell-cycle analyses. The percentage of control-CLPF and cytokine-incubated control-CLPF, CD-, and UC-CLPF in the S-phase was determined by FACS. (A) Percentage of cells in the S-phase of control-CLPF incubated for 3 days with IFN-γ (10 ng/mL) or TNF (20 ng/mL) or with both cytokines simultaneously and of CD-CLPF and UC-CLPF cultured in parallel. (B) Shows percentage of cells in the S-phase of cytokine-treated control-CLPF cultured for another 7 days without any cytokine and of CD-CLPF and UC-CLPF cultured in parallel. CLPF of 4 different control and 3 different CD and UC patients were used in the experiments. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 7 Decrease of FAK protein and FAK phosphorylation in migrating CD- and UC-CLPF. Cells were lysed 4 hours and 8 hours after the induction of migration by wounding a confluent CLPF monolayer. Resting cells (0 hours) were lysed and used as control. FAK protein and its autophosphorylation were determined by Western blotting. (A) Shows exemplary blots for CLPF of one control, Crohn’s disease, or ulcerative colitis patient. The quantitative analyses of all blots were standardized to β-actin and are given as the percentage of the 0 hours time point. (B) Shows total FAK protein and (C) FAK autophosphorylation at tyrosine 397. CLPF of 3 different control and CD patients and 2 different UC patients were used in the experiments. Data were not statistically significant. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 8 Regulation of FAK protein and FAK phosphorylation in migrating cytokine-treated control-CLPF. Control-CLPF were preincubated for 3 days with IFN-γ (10 ng/mL) or TNF (20 ng/mL) or with both cytokines simultaneously. Cells were lysed 4 hours and 8 hours after the induction of migration by wounding a confluent CLPF monolayer. Resting cells (0 hours) were lysed and used as control. Pretreatment with IFN-γ and TNF induced a reduction of FAK protein and phosphorylation in control-CLPF. One exemplary Western blot for each assay group is shown in A. The quantitative analyses of all blots were standardized to β-actin and are given as the percentage of the 0 hours time point. (B) Shows total FAK protein and (C) FAK autophosphorylation at tyrosin 397. CLPF of 3 different control, CD, and UC patients were used in the experiments. Statistics: (C) ∗Zero hours time point of TNF-treated CLPF vs. 8 hours time point of TNF-treated CLPF, Student t test, P = 0.0021. Not indicated in the Figure: zero hours time point of untreated CLPF vs. 0 hours time point of TNF-treated CLPF; Student t test, P = 0.0372. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 9 Regulation of FAK protein and FAK phosphorylation in migrating control-CLPF exposed for 3 days to IFN-γ and TNF and subsequent culture for another 7 days under normal conditions without IFN-γ and TNF supplementation. Cells were lysed 4 hours and 8 hours after the induction of migration by wounding a confluent CLPF monolayer. Resting cells (0 hours) were lysed and used as control. Pretreatment with both IFN-γ and TNF induced a reduction of FAK protein and phosphorylation in control-CLPF. One exemplary Western blot for each assay group is shown in A, and, again, the quantitative analyses of all blots were standardized to β-actin and are given as the percentage of the 0 hours time point. (B) Shows total FAK protein and (C) FAK autophosphorylation at tyrosine 397. CLPF of 3 different control, CD, and UC patients were used in the experiments. Data were not statistically significant. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)

Figure 10 FAK mRNA expression was determined by Northern blotting. The migration of CLPF was induced by wounding a confluent CLPF monolayer, and mRNA was isolated 4 hours and 8 hours after the induction of migration. Resting cells (0 hours) were lysed and used as control. No differences in FAK mRNA expression could be observed. CLPF mRNA (lanes 10 and 11) was investigated because the FAK bands were of similar size as the 28S bands. Therefore, mRNA was assayed together with the RNA probes to exclude a confusion of the FAK bands with the 28S bands. Gastroenterology 2003 125, 1341-1354DOI: (10.1016/j.gastro.2003.07.004)