Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury

Abstract

Background: Administration of granulocyte-macrophage colony stimulating factor (GM-CSF) relieves symptoms in Crohn's disease (CD). It has been reported that reduced GM-CSF bioactivity is associated with more aggressive ileal behaviour and that GM-CSF-null mice exhibit ileal barrier dysfunction and develop a transmural ileitis following exposure to non-steroidal anti-inflammatory drugs (NSAIDs). STAT5 signalling is central to GM-CSF action. It was therefore hypothesised that GM-CSF signalling in non-haematopoietic cells is required for ileal homeostasis.

Methods: Bone marrow (BM) chimeras were generated by reconstituting irradiated GM-CSF receptor (gm-csfr) beta chain or GM-CSF (gm-csf) deficient mice with wild type BM (WTBM-->GMRKO and WTBM-->GMKO). Intestinal barrier function and the response to NSAID-induced ileal injury were examined. Expression of gm-csf, gm-csfr or stat5 in Caco-2 and HT-29 intestinal epithelial cell (IEC) lines was knocked down and the effect of GM-CSF signalling on IEC survival and proliferation was determined.

Results: Elevated levels of GM-CSF autoantibodies in ileal CD were found to be associated with dysregulation of IEC survival and proliferation. GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras exhibited ileal hyperpermeability. NSAID exposure induced a transmural ileitis in GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras. Transplantation of wild type BM into GM-CSF-deficient mice prevented NSAID ileal injury and restored ileal barrier function. Ileal crypt IEC proliferation was reduced in WTBM-->GMRKO chimeras, while STAT5 activation in ileal IEC following NSAID exposure was abrogated in WTBM-->GMRKO chimeras. Following knock down of gm-csf, gm-csfr alpha or beta chain or stat5a/b expression in Caco-2 cells, basal proliferation was suppressed. GM-CSF normalised proliferation of Caco-2 cells exposed to NSAID, which was blocked by stat5a/b RNA interference.

Conclusions: Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury; furthermore, GM-CSF signalling in non-haematopoietic cells regulates ileal epithelial homeostasis via the STAT5 pathway. The therapeutic use of GM-CSF may therefore be beneficial in chronic ileitis associated with CD.

Figure 1

Increased levels of granulocyte-macrophage colony stimulating factor (GM-CSF) autoantibody (Ab) in Crohn's disease (CD) are associated with dysregulation of survival and proliferation of intestinal epithelial cells. (A) The frequency of apoptotic ileal crypt epithelial cells was determined by cleaved caspase-3 staining in healthy controls (CON) and patients with CD with high (Hi) or low (Lo) serum GM-CSF Ab. Results are expressed as mean±SEM (n=7); original magnification ×200; bar=50 μm. (B) Frequency of proliferative ileal crypt epithelial cells was determined by Ki67 staining in healthy controls and patients with CD with high or low serum GM-CSF Ab. Results are expressed as mean±SEM (n=7); original magnification ×400; bar=50 μm.

Figure 2

Loss of granulocyte-macrophage colony stimulating factor (GM-CSF) signalling in non-haematopoietic cells increases susceptibility to non-steroidal anti-inflammatory drug (NSAID)-induced ileitis. (A–C) WT, GMKO, GMRKO, WTBM, WTBM→GMKO, GMRBM→WT and WTBM→GMRKO mice received piroxicam (PIR) in the chow for 2 weeks and the effects on ileal histopathology and ulceration were determined. Original magnification 3100, bar=50 mm. Open circles represent the ulcer area. *p<0.01 vs WTBM; #p<0.01 vs GMKO (n=10). (D) Bacterial translocation in mesenteric lymph nodes (MLN) was determined in WT, WTBM, GMKO, GMRKO, WTBM→GMKO, GMRBM→WT and WTBM→GMRKO mice with and without piroxicam administration. *p<0.05 vs non-PIR WT mice; #p<0.05 vs non-PIR WTBM and WTBM→GMKO mice (n=10). Results are expressed as mean±SEM.

Figure 3

Loss of granulocyte-macrophage colony stimulating factor signalling in non-haematopoietic cells leads to intestinal barrier dysfunction. (A) Ileal paracellular permeability was determined with everted gut sac in wild type (WT), WTBM, GMKO, WTBM→GMRKO and WTBM→GMKO mice (n=10). (B) Ileal epithelial electric resistance (TEER) was determined by clamping the voltage across the epithelium layer mounted in Ussing chambers (n=5). Results are expressed as mean±SEM.

Figure 4

Granulocyte-macrophage colony stimulating factor (GM-CSF) is required for intestinal epithelial survival response to injury. (A) TUNEL and cleaved caspase-3 immunohistochemistry staining were performed on ileal sections from WT, GMKO, WTBM, WTBM→GMKO and WTBM→GMRKO mice under basal conditions and following exposure to piroxicam (PIR). Average apoptotic intestinal epithelial cells (IEC) per crypt was determined (original magnification 3400, bar=50 mm). Ileal epithelial cells were isolated with 1 mM EDTA from GMKO, WTBM, WTBM→GMKO and WTBM→GMRKO mice with and without piroxicam treatment. (B, C) IEC apoptosis was determined by cleaved caspase-3 western blot analysis (n=7). (D) The frequency of apoptotic IEC was determined as the 7-AAD-, CD3-, Annexin V+ population (n=7). (E) Bax and Bcl-2 expression were examined by western blot analysis (n=7). Signal intensity was determined by densitometry. Results shown as mean±SEM. NSAID, non-steroidal anti-inflammatory drug.

Figure 4

Granulocyte-macrophage colony stimulating factor (GM-CSF) is required for intestinal epithelial survival response to injury. (A) TUNEL and cleaved caspase-3 immunohistochemistry staining were performed on ileal sections from WT, GMKO, WTBM, WTBM→GMKO and WTBM→GMRKO mice under basal conditions and following exposure to piroxicam (PIR). Average apoptotic intestinal epithelial cells (IEC) per crypt was determined (original magnification 3400, bar=50 mm). Ileal epithelial cells were isolated with 1 mM EDTA from GMKO, WTBM, WTBM→GMKO and WTBM→GMRKO mice with and without piroxicam treatment. (B, C) IEC apoptosis was determined by cleaved caspase-3 western blot analysis (n=7). (D) The frequency of apoptotic IEC was determined as the 7-AAD-, CD3-, Annexin V+ population (n=7). (E) Bax and Bcl-2 expression were examined by western blot analysis (n=7). Signal intensity was determined by densitometry. Results shown as mean±SEM. NSAID, non-steroidal anti-inflammatory drug.

Figure 5

Granulocyte-macrophage colony stimulating factor (GM-CSF) signaling in non-haematopoietic cells regulates cellular proliferation. (A) Proliferation of ileal intestinal epithelial cells (IEC) was determined using BrdU incorporation measured by immunohistochemistry staining (n=7 mice, ≥200 crypts counted per group). Original magnification ×400, bar=50 μm. (B) Ileal IEC were isolated with 1 mM EDTA from WTBM and WTBM→GMRKO mice with and without piroxicam (PIR) treatment, cyclin D1 and p21 were determined by western blot analysis (n=5). Results shown as mean±SEM. NSAID, non-steroidal anti-inflammatory drug.

Figure 6

Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates survival and proliferation of intestinal epithelial cells in vitro. (A) Subconfluent Caco-2 and HT-29 cells were incubated with rhGM-CSF (1 ng/ml) and indomethacin (IN, 10^-4 M) for 24 h, cell proliferation was measured using BrdU incorporation. *p<0.01 vs control (CON) group; #p<0.01 vs IN group (n=10). (B, C) gm-csf siRNA was used to knock down the expression of GM-CSF in subconfluent Caco-2 cells in the absence and presence of rhGM-CSF (1 ng/ml) administration for 24 h. Proliferation of Caco-2 cells was detected by BrdU incorporation and apoptosis of Caco-2 cells by cleaved caspase-3 western blot analysis. Control (CON) group: transfection reagent without siRNA; *p<0.05 vs control or GM-CSF group (n=10). (D) Proliferation of Caco-2 cells was detected under gm-csfαc or βc knockdown. *p<0.01 vs control group (n=10). Percentage of knock-down was determined by western blot analysis. (E) Cyclin D1 and p21 were determined by western blot analysis in subconfluent Caco-2 and HT-29 cells with and without gm-csfrβc knock-down. (F) After 24 h tumour necrosis factor α (TNFα) (10 ng/ml) administration, cleaved caspase-3 was measured by western blot analysis in subconfluent Caco-2 cells with and without gm-csfrβc knock-down. Results representative of five independent experiments are shown. BrdU incorporation results are shown as mean±SEM.

Figure 7

Granulocyte-macrophage colony stimulating factor (GM-CSF) activates STAT5 in intestinal epithelial cells (IEC) in response to injury. (A) WTBM and WTBM→GMRKO mice received piroxicam (PIR) in the chow for 2 weeks and STAT5 activation was examined by immunohistochemistry staining (original magnification 3100, bar=50 mm, inset is negative control). Ileal epithelial cells in WTBM, WTBM→GMKO and WTBM→GMRKO mice with and without proxicam treatment were isolated. (B) pSTAT5 and STAT5 in nuclear proteins (NE) was determined by western blot analysis. (C, D) Ileal IEC were isolated from WT mice and then stimulated with rmGM-CSF (1 ng/ml) for 30 min; pSTAT5 in NE and Bcl-2 in cytosolic protein (CE) were detected by western blot analysis. Signal intensity was determined by densitometry and results are shown as mean±SEM (n=5).

Figure 8

Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates the survival and proliferation of intestinal epithelial cells through activation of STAT5. (A) Subconfluent Caco-2 cells were stimulated with rhGM-CSF (1 ng/ml) for 15, 30 and 60 min and the activation of STAT5 (pSTAT5) was examined in nuclear proteins (NE) by western blot analysis. (B) STAT5, Bid, Bax and cleaved caspse-3 expression were measured by western blot analysis with and without STAT5 a and b siRNA (STAT5 siRNA). Control (CON) group: transfection reagent without siRNA. (C) Caco-2 cells were stimulated by rhGM-CSF (1 ng/ml) with and without STAT5 siRNA and cell proliferation was measured using BrdU incorporation. *p<0.05 vs control group. (D) Cyclin D1 and p21 were identified by western blot analysis with and without STAT5a and b RNAi. Signal intensity was determined by densitometry and is shown as the mean±SEM. Results representative of five independent experiments are shown.