Tumor necrosis factor alpha derived from classically activated M1 macrophages reduces interstitial cell of Cajal numbers

Background: Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal (ICC) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Methods: Cultures of gastric ICC were treated with conditioned medium (CM) from activated bone marrow-derived macrophages (BMDMs) and the effect of CM was quantified by counting ICC per high-powered field. Key Results: Bone marrow-derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT-PCR. Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2-CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1-CM, including tumor necrosis factor alpha (TNF-alpha). ELISA detected 0.697 +/- 0.03 ng mL(-1) TNF-alpha in M1-CM. Recombinant mouse TNF-alpha reduced Kit expression and ICC numbers in a concentration-dependent manner (EC50 = 0.817 ng mL(-1)). Blocking M1-CM TNF-alpha with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z-VAD. fmk partly preserved ICC numbers (cells/field; 6.63 +/- 1.04, 9.82 +/- 1.80 w/Z-VAD. fmk, n=6, P<.05). Conclusions & Inferences: This work demonstrates that TNF-alpha secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase-dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis.