title: Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.
abstract: 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.
  
  
  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-α). ELISA detected 0.697±0.03 ng mL-1TNF-α in M1-CM. Recombinant mouse TNF-α reduced Kit expression and ICC numbers in a concentration-dependent manner (EC50 = 0.817 ng mL-1). Blocking M1-CM TNF-α 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
  This work demonstrates that TNF-α 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.
authors: Enteric NeuroScience Program, Mayo Clinic, Rochester, MN, USA.
authors: S T
authors: ST
authors: Eisenman
authors: Enteric NeuroScience Program, Mayo Clinic, Rochester, MN, USA.
authors: S J
authors: SJ
authors: Gibbons
authors: Enteric NeuroScience Program, Mayo Clinic, Rochester, MN, USA.
authors: P-J
authors: PJ
authors: Verhulst
authors: Enteric NeuroScience Program, Mayo Clinic, Rochester, MN, USA.
authors: G
authors: G
authors: Cipriani
authors: Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, München, Germany.
authors: D
authors: D
authors: Saur
authors: Enteric NeuroScience Program, Mayo Clinic, Rochester, MN, USA.
authors: G
authors: G
authors: Farrugia
mesh_terms: D000818:Animals; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D055879:Gene Knock-In Techniques; D056885:Interstitial Cells of Cajal / Q000187:drug effects / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D014409:Tumor Necrosis Factor-alpha
keywords: caspase-mediated apoptosis; cell death; cytokines; diabetic gastroparesis; gastrointestinal motility