SOLUBLE TUMOR-NECROSIS-FACTOR-ALPHA (TNF-ALPHA) RECEPTORS IN HUMAN COLOSTRUM AND MILK BIND TO TNF-ALPHA AND NEUTRALIZE TNF-ALPHA BIOACTIVITY

We used column chromatography, affinity binding, and bioassay methods to address whether the soluble tumor necrosis factor (TNF)-alpha recep tors present in human colostrum and milk bind to and modify TNF-alpha bioactivity. In gel chromatography experiments, soluble TNF-alpha rece ptor I (sTNFRI) and sTNFRlI in human colostrum sequentially increased their molecular sizes from 49 kD to 71 kD and 60 kD, respectively, aft er addition of increasing molar excesses of recombinant TNF-alpha. App lication of colostrum to a TNF-alpha affinity matrix followed by washi ng and elution resulted in 2925-fold enrichment of sTNFRI, consistent with sTNFRI binding to the TNF-alpha affinity matrix. In other samples of colostrum and milk, the content of both sTNFRI and sTNFRII decreas ed significantly after passage over the matrix, but the material elute d from the matrix lost the ability to rebind to the TNF-alpha and was not active in a WEHI-13var bioassay for TNF-alpha. Specimens of human colostrum and milk diluted 1:16 shifted the LD50 for TNF-alpha 4-fold in this bioassay, and milk protection of WEHI-13var cells against TNF- alpha was significantly diminished after passage down the TNF-alpha af finity matrix (p < 0.001). Affinity purification of milk sTNFRI using polyclonal anti-sTNFRI produced fractions containing proteins of 30 kD , which could be visualized by Western blot using polyclonal anti-sTNF RI. Addition of this fraction to the WEHI-13var bioassay reversed the effects of 10 pg/mL TNF-alpha in the assay. These data demonstrate tha t sTNFRI and LT from human colostrum and milk bind to TNF-alpha, that both colostrum and mill; interfere with the bioactivity of TNF-alpha, and that affinity-purified sTNFRI from human milk blocks the bioactivi ty of TNF-alpha. These effects may contribute to the anti-inflammatory character of human colostrum and milk.