Abstract
We used column chromatography, affinity binding, and bioassay methods to address whether the soluble tumor necrosis factor (TNF)-α receptors present in human colostrum and milk bind to and modify TNF-α bioactivity. In gel chromatography experiments, soluble TNF-α receptor I (sTNFRI) and sTNFRII in human colostrum sequentially increased their molecular sizes from 49 kD to 71 kD and 60 kD, respectively, after addition of increasing molar excesses of recombinant TNF-α. Application of colostrum to a TNF-α affinity matrix followed by washing and elution resulted in 2925-fold enrichment of sTNFRI, consistent with sTNFRI binding to the TNF-α affinity matrix. In other samples of colostrum and milk, the content of both sTNFRI and sTNFRII decreased significantly after passage over the matrix, but the material eluted from the matrix lost the ability to rebind to the TNF-α and was not active in a WEHI-13var bioassay for TNF-α. Specimens of human colostrum and milk diluted 1:16 shifted the LD50 for TNF-α 4-fold in this bioassay, and milk protection of WEHI-13var cells against TNF-α was significantly diminished after passage down the TNF-α affinity 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-sTNFRI. Addition of this fraction to the WEHI-13var bioassay reversed the effects of 10 pg/mL TNF-α in the assay. These data demonstrate that sTNFRI and II from human colostrum and milk bind to TNF-α, that both colostrum and milk interfere with the bioactivity of TNF-α, and that affinity-purified sTNFRI from human milk blocks the bioactivity of TNF-α. These effects may contribute to the anti-inflammatory character of human colostrum and milk.
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Abbreviations
- TNF :
-
tumor necrosis factor
- sTNFR :
-
soluble TNF-α receptor
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Acknowledgements
The authors thank Iwona Malinowska, M.D., for initial work on this project, Rosalee Hewitt, R.N., and Patti Lundy, R.N., for significant efforts collecting specimens of human colostrum and milk, and Medela, Inc., for the use of the portable electric breast pump so critical to the performance of these studies.
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Supported by National Institutes of Health NICHD Grant HD 13021-18.
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Buescher, E., McWilliams-Koeppen, P. Soluble Tumor Necrosis Factor-α (TNF-α) Receptors in Human Colostrum and Milk Bind to TNF-α and Neutralize TNF-α Bioactivity. Pediatr Res 44, 37–42 (1998). https://doi.org/10.1203/00006450-199807000-00006
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DOI: https://doi.org/10.1203/00006450-199807000-00006
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