Chemokinetic Agents for Monocytes in Human Milk: Possible Role of Tumor Necrosis Factor-α

Abstract

ABSTRACT. Human milk was found to contain chemokinetic agents for human blood monocytes. The chemokinetic agents were whey proteins that were inactivated by heating at 56" C for 20 min or treatment with trypsin. Three peaks of chemokinetic activity less than 60 kD in size were found by gel filtration chromatography. The chemokinetic activity of each peak obtained by gel filtration was partially blocked by polyclonal rabbit antibodies to recombinant human tumor necrosis factor-a (TNF-a). TNF-a, or a protein that immunologically cross-reacted with it, was also detected in human milk by blockage of the cytotoxicity of human milk by anti-TNF-a. Such proteins or others that elicit the release of TNF-a from the target cells may be responsible for the enhanced motility of human milk macrophages, and it is possible that they may alter the immunologic or metabolic activities of the alimentary tract of the recipient infant. (Pediatu Res 25:629-633) Abbreviations mammary gland or that blood monocytes are modified in the mammary gland or by factors in human milk. Additional experimental observations supported those possibilities. In contrast to the lack of movement of neutrophils in human milk (12)(13)(14), it was discovered that HMM spontaneously moved more rapidly than blood monocytes in a three-dimensional, type I collagen matrix (1 5).In this study, the genesis of the increased motility of human milk macrophages was investigated. We tested whether circulating systemic factors produced early in lactation, mammary gland tissue factors, or constituents in human milk up-regulated the motility of blood monocytes. The investigations revealed that human milk contains chemokinetic agents for blood monocytes and that one of those chemokinetic agents may be TNF-a or a protein that immunologically cross-reacts with it. MATERIALS AND METHODSResearch subjects-leukocyte preparations. The use of human subjects in this iesearch was ~p p~o v e d by the Institutional Review HML, human milk leukocytes HMM, human milk macrophages Board of the Medical School. Donors were healthy adults or MEME, minimum essential medium, Eagle-modified healthy women who delivered full-term, normal infants vaginally PBML, peripheral blood mononuclear leukocytes 2 to 3 days before the collections (16). Milk was collected into sterile polypropylene tubes by a low pressure electric pump TNF-a, tumor necrosis factor-a (Egnell, Inc., Cary, IL). Unfractionated HML were prepared as follows. Human colostrum was diluted 1 :4 in RMPI 1640 (Whittaker M.A. Bioproducts, Walkersville, MD) and centrifuged at 4000 x g for 10 min at 4" C. The cellular pellets were then Neutrophils, macrophages, and lymphocytes are found in hu-resuspended in RPMI 1640 to achieve a final concentration of 1 man milk during the first few months of lactation and the X lo7 leukocytes/ml. Heparinized venous blood was subjected numbers of macrophages in milk are particularly high as com-to Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) density pared to the numbers of monocytes in human blood (1, ...

Activated and memory T lymphocytes in human milk

“…These factors proved to be proteins whose approximate M r as determined by gel filtration were 50-60, 25, and 10-17 kDa 1311. The chemokinetic activities in HM were partially blocked by polyclonal antibodies to human recombinant tumor necrosis factor-a (TNF-a) [31]. Since then, we have found immunochemical evidence for TNF-a in HM 1361.…”

“…Recently we reported that chemokinetic agents for monocytes are present in HM and that the increased motility of HM macrophages may be due to these agents [31]. These factors proved to be proteins whose approximate M r as determined by gel filtration were 50-60, 25, and 10-17 kDa 1311.…”

Tumor Necrosis Factor-α in Human Milk

“…When blood mononuclear cells were incubated in whole human colostrum or its whey protein fraction, the motility of the blood monocytes increased to that of milk macrophages (8). The activity was abrogated by trypsin and considerably decreased by polyclonal antibodies to rhTNFa.…”

“…The RNA was denatured in a loading buffer containing Sephacryl S-200 gel filtration column (Pharmacia LKB Biotech-formamide and formaldehyde. The concentration of RNA was nology, Piscataway, NJ) (8). The resultant fractions were concen-estimated spectrophotometrically at 260 nm.…”

“…The chemokinetic activity peaks in human milk were blocked by antibodies to rhTNF-a. In addition, the studies suggested that some of the limited cytotoxic activity of human milk against murine L-929 cells was blocked by antibodies to rhTNF-a (8).…”