Dani-Louise Bryan

Cytokines (IL-1β, IL-6, TNF-α, TGF-β1, and TGF-β2) and prostaglandin E2 in human milk during the first three months postpartum

Postpartum changes in the concentrations of IL-1β, IL-6 tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), TGF-β2, and prostaglandin E2 in 257 human milk samples collected longitudinally from 49 healthy mothers during the first 12 wk of lactation were determined by ELISA or RIA. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were present in only a proportion of samples, and there was a wide range of concentrations detected at each time in the present study (IL-1β, <15-400 pg/mL; IL-6, <15-1032 pg/mL; TNF-α, <15-2933 pg/mL). Concentrations of prostaglandin E2 increased after the first week and remained elevated for the remainder of the study (range, <10-9966 pg/mL). The antiinflammatory cytokines TGF-β1 (range, 43-7108 pg/mL) and TGF-β2 (range, 208-57935 pg/mL) were present in substantial quantities in all samples, and there was little change in the mean concentration during 12 wk of lactation. The present study shows that immunomodulating agents are normally present in human milk in physiologically relevant quantities for at least the first 3 mo of the breast-fed infants life.

Interleukin-12 in Human Milk

The presence of interleukin-12 (IL-12) in 39 samples of human milk was investigated using the enzyme-linked immunosorbent assay. IL-12 (>40 pg/mL) was detected in 24 of the 39 samples collected (1408 ± 2256 pg/mL, mean ± SD, n = 24). A range of concentrations of IL-12 was observed in colostrum, transitional, and mature milk, with an apparent decrease in the mean concentration over time postpartum.

Immunomodulatory constituents of human milk change in response to infant bronchiolitis.

Although epidemiological evidence is generally supportive of a causal association between respiratory syncytial virus (RSV) bronchiolitis during infancy and the development of persistent wheeze/asthma, if not allergy, the mechanism by which this occurs and an explanation for why all children do not succumb remains to be elucidated. Breast feeding has been found to confer a protective effect against respiratory infections such as RSV bronchiolitis and allergy; however, again there is little direct evidence and no clear mechanism. In this study, we examined whether human milk immunomodulatory factors (cells, cytokines) change in response to clinically diagnosed, severe bronchiolitis in the recipient breast‐fed infant. We examined milk from 36 breast feeding mothers of infants hospitalized with bronchiolitis and compared them with milk from 63 mothers of postpartum age‐matched healthy controls. Milks from mothers of infants hospitalized with bronchiolitis had significantly greater numbers of viable cells when compared with the milks obtained from mothers of healthy infants (1.3 ± 0.4 vs. 0.3 ± 0.03 × 106 cells/ml, mean ± s.e.m.; p ≤ 0.001). Further, the cells obtained from the mothers of infants hospitalized with bronchiolitis were found to produce a skewed cytokine profile ex vivo in response to stimulation by live RSV but not when cultured with a non‐specific mitogen (concanavalin A). This study provides preliminary evidence for an immunological link between mothers and their breast‐fed infants during severe respiratory infections as well as a possible contributing factor to the development of persistent wheeze in these infants.

Cytokine Production by Human Milk Cells and Peripheral Blood Mononuclear Cells from the Same Mothers

Samples of milk (n = 80) and venous blood were collected at 5 weeks postpartum from 82 lactating mothers. Human milk cells and peripheral blood mononuclear cells were isolated and the production of interleukin-1β, interleukin-6, and tumor necrosis factor-α in the absence and presence of lipopolysaccharide was determined by enzyme-linked immunosorbent assay. Human milk cells spontaneously produced significantly less interleukin-1β, interleukin-6, and tumor necrosis factor-α than peripheral blood mononuclear cells in the absence of stimulation. In vitro stimulation of human milk cells with lipopolysaccharide (500 ng/ml) for 24 hr increased cytokine production by approximately 40–50%, whereas peripheral blood mononuclear cells responded to lipopolysaccharide (200 ng/ml) with increased cytokine production of up to 350%. These observations suggest that cells in milk are capable of active involvement in the production of the interleukin-1β, interleukin-6, and tumor necrosis factor-α in the mammary gland and have the capacity to respond to further stimulation after leaving the breast.

Transforming growth factor beta in human milk does not change in response to modest intakes of docosahexaenoic acid.

Long-chain polyunsaturated fatty acids have been associated with aspects of immune regulation including cytokine production. The purpose of this study was to investigate the effect of maternal dietary supplementation with tuna oil, rich in docosahexaenoic acid (DHA), on the concentration of transforming growth factor beta 1 (TGFβ1) and TGFβ2 in breast milk. In this randomized, dietary intervention trial, mothers of term infants consumed a daily supplement of 2000 mg oil containing either placebo (n=40), 300 mg DHA (n=40), or 600 mg DHA (n=40). The DHA increase in milk and plasma was proportional to dietary DHA. There was no relationship between milk DHA status and TGFβ1 and TGFβ2 levels.

Modulation of respiratory syncytial virus-induced prostaglandin E2 production by n-3 long-chain polyunsaturated fatty acids in human respiratory epithelium

Infection with respiratory syncytial virus (RSV) results in substantial infant morbidity and has been associated with the subsequent development of childhood asthma. Inflammatory mediators produced by both the epithelium and tissue leukocytes during RSV infection stimulate the release of chemotactic factors by the respiratory epithelium and the subsequent influx of inflammatory cells, predominantly neutrophils. We investigated the production of inflammatory mediators [prostaglandin E2 (PGE2), interleukin (IL)-1β, tumor necrosis factor α] and chemokines [IL-8, RANTES (regulation on activation, normal T cell expressed and secreted)] by alveolar epithelial cells in response to RSV infection. Infection of a human alveolar epithelial transformed cell line (A549 cells) with live RSV substantially increased production of PGE2, IL-8, and RANTES. By altering cell membrane FA through incorporation of the long-chain PUFA (LCPUFA) arachidonic acid, EPA, and DHA, we were subsequently able to significantly modulate PGE2 production by the infected epithelium. Because of the dynamic nature of the effects of PGE2 on lung function, regulation of this prostaglandin during RSV infection by n−3 LCPUFA has the potential to significantly alter the disease process.

Polyunsaturated fatty acids regulate cytokine and prostaglandin E2 production by respiratory cells in response to mast cell mediators

A protective association between breastfeeding and the development of bronchial asthma has been demonstrated. However, a mechanism remains unclear. FA present in human milk but rare in infant formula have been associated with marked immunological modulation as well as some indications of protection from asthma development. We examined the effect of in vitro manipulation of membrane phospholipid on the production of cytokines and prostaglandin (PG)E2 by respiratory epithelial cells (A549) in response to stimulation by mast cell mediators of allergic disease [histamine, tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-5]. DHA and CLA significantly decreased the production of IL-8 in response to stimulation by TNF-α [2907±970 (DHA) and 6471±1203 (CLA) vs. 12,287±2309 (control) pg/ml; P≤0.05, mean±SEM], whereas both EPA and DHA reduced histamine-stimulated RANTES (regulation on activation, T cell-expressed and-secreted) production [2314±861 (EPA) and 877±326 (DHA) vs. 8526±1118 (control) pg/ml; P≤0.03]. PGE2 released in response to histamine was decreased by n−3 [1305±399 (α-linolenic acid), 406±73 (EPA), and 265±32 (DHA) vs. 9324±3672 (control) pg/mL; P≤0.05] and increased by n−6 [18,843±4439 (arachidonic acid) vs. 9324±3672 (control) pg/mL; P=0.02], with CLA producing a decrease of the same magnitude as DHA [553±126 (CLA) vs. 9324±3672 (control) pg/mL; P=0.03]. This study demonstrates the potential for immunological manipulation of the respiratory epithelium by FA in situ during allergic responses and suggests that further investigation into FA intervention in infants via human milk or supplemented infant formula, to prevent the development of allergic disease, may be worthwhile.

Incorporation of α-linolenic acid and linoleic acid into human respiratory epithelial cell lines

Animal and human studies designed to examine the effects of α-linolenic acid (ALA) and linoleic acid (LA) supplementation on the fatty acid composition of plasma and tissues have demonstrated a marked difference in incorporation into phospholipids of these 18-carbon precursors of the long-chain polyunsaturates. Whereas tissue phospholipid levels are linearly related to dietary ALA and LA, the levels of tissue LA can be 10-fold higher than tissue ALA even when dietary levels are equivalent. There is some dispute whether this disparity is due to ALA being more rapidly metabolized to its products or substantially oxidized by the liver, or whether LA but not ALA is readily incorporated into cellular phospholipids. We examined the level of incorporation of polyunsaturated fatty acids into human respiratory epithelial cell lines (A549, 16HBE) by determining the dose-dependent incorporation of ALA and LA as free fatty acid (5–150 μg FFA/mL). Cell membrane phospholipid ALA and LA were both increased up to ∼20–30% total fatty acids, with a concomitant decrease predominantly in monounsaturated membrane fatty acids, before significant toxicity was observed (50 μg/mL). Our data support the concept that rather than any inherent inability by human cells to incorporate ALA into membrane phospholipids, the lack of ALA content in human and animal tissues in vivo is due to the rapid metabolism or oxidation of this fatty acid in the liver.

Cells from Mature Human Milk are Capable of Cytokine Production Following in Vitro Stimulation

Leukocytes in milk may contribute to development of mucosal defense mechanisms in the recipient infant (Hanson 1999). The principal types of leukocytes found in human milk are granulocytes, monocytes/macrophages, and lymphocytes. Each of these cell types could be responsible for production of a variety of cytokines, and a number of investigators have examined this possibility using cells isolated from colostrum (Rudloff et al. 1992; Subiza et al. 1988; Skansen-Saphir et al. 1993). In contrast, little information is available regarding cytokine production by cells obtained from mature milk or the relationship between milk cell and peripheral blood cell cytokine production. The aim of this study was to determine the capacity of a mixed population of human milk cells (HMC) to produce the potentially pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and compare the cytokine profile with that obtained from peripheral blood mononuclear cells (PBMC) collected from the same mothers.

The Effect of Dietary DHA Supplementation on Human Milk Cytokines

Some expert nutrition committees have recommended LCPUFA supplementation for lactating women. As the n-3 PUFA have been recognized as regulatory agents for cytokine and eicosanoid production it is important to define the effect such dietary changes have on the immunomodulating potential of human milk The aim of this study was to examine the effects of dietary docosahexaenoic acid (DHA) supplementation on the human milk cytokines IL-6, TNFα, TGFβ1 and TGFβ2.