Professional Documents
Culture Documents
The majority of infants encounter respiratory profile produced by immunoactive cells. These
syncytial virus (RSV) during the first 12 months changes affect cells not only at sites of activation,
of life, often manifesting as severe bronchiolitis such as the respiratory mucosal surfaces, but also
requiring hospitalization. Epidemiological stud- systemically in peripheral blood. The peripheral
ies demonstrate a link between RSV bronchiolitis blood mononuclear cells (PBMC) of infants
in infants and the development of persistent hospitalized with RSV-mediated respiratory tract
wheeze or asthma during childhood (1, 2). infections demonstrate a significantly elevated
However, the mechanism by which this occurs, interleukin (IL)-4 to interferon (IFN)-c ratio
as well as an explanation for why all infants upon mitogen stimulation in vitro (3). Similarly,
contracting RSV bronchiolitis do not progress to the PBMC of atopic adults produce higher levels
asthma has yet to be elucidated. of T helper (Th) 2 cytokines, IL-4 and IL-5, and
Activation of the immune system by infections reduced levels of Th1 cytokines, IL-12 and IFN-c
such as RSV significantly alters the cytokine (3, 4), demonstrating a Th2 bias. This Th2 bias,
495
Bryan et al.
as well as increases in chemokines are also found for viral identification. A separate group of 63
in the aqueous fraction of human milk from postpartum age-matched women who had a
atopic mothers (4–8). healthy breast feeding child also provided a
There is some debate about the protective effect breast milk sample. A series of questions were
of breast feeding against respiratory infections asked relating to socio-economic status, number
such as RSV-mediated bronchiolitis. Although the of children in the household, exclusivity of breast
epidemiological evidence is generally supportive feeding and consumption of any alcohol or
of this concept there are few specific examples of cigarettes (yes/no). Socio-economic status was
protection and the potential underlying mecha- defined through two parameters. The first being a
nisms are unclear (9). Human milk provides scale of highest level of education achieved with a
protection for the recipient infant by the transfer score of 1 being the lowest (less than or equal to
of a number of factors including anti-infectious year 11 of secondary school) and 6 being the
agents such as immunoglobulins, lactoferrin and highest (postgraduate degree). The second meas-
oligosaccharides (7). However, it may be specula- ure ranked occupation according to the scale
ted that long-term benefits must be derived from developed by Daniel (15) which assigns a score
human milk factors capable of modulating the based on power, privilege and prestige of occu-
developing infant immune system. A number of pations in Australia ranging from 1 as the highest
potentially immunomodulating factors have been level (e.g. 1.2 Judge) to 6 as the lowest level (e.g.
described in human milk, including many cyto- 6.0 unemployed). Data relating to maternal
kines and maternally derived human milk cells. illness (yes/no) and self-reported atopic status
Cells derived from human milk produce a [allergic rhinoconjunctivitis, atopic asthma, atop-
number of cytokines ex vivo both constitutively ic dermatitis or food allergy and as well as total
and after stimulation with mitogens (10, 11). As serum immunoglobulin (Ig) E; Imx Total IgE
viable human milk cells have been isolated from Kit; Abbott Laboratories, Abbott Park, IL,
the stools of human milk-fed infants, it can be USA] were used to determine maternal atopic
postulated that the immature digestive system of status. All samples were collected with the
the recipient infant does not digest or disable a approval of the Flinders Clinical Research Ethics
substantial proportion of these cells (12). While Committee of Flinders Medical Centre, South
the source of human milk cells has not been Australia and with the informed consent of each
adequately identified, there is evidence that at of the participants.
least some of the cells have homed directly from
mucosal sites such as the gastrointestinal tract
Collection and processing of human milk samples
following in situ activation (13, 14). As the
cytokines known to be affected by RSV infection Mothers were asked to provide a complete
are also produced by human milk cells, it is expression from one breast at least 2 h after the
reasonable to postulate that these effects may be last feed from that breast, to minimize intra-feed
conferred on the cells delivered to the infant via variation and during the morning to limit diurnal
breast feeding. variation. Samples were expressed using an
In this study, we examined whether human electric pump (Ameda, Hollister, Chicago, IL,
milk cells and cytokines change in response to USA) which collects directly into a sterile infant
clinically diagnosed, severe bronchiolitis in the feeding bottle. The sample was transferred into
breast-fed infant. sterile polypropylene centrifuge tubes (Greiner
Labortechnik GmbH, Frickenhausen, Germany)
and centrifuged at 890 · g for 30 min before the
Materials and methods fat layer was removed. The aqueous fraction was
collected, aliquoted and stored at )80C until
Subjects
analysis by enzyme-linked immunosorbent assay
Thirty-six breast feeding (>80% milk feeds as (ELISA).
breast milk) mothers of term infants (‡37 wk of
gestation) provided a single breast milk sample
Human milk cell culture
while their infant was hospitalized with bron-
chiolitis. Clinical diagnosis of bronchiolitis was The resulting cell pellet was resuspended and
defined as wheezing illness in an infant transferred to a clean tube in phosphate-buffered
( £ 12 months of age) caused by a viral respirat- saline, washed x1, the supernatant discarded and
ory tract infection associated with hyperinflation. the final pellet resuspended in sterile RPMI-1640
Each infant admitted with bronchiolitis under- (2 mm l-glutamine, 10 mm HEPES, 50 U/ml
went a routine nasopharyngeal aspirate (NPA) penicillin, 37.5 U/ml streptomycin, 10%
496
Human milk cells in bronchiolitis
*p £ 0.05.
Bronchiolitic group (n ¼ 22), control group (n ¼ 21).
àBronchiolitic group (n ¼ 20), control group (n ¼ 13).
498
Human milk cells in bronchiolitis
4000 4000
2000
4000 2000
* 1000
2000
50 500
0 0 0
BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY
Fig. 1. Comparison of human milk aqueous phase cytokines (pg/ml) between mothers of bronchiolitic infants (BRONCH,
n ¼ 36) and healthy controls (HEALTHY, n ¼ 63). (a) Th1 cytokines interferon-c and interleukin (IL)-2, (b) Th2 cytokines
IL-4 and IL-10 and (c) chemokines IL-8 and RANTES. Median 10th, 25th, 75th and 90th percentiles plus outliers (•).
Significant differences (p £ 0.05) distinguished by an *.
infants when compared with those of healthy when compared with mothers of healthy infants
controls (Fig. 1a–c). (Fig. 3c).
1000 50,000
200
* 500 25,000
100
0 0 0
BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY
*
200
100
0
BRONCH CONTROL
Fig. 2. Comparison of cytokines (pg/ml) produced by human milk cells stimulated with live respiratory syncytial virus
[multiplicity of infection (MOI) ¼ 3, 24 h] between mothers of bronchiolitic infants (BRONCH, n ¼ 33) and healthy controls
(HEALTHY, n ¼ 31). (a) Th1, interferon (IFN)-c and interleukin (IL)-2, (b) Th2, IL-4 and IL-10, and (c) chemokines, IL-8
and RANTES, (d) Th2 to Th1 ratio, IL-10:IFN-c. Median 10th, 25th, 75th and 90th percentiles plus outliers (•). Significant
differences (p £ 0.05) distinguished by an *.
pg/mL (median, percentiles and outliers)
10,000 50,000
5000
*
0 0 0
BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY BRONCH HEALTHY
Fig. 3. Comparison of cytokines (pg/ml) produced by human milk cells stimulated with concanavalin A (150 lg/ml, 48 h)
between mothers of bronchiolitic infants (BRONCH, n ¼ 36) and healthy controls (HEALTHY, n ¼ 61). (a) Th1, interferon-
c and interleukin (IL)-2, (b) Th2, IL-4 and IL-10 and (c) chemokines, IL-8 and RANTES. Median 10th, 25th, 75th and 90th
percentiles plus outliers (•). Significant differences (p £ 0.05) distinguished by an *.
profile. In addition, this effect is only found with IL-10:IFN-c response. These results suggest that
live RSV, the most common causal agent for there is activation of human milk cells from this
infant bronchilitis, not in response to non-specific group of mothers of infants with bronchiolitis,
activation of the cell population, as stimulation but that this activation differs in response to RSV
with the mitogen ConA did not result in a similar in those mothers currently exposed.
500
Human milk cells in bronchiolitis
No data are available which directly addresses breast milk of atopic and nonatopic women. Allergy
this issue. However, there was no difference 1999: 54: 206–11.
7. Bottcher MF, Jenmalm MC, Garofalo RP, Bjork-
found in the volume of milk obtained from sten B. Cytokines in breast milk from allergic and
complete expressions in each group indicating nonallergic mothers. Pediatr Res 2000: 47: 157–62.
little effect of infant illness on total milk volume. 8. Bottcher MF, Jenmalm MC, Bjorksten B, Garofalo
By selecting infants hospitalized with a speci- RP. Chemoattractant factors in breast milk from aller-
fic, clinically diagnosed condition, we have been gic and nonallergic mothers. Pediatr Res 2000: 47:
able to identify a highly significant change in the 592–7.
9. Oddy WH. A review of the effects of breastfeeding on
milk that is being received by the infant who is ill respiratory infections, atopy, and childhood asthma.
as opposed to a healthy infant. This research J Asthma 2004: 41: 605–21.
provides some evidence for a link between the 10. Jarvinen KM, Laine S, Suomalainen H. Defective
cells found in human milk and illness in the tumour necrosis factor-alpha production in mother’s
recipient infant. The significant alteration in milk is related to cow’s milk allergy in suckling infants.
the cytokines produced by these cells in response Clin Exp Allergy 2000: 30: 637–43.
11. Hawkes JS, Bryan DL, Gibson RA. Cytokine pro-
to secondary exposure to RSV also suggests a duction by human milk cells and peripheral blood
specific immunological outcome resultant from mononuclear cells from the same mothers. J Clin
maternal viral exposure. This work may contrib- Immunol 2002: 22: 338–44.
ute some explanation for the inconsistent reports 12. Michie CA, Tantscher E, Rot A. The long term effects
of protection by human milk feeding against of breastfeeding: a role for the cells in breast milk?
J Trop Pediatr 1998: 44: 2–3.
both RSV bronchiolitis and the development of 13. Roux ME, McWilliams M, Phillips-Quagliata JM,
asthma. Weisz-Carrington P, Lamm ME. Origin of IgA-
secreting plasma cells in the mammary gland. J Exp
Acknowledgments Med 1977: 146: 1311–22.
This study was supported by the Channel 7 Children’s 14. McDermott MR, Bienenstock J. Evidence for a
Research Foundation of South Australia and Flinders common mucosal immunologic system: I. Migration of
Medical Centre Foundation. RAG was partly supported by B immunoblasts into intestinal, respiratory, and genital
the MS McLeod Research Trust; PHH by the National tissues. J Immunol 1979: 122: 1892–8.
Health and Medical Research Council. The authors would 15. Daniel A. Power, Privilege and Prestige: Occupations
like to thank Mandy O’Grady, Judy Bettess and Kathy Byrt in Australia. Melbourne: Longman-Cheshire, 1983.
for their assistance in enrolling mothers and collecting milk 16. Bryan D-L, Hart PH, Forsyth KD, Gibson RA.
samples and Dr Peter Macardle for technical assistance with Modulation of respiratory syncytial virus induced
flow cytometry. We would also like to thank Prof. Ari prostaglandin E2 production by n-3 LCPUFA in human
Verbyla and Dr Adrian Esterman for their assistance with respiratory epithelium. Lipids 2005: 40: 1007–11.
statistical analyses. 17. Waris M, Meurman O, Mufson MA, Ruuskanen O,
Halonen P. Shedding of infectious virus and virus
antigen during acute infection with respiratory syncytial
References
virus. J Med Virol 1992: 38: 111–6.
1. Openshaw PJ, Dean GS, Culley FJ. Links between 18. Henderson FW. Pulmonary infections with respiratory
respiratory syncytial virus bronchiolitis and childhood syncytial virus and the parainfluenza viruses. Semin
asthma: clinical and research approaches. Pediatr Infect Respir Infect 1987: 2: 112–21.
Dis J 2003: 22: S58–64. 19. Jain L, Vidyasagar D, Xanthou M, Ghai V,
2. Henderson J, Hilliard TN, Sherriff A, et al. Shimada S, Blend M. In vivo distribution of human
Hospitalization for RSV bronchiolitis before 12 months milk leucocytes after ingestion by newborn baboons.
of age and subsequent asthma, atopy and wheeze: Arch Dis Child 1989: 64: 930–3.
a longitudinal birth cohort study. Pediatr Allergy 20. Zhou L, Yoshimura Y, Huang YY, et al. Two inde-
Immunol 2005: 16: 386–92. pendent pathways of maternal cell transmission to
3. Roman M, Calhoun WJ, Hinton KL, et al. Respirat- offspring: through placenta during pregnancy and
ory syncytial virus infection in infants is associated with by breast-feeding after birth. Immunology 2000: 101:
predominant Th-2-like response. Am J Respir Crit Care 570–80.
Med 1997: 156: 190–5. 21. Koopman LP, Savelkoul H, van Benten IJ, et al.
4. Bullens DM, van Den KC, Dilissen E, Kasran A, Increased serum IL-10/IL-12 ratio in wheezing infants.
Ceuppens JL. Allergen-specific T cells from birch-pol- Pediatr Allergy Immunol 2003: 14: 112–9.
len-allergic patients and healthy controls differ in T 22. Bartz H, Buning-Pfaue F, Turkel O, Schauer U.
helper 2 cytokine and in interleukin-10 production. Clin Respiratory syncytial virus induces prostaglandin E2,
Exp Allergy 2004: 34: 879–87. IL-10 and IL-11 generation in antigen presenting cells.
5. Van der Pouw Kraan TC, Boeije LC, de Groot ER, Clin Exp Immunol 2002: 129: 438–45.
et al. Reduced production of IL-12 and IL-12-depend- 23. Bont L, Heijnen CJ, Kavelaars A, et al. Monocyte
ent IFN-gamma release in patients with allergic asthma. IL-10 production during respiratory syncytial virus
J Immunol 1997: 158: 5560–5. bronchiolitis is associated with recurrent wheezing in a
6. Rudloff S, Niehues T, Rutsch M, Kunz C, one-year follow-up study. Am J Respir Crit Care Med
Schroten H. Inflammation markers and cytokines in 2000: 161: 1518–23.
502