Gastroenterology Unit, The Queen Elizabeth Hospital. Woodville South. Adelaide and' Department of Paediatrics,. University of Adelaide and Women's and ...
Immunology and Cell Biology (1994) 72, 306-313
Phenotype of T cells, their soluble receptor levels, and cytokine profile of human breast milk BRETT A. EGLINTON, DONAL M. ROBERTON' and ADRIAN G. CUMMINS Gastroenterology Unit, The Queen Elizabeth Hospital. Woodville South. Adelaide and' Department of Paediatrics, University of Adelaide and Women's and Children's Hospital. North Adelaide. South Australia. Australia Summar)' Human breast milk has important immunoprotective and immunosuppressive functions for an infant. The purpose of this study was to extend the phenotype of milk cells and to measure soluble T cell receptor levels and cytokines in milk, and to compare these with neonatal and adult blood. Milk T cells had a more equivalent CD4 : CD8 ratio than blood; milk CD4 T cells mainly expressed the CD45RO (antigen primed/memory) phenotype; milk CD8 cells had an equivalent C D l l b : C D 2 8 suppressor; cytotoxic phenotype; and milk T cells had 2-3-fold higher percentages of activated CD4 IL-2R and CD8 HML-1 or CD8 VLA-1 cells than blood. Soluble IL-2R, CD4 and CD8 concentrations were lower in milk than adult blood, although relatively increased when compared to the lower T cell concentration in milk. Breast milk contained high levels of IFN-y but low levels of other measured cytokines compared to blood. These distinct differences of T cells and their soluble products are likely to influence an infant's immune system. Keywords: breast milk, cytokines, infant, T cells.
During breast feeding, immune milk cells and soluble factors are ingested and have the potential to influence an infant's immune system. A breast-fed baby ingests about 10^ milk cells per day with breast feeding often continuing for several months.' These milk cells consist of macrophages and colostral corpuscles (large lipid-laden macrophages), neutrophils, and 5-27% lymphocytes of which the majority are T cells.'- Not only is it possible that these maternally derived lymphocytes reside in the neonatal gut and transfer immune responses,^'* but they are likely to secrete cytokines, although mainly macrophage-derived factors have been described,^'^ as well as the immunosuppressive cytokine, transforming growth factor-p (TGF-p).^ The relatively neutral pH ofthe stomach in early infancy, together with the buffering capacity of milk, may allow survival of milk lymphocytes as well as cytokines during gastrointestinal transit. The role of milk-derived T cells and their soluble products, such as soluble T cell receptors and cyto-
kines, remains poorly defined, although milk feeding is known to serve such functions as transfer of cellmediated immune responses,"* protection against respiratory and gut infections,^ and suppression of certain diseases such as atopy and coeliac disease.^"^ We have also observed that withdrawal of milk feeding during weaning results in physiological activation ofthe immune system in rats," in guinea-pigs (unpubl. data) and in healthy human infants.'- Taken together, these effects may be related to the delayed-type hypersensitivity function (DTH) of CD4 T cells, and to the cytotoxic and suppressor functions of CD8 T cells. Indeed, we have found that human breast milk contains more activated CD4 1L-2R and CD8 HML-1 T cells than peripheral blood lymphocytes from adults.'-^ Soluble T cell factors, such as receptor levels or cytokines, have not been described. Soluble Tcell receptors (sIL-2R and sCD8) indicate activation (and by analogy possibly sCD4),'''-'-'^ and cytokines (IFN-y or IL-2) may indicate DTH of CD4 or cytotoxic function of CD8 cells, and IL-4 (as well as TGF-p) suppressor function.'*"
Correspondcnce: Dr A. G. Cummins, Gaslrocnterology Unit, The Queen Elizabeth Hospital, Woodville South. SA 5011, Australia. Received 21 August 1993; accepted 16 December 1993.
Therefore, we were interested in further the phenotyping of milk T cells, particularly ating CDl lb ; CD28 suppressor ; cytotoxic CD8 T cells,"' and in further studying the
Introduction
extending differentisubsets of activation
307
T cells and soluble immune factors in milk status of CD4 and CD8 T cell subsets, as well as tneasuring soluble T cells receptor levels and cytokines. Values obtained for milk samples were compared with neonatal and adult blood samples. Materials and methods Collection of milk and blood Breast milk samples were obtained with consent from healthy mothers of term infants within 9 days of parturition, while these mothers were resident in the Maternity Division of The Queen Elizabeth Hospital. The mean ± s.d. post partum day of sample coUeetion was 4.6 ± 1.4 days. Neonatal blood samples from 28 term infants with a mean ±s.d. age of 4.6 ±4.1 days were obtained from the residuum remaining after biochemical analysis for physiologieal hyperbilirubinaemia in the Department of Clinical Chemistry of The Queen Elizabeth Hospital. Samples were used only from those infants who were otherwise healthy. The infants studied were not the infants of mothers enrolled in the study for provision of breast milk samples. Blood was collected from a total of 52 healthy adult volunteers. Subjects were excluded if they had a history of infection during the previous four weeks. Some data from the neonatal and adult blood samples were used for comparison in a concurrent study of immune activation during infancy. Guidelines for human experimentation of the National Health and Medical Research Council of Australia were followed, and the study was approved by the Human Ethics Committee of The Queen Elizabeth Hospital.
Separation and purification of milk lymphocytes Freshly expressed milk samples were collected between 0900 and 1200h, and mixed with an equal volume of washing buffer at 4°C (PBS/1% w/v BSA/0.02% w/v sodium azide) to solidify the milk fat. Milk cells were sedimented by centrifugation (600^, 15 min) and resuspended in cold washing
buffer. For some samples, a small aliquot was taken to determine the total milk cell count and a cytospin preparation made and stained with Giemsa stain for a differential count. The absolute lymphocyte concentration was calculated for 10 milk samples from the differential and total cell counts. Milk cells were shaken with an equal volume of light mineral oil (M3616. Sigma Chemical Co., Sydney. NSW, Australia) to dissolve residual milk fat.'"* The mixture was centrifuged (600^, 15 min), the clear oily layer removed, and the sedimented cells resuspended in 5 mL of PBS. The milk cells were layered above 3 mL of NycoPrep (density 1.077 g/mL. Nycomed, Oslo. Norway), and centrifuged at 400 g for 25 min. Cells at the interface were collected, washed and counted. In order to compare the T cell concentration of milk with that of blood, an average Tcell concentration in blood was calculated using the conventional mean (range) of 2100 (13003300) cells/mL (Department of Haematology, The Queen Elizabeth Hospital).
Flow cytometry of milk and blood cells Purified milk cells or whole blood were incubated in the dark (1 5 min. 4°C) with saturating concentrations of monoclonal antibodies as listed in Table 1. Red cells in blood samples were lysed with 2 mL of FACS lysing solution (Becton Dickinson, San Jose, CA, USA). Some samples of blood were also purified using the method for milk; results obtained after flow cytometry were similar to those ofthe direct lysis technique. Milk and peripheral blood lymphocytes were labelled with directly conjugated phycoeothrin (PE) or FITC mouse monoclonal igG antibodies. An indirect technique was used to detect HML-1 labelling using either goat FITC F(ab')2 anti-mouse antibody (Cappel. Organon Teknika. Veedijk, Belgium), or rabbit anti-mouse PE antibody (Serotec, Oxford, UK). Contaminating macrophages/colostral corpuscles in milk were labelled with biotin conjugated antiCD 14 followed by a streptavidin-duocHROME second step (Becton Dickinson). After cell labelling, excess antibody was removed by washing and the cells were fixed in 0.2%
Table 1. Monoclonal antibody nomenclature and specificity. Determinant
Clone
Distribution
Source
CD3 aP-TcR
SK7 WT31 11F2 SK3 SKI D12 CD28.2
Pan T cell aP Tcell receptor y5 Tceli receptor T helper/inducer T cytotoxic/suppressor CD8 suppressor CD8 cytotoxic Monocytes/macrophages Activated T cells Unprimed/naive cells Primed/memory cells Intra-epithelial lymphocytes Late activated T cells
Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Immunotech Coulter Becton Dickinson Becton Dickinson Dako Immunotech T Cell Sciences
Y5-TCR
CD4 CD8 CDUb CD28 CD14 CD25 (IL-2R) CD45RA CD45RO HML-1 VLA-1
116
2A3 L48 UCHLl 2G5.1 TS2/7
308
B. A. Eglinton et al.
paraformaldehyde/PBS/0.02% sodium azide before analysis on a FACScan flow cytometer (Becton Dickinson). To improve detection of milk lymphocytes, contaminating CD14* macrophages/colostral corpuscles were excluded during data acquisition. Cells were displayed using sidescatter versus FL3, and positive cells on FL3 were excluded from acquisition. Data were collected for 10 000 events for later analyses. A lymphocyte gate was set using forward and sidescatter and two-colour fluorescence was assessed. For dual colour analyses, cursors were set at the same values as those determined using the relevant single labels. Single label analysis was performed on all specimens, but dual label analysis was performed on a smaller number of sample. Dual analyses were performed with CD3 versus IL-2R: CD45RA, CD45RO. IL2R, VLA-1 and H M H versus CD4; CD28, CDl lb. IL-2R, VLA-1 and HML-1 versus CD8; ap TcR or 78 TcR venis HML-1, and IL-2R versus VLA-l.
used to test for significant differences between means using a 95% experiment-wise confidence interval."
Results General features of milk samples The mean ± s.d. total cell count of 20 samples of milk was 6.7 ±5.2 x lOVmL. A differential mean ±s.d. count of 10 milk samples yielded 7.5±6.5% lymphocytes, 61.5± 12.6% macrophages/colostral corpuscles, 28.5± 15.1% neutrophils and 2.4±3.5% eosinophils. Thus, an estimated mean lymphocyte count for milk is
Comparison of phenotypes of lymphocytes in milk, and in neonatal and adult blood Soluble receptor and cytokine assays sIL-2R concentrations were measured in milk and plasma using an ELISA (CellFree IL-2R; T Cell Sciences, Cambridge, MA, USA). Plasma aliquots of 25 ^iL were diluted I : 2 before being measured. Because adult plasma samples were collected in EDTA anti-coagulant, which interferes with the peroxidase detection system, the assay was modified for all samples to include a wash with PBS after incubation ofthe samples and before addition of peroxidase-conjugated detecting antibody. The lower limit of assay sensitivity is 50 U/mL. sCD4 and sCD8 were measured using ELISA (CellFree CD4 and CellFree CD8, T Cell Sciences). The lower limit of sensitivities ofthe sCD4 and sCD8 assays are 12 and 50 U/mL, respectively. The soluble T cell receptor concentrations were also related to the lymphocyte count in milk using the results ofthe total and differential cell counts and values from flow cytometry. These were compared to reference data of adult blood (Department of Hematology, The Queen Elizabeth Hospital). Plasma GM-CSF, IFN-7, IL-2. IL-4 and IL-6 were measured using ELISA (Factor-Test, InterTest-y. InterTest2X, InterTest-4, Predicta IL-6; Genzyme, Cambridge, MA, USA). The lower limit of sensitivities ofthe GM-CSF, IFN-y, IL-2, IL-4 and IL-6 assays were 4, 100, 100, 45 and 35 pg/ mL, respectively. TNF-p was also measured by ELISA (Amersham, Amersham, UK) and had a lower limit of sensitivity of 31 pg/mL. Plasma of neonates was diluted 1 : 4 for the IL-4 and TNF-P assays. Concentrations of GM-CSF and IFN-y in neonatal plasma were measured using pooled samples of equal aliquots from 28 infants.
Statistics Results were summarized as mean ± s.d. for data that were normally distributed. Cytokine concentrations were summarized as the median and range because of their skewed distribution. Cytokine concentrations were transformed to log|o(J^+ 1) to stabilize the variance and normalize their distribution before data analysis. Peritz's multiple F test was
The mean ± s.d. (n) percentages of CD3 lymphocytes detected in milk, neonatal and adult blood were 68.6±18.3 (/i = 27), 82.6±6.9 (/7 = 28), 73.0±7.1 (« = 52), respectively. Neonatal blood had a higher proportion of T cells than adult blood ( P < 0 . 0 5 ) . The mean proportion of T cells expressing the y5-receptor were higher in milk than neonatal blood (5.2 vs 2.5%, /*=0.04), but did not differ in comparison to adult blood. In order to allow comparison of milk and neonatal and adult blood, other data were related to denominators of CD3. CD4 and CD8 and are given in Tables 2 and 3 and Fig. 1. Some CD4 cells expressed both CD45RA and CD45RO on dual labelling with values of up to 30% in adult blood. The remaining CD8 T cells (--10%) in milk and blood were double negative for C D l l b and CD28. and dual positive staining was < 3%. Milk samples contained a higher proportion of activated T cells than blood samples (Table 3, Fig. 1). The majority of HML-1 * T cells had the CD8 rather than the CD4 phenotype. HML-1 ^ T cells had a ratio of the ap to y6 TcR for 12 milk samples of 2.6 : I. Only 13% of VLA-1 positive cells co-expressed IL-2R.
Soluble receptor concentrations of T cells in milk, and in neonatal and adult blood Concentrations of sIL-2R, sCD4 and sCD8 are shown in Table 4. sIL-2R and sCD8 concentrations were lower in milk than in either neonatal or adull blood due to the lower cell concentrations, and sCD4 levels were similar in milk and blood. However, soluble T cell receptors in milk were increased in comparison to the milk lymphocyte concentration. Assuming an average blood lymphocyte count of 2.1 x lOVmL, of
309
T cells and soluble immune factors in milk
Table 2. Phenotype of milk-derived, and neonatal and adult blood CD3, CD4 and CD8 lymphocytes byflowcytometry. Determinant
Milk
Neonatal blood
Adult blood
96.5 + 5.9 2.5 + 1.6 77.5±5.7 24.9 ±6.3
96.7±5.0 3.6±3.3
fl = 27)
aP-TcR yS-TcR CD4 CD8
94.1+31.2 5.2±6.0 46.5±15.6 46.4±17.3
63.9 ±10.5 36.6±11.8
In milk. CDl4^ macrophages/colostral corpuscles were excluded using the third fluorescent channel. Cells were selected on forward and sidescatter for lymphocytes. Values are expressed as mean percentage ± s.d. of CD3 cells.
which 60% are CD3 T cells, adult blood contains approximately 150x lO"* CD3 Tcells/mL compared to approximately 3 x IO"'/mL in milk. Thus. sIL-2R and sCD8 were relatively increased by 7-fold and sCD4 by 30-fold in relationship to the CD3 concentration. SIL-2R was elevated 2-fold in neonatal blood compared to adult blood, indicating increased T cell activation in neonates.
•P Pooled
600
ft
6-
U
4-
A AM
S
2-
200-
n-27
n - 18
n-26
0-
1000800-
P
