Original ArticlesInterleukin 2 receptor regulation and IL-2 function in the human infant
Introduction
We have shown previously that cord blood lymphocytes express lower resting levels of IL-2R than adult lymphocytes [1]. The analysis was performed with a highly sensitive method that detects low expression levels (100 molecules per cell) [2], and the difference was statistically significant, with p = 0.0001 for α chain (CD25) expression on CD4 and CD8 T cell subsets and on B cells [1]. IL-2 receptor β chain (CD122) was reduced on cord T cells but not significantly on cord B cells [1], and γ chain (CD132) expression was reduced on cord T and B cells, compared to adult cells [3]. The reduction of γ chain in cord compared with adult cells has been confirmed independently [4]. The present study extends these investigations to include infants and young children up to 18 months of age, and shows a gradual rise in IL-2R α and β levels.
While membrane CD25 expression is low on neonatal cells, soluble CD25 has been reported to be either equivalent in cord and adult blood or higher in neonatal blood 5, 6. Soluble CD25 has also been reported to be produced at equivalent levels from cord and adult cells in culture [7]. Soluble CD25 is not necessarily shed from circulating lymphocytes; it may derive from sources such as the thymus, which contains CD25-positive T cell precursors [8]. Nevertheless, the contrast between membrane CD25 expression (low in the neonate) and soluble CD25 concentration (high in the neonate) raises the question whether IL-2R production is low in the neonatal period or whether it is produced but is shed from the membrane. This question was addressed by testing cord and adult cells for IL-2 receptor mRNA by reverse transcription followed by quantitative polymerase chain reaction (RT-PCR), and by testing cord plasma for ability to down-regulate IL-2 receptor expression on adult cells.
Low expression of IL-2R on neonatal cells may reflect a relative inability of neonatal cells to make IL-2R in response to stimulation, or may simply reflect a lack of stimulation. Several groups 7, 9, 10, 11 found that cord blood cells can respond to PHA or to CD3 with levels of CD25 expression comparable to or higher than those attained by adult cells treated in the same way. Bessler et al. [12] showed that term (but not premature) neonatal cells could make IL-2R mRNA in response to PHA activation at levels comparable to those found in adult cells. However, some reports 13, 14 showed lower responses to PHA, CD3 and CD2 stimulation of cord as compared with adult cells. Kawano et al [15] found that cord cells were unresponsive in the autologous mixed lymphocyte reaction, and that this correlated with a lack of upregulation of CD25, in contrast to adult cells. In the light of these conflicting findings in the literature, we examined the ability of cord and adult lymphocytes to express all three IL-2 receptor chains, using a high sensitivity staining procedure to detect subtle differences that might be missed with conventional immunofluorescence techniques, which would not detect the receptor until it reached a high concentration.
We considered the functional consequences of a deficit in IL-2 receptor expression in the neonate. We have previously shown that IL-2-stimulated T cell proliferation is reduced in cord compared with adult cells under certain conditions [3]. Other functional studies have shown a greater proliferative response to IL-2 in cord compared with adult cells 10, 16, although Kawano et al [15] attributed the lower autologous mixed lymphocyte reaction in neonatal blood to a lack of IL-2 responsiveness. Watson et al [17] found neonatal B cells to be less responsive to IL-2 than adult B cells. Splawski and Lipsky [18] found that the ability of neonatal B cells to secrete immunoglobulin could be restored to adult levels by IL-2. With this background of contradictory findings in different in vitro systems, we explored in vivo activation, examining IL-2R levels on mononuclear cells from peripheral blood of infants suffering acute infection.
Section snippets
Cells
Adult blood mononuclear cells were isolated by centrifugation on Ficoll-hypaque. Cord blood cells were fractionated using a dual Ficoll-hypaque procedure, to remove contaminating nucleated red cells [19], or, in the activation experiments, using Ficoll-hypaque followed by magnetic beads coated with anti-glycophorin antibody [20]. Cells were >95% CD45 positive after purification. Blood samples were obtained with informed consent and under the guidelines of the Research Ethics Committee of the
IL-2 receptor expression on infant cells — variation with age
Membrane expression of IL-2R chains was analysed by high-sensitivity immunofluorescence on whole blood. Fig. 1A shows expression of α, β and γ chains, plotted as percent of cells positive against age, for infants and young children up to 18 months of age. There is a gradual increase in levels of α and β chain with age (r2 = 0.236 (n = 28) and 0.276 (n = 27), respectively). Adult controls studied in the same series gave mean ±se values of 44 ± 3 for α chain and 47 ± 6 for β chain (n = 12). The
Discussion
We previously showed low expression of all three chains of the IL-2 receptor in cord blood cells, compared to adult cells 1, 3. Cummins et al. [5] reported an increase in CD25 membrane (and soluble) expression at weaning, but their study did not use a high-sensitivity procedure and did not examine the β and γ receptor chains. The high sensitivity procedure utilises selected batches of reagents, a three-layer amplification and phycoerythrin as the fluorochrome, and gives a distinctly different
Acknowledgements
This work was supported by a Project grant from the Australian National Health and Medical Research Council, and an equipment grant from the Variety Club, South Australia. We are grateful to Ms Sue Brandford, Institute of Medical and Veterinary Science (IMVS, Adelaide) and Ms Justine Elliott and Dr. Brant Bassam (Perkin Elmer Cetus, Melbourne) for help with the quantitative PCR, and to the IMVS for time on the ABI Prism 7700 sequence detector.
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