Scand. J. Immunol.

53, 7278, 2001

Cord Blood Leucocyte Expression of Functionally Significant Molecules Involved in the Regulation of Cellular Immunity
S. HODGE,* G. HODGE* & R. FLOWER & P. HAN*
*Haematology Department, Women's and Children's Hospital, and University of South Australia, 72 King William Street, Adelaide, South Australia (Received 12 July 2000; Accepted in revised form 20 October 2000)

Hodge S, Hodge G, Flower R, Han P. Cord Blood Leucocyte Expression of Functionally Significant Molecules Involved in the Regulation of Cellular Immunity. Scand J Immunol 2001;53:7278 The cellular immune system of the newborn infant is immature and hypo-responsive when compared with adults. The extent to which immaturity of the leucocyte function underlies hyporesponsiveness in the newborn is incompletely understood. In this study flow cytometric techniques were applied to investigate the concurrent expression of a range of surface and intracellular leucocyte functional molecules and cytokines in resting and stimulated cord and adult blood. Production of interleukin (IL)-2 and expression of the components of its receptor, IL-2Ra/b/g, were investigated. No differences in the proportion of leucocytes producing IL-2Ra and IL-2Rg were observed for newborns and adults. A lower proportion of T cells and natural killer (NK) cells from newborns expressed IL-2Rb and upregulation of expression was slower. We hypothesize that reduced IL-2Rb may curtail early autocrine IL-2 activation of immune responses in the newborn. This hypothesis was supported by the observation that an increased proportion of stimulated T cells from newborns produced IL-2 at 4 h poststimulation, but at 24 h the proportion was lower than for adult T cells. The very low levels of interferon (IFN)-g produced by neonatal T cells and NK cells may also be partly explained by a curtailment of early autocrine activation of T cells. Expression and kinetics of upregulation for other functional molecules were studied. CD71, HLA-DR, tissue factor and CD152 levels were not significantly different for adults and newborns, suggesting that cord blood leucocytes, in some respects, may demonstrate functional maturity. IL-6 secretion by stimulated monocytes was also comparable in cord and adult blood. However, IL-1a and IL-1b were produced by a lower proportion of monocytes from newborns than adults. Similarly, tumour necrosis factor (TNF)-a production for monocytes and T cells was lower in cord blood. The mean fluorescence intensity for IL-1a, IL-1b and TNF-a was also lower for leucocytes from cord blood. These findings are significant in relation to the inability of newborn infants to mount a febrile response to infection. The findings of lower expression of IL-2Rb and lower production of inflammatory cytokines IL-1a, IL-1b and TNF-a is a basis for improved understanding of the immunological immaturity of leucocytes in the newborn. Dr S. Hodge, Haematology Department, Women's and Children's Hospital, 72 King William Street, North Adelaide, South Australia 5006. E-mail: sandy.hodge@imvs.sa.gov.au

INTRODUCTION The cellular immune system of the newborn infant is immature and hyporesponsive in comparison to adults. Neonates are more highly susceptible to infection than adults, and exhibit more severe or prolonged symptoms when infected [1]. Lower leucocyte function in newborns when compared with adults, especially impaired production of some cytokines, has been widely reported [14]. IFN-g has been shown to be
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important for the regulation of immune responses. IFN-g inhibits virus replication and growth of intracellular parasites and enhances the phagocytic activity of macrophages [5, 6]. Immaturity of a newborn's ability to produce IFN-g would therefore contribute to a functional deficiency of the immune system. The inability of the neonate to mount a febrile response to infection is also thought to be a factor in the immunological immaturity in the newborn. IL-1, IL-6 and TNF-a are involved

Markers of Cellular Immunity in Cord Blood 73

Table 1. MoAbs used for three colour analysis of surface and intracellular markers and intracellular cytokines Panel of monoclonal antibodies Cell type T cell FITC TNF-a IFN-g CD25 CD71 HLA-DR CD122 CD69 TNF-a IL1-a CD25 Tissue factor CD71 CD122 CD69 CD3 CD3 CD3 CD3 CD3 CD3 CD3 CD3 CD3 IgG1 i/c (BD) i/c (BD) s (BD) s (D) s (BD) s (BD) s (BD) i/c (BD) i/c (BD) s (BD) s (AD) s s s s s s s s s s s s s (D) (BD) (BD) (BD) (BD) (BD) (BD) (BD) (BD) (BD) (BD) (BD) (BD) PE IL-2 CD69 CD25 CD132 CTLA-4 CD122 CD132 CD69 IL-6 IL1-b CD25 CD132 CD132 CD122 CD69 IFN-g CD25 CD25 CD69 HLA-DR CD122 CD122 CD132 CD132 IgG1 i/c (BD) s (BD) i/c (BD) s (P) s (D) i/c (P) i/c (P) i/c (BD) i/c (BD) i/c (BD) i/c (BD) s (P) i/c (P) i/c (P) i/c (BD) i/c (BD) s (BD) i/c (BD) s (BD) s (BD) s (P) i/c (P) s (P) i/c (P) s (BD) PECy5 CD3 (IT) CD3 (IT) CD3 (IT) CD3 (IT) CD3 (IT) CD3 (IT) CD3 (IT) CD3 (IT) CD14 (IT) CD14 (IT) CD14 (IT) CD14 (IT) CD14 (IT) CD14 (IT) CD14 (IT) CD56 (IT) CD56 (IT) CD56 (IT) CD56 (IT) CD56 (IT) CD56 (IT) CD56 (IT) CD56 (IT) CD56 (IT) CD3, CD14 or CD45 (IT)

Monocyte

NK cell

Control

s, antibody used for surface staining; i/c, antibody used for intracellular staining; BD, Becton Dickenson; P, Pharmingen; IT, Immunotech; D, Dako; AD, AmericanDignostica.

in the induction of fever and the acute phase response [7]. IL-6 is a cytokine produced primarily by monocytes and macrophages in response to stimulation by IL-1 [8]. Conflicting data in relation to IL-6 expression by resting and stimulated cord and adult leucocytes have been reported. Schibler et al. [9] reported a lower IL-6 production by neonates when compared with adult production. At birth, normal production of IL-6 in response to stimuli has also been reported [2, 10]. The kinetics of the production of IL-1 by leucocytes from adults and newborn babies has been reported to be similar [2, 7, 912], although the expression was significantly lower for leucocytes from cord blood. Reports of levels of expression of TNF-a by cord blood T cells have also contained conflicting data [3, 7, 13]. An investigation of levels of production of various cytokines by stimulated cord blood leucocytes may clarify differences in leucocytes in the newborn that relate to their inability to mount a febrile response to infection. There have also been reports of significantly lower expression of some cell-surface molecules important for the regulation of leucocyte function, when compared with levels expressed in

adults [14]. Zola et al. [14] reported a significantly lower expression of the b and g chains of the IL-2 receptor on the surface of resting cord blood leucocytes than for adult leucocytes, and reduced IL-2 dependent T-cell activation in vitro. Zola et al. [14] suggested that, in the newborn, the IL-2 receptor may display a `functional immaturity', and therefore, fail to mediate the transduction of the ligand-induced signal. Other cytokine receptors also may not be fully functional in newborns, resulting in a lack of mature function of some immunological pathways. The extent to which immaturity of leucocyte function underlies immunological hyporesponsiveness in the newborn is incompletely understood. We have applied flow cytometric techniques to investigate concurrent expression of a range of surface and intracellular leucocyte activation markers and cytokines in resting and stimulated cord blood. Concurrent surface and intracellular expression of functional molecules for a variety of cell types has been investigated in the newborn. The data presented further elucidates the basis of immunological immaturity in the newborn infant.

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74 S. Hodge et al.
Table 2. Cytokine production by stimulated cord and adult whole blood. The percentage of cells producing cytokine are shown, with the MFI at 24 h shown in brackets T-cell IFN-g Adult 0h 4h 24 h (5.8) Cord 0h 4h 24 h Monocyte TNF-a NK cell IFN-g

IL-2

TNF-a

IL-1a

IL-1b

IL-6

0.8 12.3 20.4 ^ 10.7 (7.6) 2.1 2.5* 2.0* ^ 2.0 (0.8)***

3.8 11 9.9 ^ 6.6 (7.7) 1.6 18** 3.5* ^ 6.5 (2.8)***

0.7 20.7 8.5 ^ 13.3 (10.4) 0.6 14* 4* ^ 6.7 (3.8)***

0.9 12.4 61.6 ^ 19.0 (15.4) 2.1 9.2 32.0* ^ 4.0 (3.1)***

2.9 30.8 74.3 ^ 22.5 (22.3) 2.8 35.9 52.0* ^ 13.0 (10.1)**

6 50 58.1 ^ 20.4 (6.0) 2.3 34.9 45* ^ 16.0 (12.6)***

4.0 22.0 30.0 ^ 9.9 (2.7) 4.0 14.0 25.0 ^ 10.2 (6.3)

1.6 5.7 15 ^ 8.4

0.9 0.5* 0.5** ^ 0.2 (0.1)***

Results expressed as mean of five experiments. Standard deviations did not change markedly with time, therefore SD shown for 24 h experiments only. * Significantly less proportion of cord cells expressing cytokine at specified time (P , 0.05). ** Significantly higher proportion of cord cells expressing cytokine at specified time (P , 0.05). *** Significantly lower MFI for cord cells (i.e. amount of cytokine produced) at 24 h (P , 0.05).

MATERIALS AND METHODS Collection and stimulation of whole blood

For investigation of the expression of functional molecules for unstimulated leucocytes, blood from 25 healthy adults, or cord blood from 25 normal deliveries, was collected into sodium heparin (20 U/ml, Faulding, Adelaide, Australia) and processed within 3 h of collection. For investigation of the kinetics of expression of surface and intracellular functional molecules for stimulated leucocytes, blood from a healthy adult, or cord blood from a normal delivery, was collected into sodium heparin (20 U/ml, Faulding) and processed within 3 h of collection. Five experiments were carried out on separate days using cord and adult blood, stimulated and tested in parallel. Before stimulation, whole blood was diluted 1 : 2 with RPMI. Incubation was carried out at 37 8C in 5% CO2-in-air, and testing carried out at 0, 4, and 24 h for T cells and 0, 4, 24 and 48 h for monocytes and NK cells, as previously described [15]. Stimuli for investigation of expression of functional molecules were: SEB (Sigma, St. Louis, MO, USA) (10 mg/ml), E. coli lipopolysaccharide (LPS, Sigma) (100 ng/ml) as well as IL-12 (R & D Systems Inc., Minneapolis, USA) (50 ng/ml) and PHA (Wellcome, Beckenham, UK) (5 mg/ml), CD28 (Becton Dickinson, San Jose, CA, USA) (10 mg/ml) was used as a comitogen with SEB in this study. Stimuli for investigation of cytokine production by T cells were PMA (Sigma) (25 ng/ml) plus Ionomycin (Sigma) (1 mg/ml). The stimuli for investigation of cytokine production by monocytes and NK cells were the same as those described above. The concentrations of stimuli were previously found to be optimal for the various cell types (data not shown). Staining of surface markers on leucocytes. Staining of surface markers was carried out using the `whole blood lysis' method [16]. Table 1 presents the combinations of monoclonal antibodies (MoAbs) used in this study, and the corresponding fluorescent dyes. A 5-ml volume of the MoAb was added to 100 ml of whole blood mixture then incubated in the dark at room temperature, for 15 min. In order to lyse

red blood cells, 2 ml FACSlyse (Becton-Dickinson) was added for exactly 10 min at room temperature in the dark. The cells were then centrifuged for 5 min at 500 g, the supernatant discarded, and the cells washed once in 2 ml of 1% bovine serum albumin (BSA) (Sigma) in Isoton 11 (Coulter Electronics, Hialeah, FL, USA). The cell pellet was resuspended in 0.3 ml of 1% paraformaldehyde in Isoton 11. The suspensions of fixed cells were kept at 4 8C in the dark and analyzed within 24 h on a fluorescent activated cell scan (FACScan, Becton Dickinson) using Lysis II software (Becton-Dickinson). Staining of intracellular cytokines and intracellular functional molecules. Staining of surface markers with fluorescent-conjugated MoAbs was carried out immediately prior to staining of intracellular cytokines, as described above, for identification of cell subsets. To block Fc receptors and reduce nonspecific staining, 20 ml human Ig (Intragam, CSL, Sydney, Australia) was added to each tube for 10 min at room temperature. In addition, 50 ml of cold 20 mm EDTA (Sigma) was added to each tube to prevent loss of monocytes by sticking and clumping. This step was incorporated into the procedure before surface staining and also after permeabilization of the leucocytes. For intracellular staining, following the above processing, cells were permeabilized with 500 ml FACSpermTM (Becton-Dickinson) for 10 min at room temperature, then washed with 0.5% BSA in Isoton 11, centrifuged at 500 g, and the supernatant discarded. The cells were then incubated at room temperature for 30 min with an appropriate fluorescent-conjugated antibody to the intracellular marker of interest. After a further wash, the cells were resuspended in 1% paraformaldehyde in Isoton 11. For investigation of the cytokine production, activation was carried out in the presence of monensin (Sigma) which acted as a `golgi block', inhibiting intracellular transport, and retaining the cytokines produced during activation inside the cell. For simultaneous analysis of surface and intracellular functional molecules (i.e. IL-2Ra/b/g), the `golgi block' was omitted and the percentage of positive staining cells calculated as previously described [17]. The MoAbs were obtained commercially, conjugated to fluorescein

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Markers of Cellular Immunity in Cord Blood 75

Fig. 1. Representative experiment showing kinetics of cytokine expression by: (A) T cells from adult whole blood stimulated with PMA 1 ionomycin for 24 h; (B) T cells from cord blood; (C) monocytes from adult blood stimulated with LPS for 48 h; (D) monocytes from cord blood; and (E) T cells and NK cells from adult (solid line) and cord blood (broken line). isothiocyanate (FITC), phycoerythrin (PE), PerCP or Cy5. Different cell types were identified using third colour labelled MoAbs directed against CD3, CD4, CD56 or CD14. In Table 1 the combinations of MoAbs to intracellular cytokines used in the study are presented. Statistical analysis. Differences between the median values for paired data were tested using non-parametric techniques (Wilcoxon's test for paired data).

RESULTS Inflammatory cytokine production A summary of the production of various cytokines by adult and cord blood leucocytes is presented in Table 2. A significantly higher proportion of LPS-stimulated monocytes from adult blood produced IL-1a, IL-1b and TNF-a when compared with monocytes from cord blood (P , 0.05). The proportion of cord blood and adult blood monocytes expressing IL-6 was not significantly different at 4 h or 24 h (Fig. 1C, D). The percentage of cord blood T cells in which production of IL-2 was detected was significantly greater than adult T-cell expression of this cytokine at 4 h (P , 0.05). At 24 h, however, a significantly greater proportion of adult T cells than cord cells expressed IL-2 (Fig. 1B). The proportion of cord blood NK cells or T cells in which the production of IFN-g was detected was not significantly upregulated by stimulation with IL-12 and PHA. This was in

contrast to the changes observed for stimulated adult whole blood at 4 h and 24 h (Fig. 1C). The mean fluorescence intensity (MFI), a measure of the amount of cytokine produced per cell, was investigated. Significantly higher levels of production of IFN-g were found for adult CD31 T cells and CD561 CD3NK cells in comparison to levels in cord cells. IL-1a, IL-1b and TNF-a production by monocytes from stimulated adult blood was also significantly higher than for cord blood cells (Table 2). Expression of IL-2 receptor components Levels of surface and intracellular expression for the three known IL-2R components (IL-2Ra/b/g), for resting T cells, monocytes and NK cells from adult and cord blood was compared by testing 25 healthy adults and cord blood from 25 uninfected newborn babies. A summary of data for IL-2Ra/b/g expression for resting adult and cord whole blood is presented in Table 3. A higher proportion of T cells from adults expressed IL-2Rb (CD122), both on the cell surface and intracellularly, when compared to T cells from cord blood (surface expression 8.2% (adult) in contrast to 6.1% (cord): intracellular expression 12.5% (adult) in contrast to 6.1% (cord). A significantly higher proportion of NK cells from adult blood expressed IL-2Rb (CD122) on the cell surface when compared to NK cells from cord blood (21.6% (adult) cf 15.4% (cord): P 0.048).

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76 S. Hodge et al.
Table 3. Expression of leucocyte activation markers by resting adult and cord whole blood leucocytes. Results expressed as mean ^ SD from testing of 25 uninfected adults and 25 uninfected newborns Functional Molecule T cell CD25 CD25i/c CD69 CD69i/c CD122 CD122i/c CD132 CD132i/c HLA-DR CD152 CD71 Monocyte CD25 CD25i/c CD69 CD69i/c CD122 CD122i/c CD132 CD132i/c CD71 Tissue factor NK cells CD25 CD25i/c CD69 CD122 CD122i/c CD132 CD132i/c HLA-DR Adult Cord (mean ^ SD) (mean ^ SD) 8.6 7.1 5.8 4.9 8.2 12.5 13.0 85.3 6.6 2.9 6.4 7.3 9.0 8.1 14.4 10.9 21.9 25.4 69.5 7.3 11.5 4.8 8.3 5.6 21.6 16.2 22.5 70.3 5.2 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 5.3 2.8 3.8 3.2 4.7 8.7 9.7 4.6 3.7 3.0 3.3 5.1 5.6 6.4 9.9 6.6 10.8 18.2 6.4 4.9 0.7 5.1 4.6 5.3 13.3 8.0 13.6 13.9 3.3 9.8 7.0 6.7 6.0 6.1 8.3 13.8 81.8 5.5 3.9 8.1 6.1 5.9 7.3 9.3 9.6 18.0 22.1 73.5 8.4 8.3 5.1 8.0 15.7 15.4 13.9 22.4 73.5 12 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 5.2 3.2 6.1 3.5 3.7 4.7 8.1 10.6 4.9 1.7 5.3 4.4 3.4 5.4 2.5 5.6 4.8 10.4 9.1 4.1 4.1 2.7 4.4 10.9 10.8 4.8 15.0 13.4 12.3

*P 0.046 *P 0.048

CD71, HLA-DR and CTLA-4) was investigated and compared for cord and adult blood. A significantly higher proportion of NK cells from cord blood expressed CD69 (both surface and intracellular) when compared to NK cells from adult blood 15.7% (cord) compared with 5.6% (adult): P , 0.05). For stimulated cord and adult blood no significant differences in the kinetics of expression for HLA-DR, CD71 or CTLA-4 were detected. (Wilcoxon test for paired data, P . 0.094 for all analyses. Data not shown). Despite the significantly higher proportion of NK cells from cord blood expressing CD69, a different kinetics of expression of CD69 was observed. Up-regulation of CD69 expression by cord blood NK cells was slower than for NK cells from adult blood (24 h for cord NK cells compared with 4 h for NK cells from adults). DISCUSSION Neonates are more highly susceptible to infection than adults. Part of the reason for this susceptibility is believed to be the immunological immaturity of leucocytes in the newborn. In this study a whole blood, flow cytometric assay was applied for investigation of the kinetics of surface and intracellular expression of functional molecules for T-cells, NK cells and monocytes from heterogenous cells populations in adult and cord blood. Flow cytometry has limited sensitivity when compared with some other techniques (10002000 receptor molecules detection limit). The technique, however, is sensitive enough to allow for comparative study of expression of functionally important molecules from resting and activated leucocytes in heterogenous cell populations from whole blood (a mode which reflects the complexity of the in vivo environment more closely). Furthermore, the use of a whole blood assay minimises handling and possible artefactual activation of cells which may occur during cell separation procedures. Using these techniques, we found some notable differences between cord blood leucocytes and adult leucocytes, including differences in the profile of cytokines produced and response to stimuli. No significant differences in the proportions of adult or cord leucocytes expressing IL-2Ra or IL-2Rg were detected. Detectable levels of surface and intracellular IL-2Rb were expressed by a significantly lower proportion of newborn T cells than adult T cells. A lower proportion of newborn NK cells, compared with adult cells expressed surface IL-2Rb. The kinetics of upregulation of the expression of IL-2Rb over 48 h was similar for cord and adult leucocytes. In various studies, the signal transduction function has been shown to be associated with the b chain of the IL-2R on T cells [18, 19]. We hypothesize that the low proportion of cord T cells expressing IL-2Rb may play a role in the relative inability of leucocytes from the newborn to produce IFN-g, by slowing assembly of the high affinity IL-2R. This would restrict early transduction of IL-2R ligand-induced signalling. Slow assembly of the high affinity IL-2R may curtail early autocrine IL-2 activation of T cells in the newborn. This

P 0.004 *P 0.048

* Marker expressed by significantly smaller proportion of resting cord blood leucocytes (P , 0.05). Marker expressed by significantly greater proportion of resting cord blood leucocytes (P , 0.05).

For IL-2Ra (CD25) and IL-2Rg (CD132), no significant differences in levels of surface or intracellular expression were observed between adult and cord blood for any of the cell types tested. The kinetics of expression of IL-2Ra (CD25), IL-2Rb (CD122) and IL-2Rg (CD132) was investigated for cord and adult blood, after stimulation for 48 h. No significant differences in the kinetics of expression for IL-2Ra/b/g were detected for cord and adult blood. (Wilcoxon test for paired data, P . 0.094 for all analyses. Data not shown). Expression of other leucocyte functional molecules Expression of additional functionally significant molecules involved in cell activation and signal transduction (CD69,

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Markers of Cellular Immunity in Cord Blood 77 hypothesis is supported by our observation that an increased proportion of stimulated T cells from newborns, compared with adult T cells, produced IL-2 at 4 h, however a reduced proportion of these cells produced IL-2 at 24 h. The data presented in this study supports our previous report [20] that a negligible proportion of stimulated T cells and NK cells from newborns produced IFN-g when compared with the production in adults. The IFN-g production by NK cells is essential in the prevention of infection by intracellular microbial pathogens [21], possibly by the contribution of IFN-g to development of a Th1 type response [6]. The lower levels of IFN-g production by neonatal T cells and NK cells may therefore partly explain the high susceptibility of newborns to fungal, viral, protozoan and certain bacterial infections. IL-2 is required to maintain a high level of IFN-g production by in vivo primed CD81 cells in viral infections [22]. In this study we found a lower production of IL-2 by neonatal CD41 cells when compared with adult cells. The low rate of production may reduce continued cytokine release by antiviral CD81 cells in the newborn. The rates of expression of various activation markers by leucocytes were compared for 48 h in adult and cord blood after incubation with various stimulating agents. CD69 was expressed on a higher proportion of resting cord blood NK cells than peripheral blood NK cells from adults. Despite this finding, delayed upregulation of the proportion of stimulated cord blood NK cells expressing CD69 was observed when compared with adult cells (24 h compared to 4 h for NK cells from adults). It has been suggested that CD69 has a functional role in redirected lysis, mediated by activated NK cells [23]. The delay in upregulation of CD69 may therefore indicate that the development of cytolytic action of NK cells from newborns may be slower than in adults following an infective stimulus. Functional maturity of leucocytes from newborns was investigated by the expression of markers which are involved in antigen presentation (HLA-DR), regulation of cell proliferation (CD71), T-cell costimulation (CD152) or coagulation activation (tissue factor). The expression of these markers or their kinetics of the upregulation, was not significantly different for adults and newborns. These data indicate that leucocytes from newborns may not be functionally immature in all areas. For both adult and cord blood monocytes, the expression of CD71 was upregulated earlier on stimulated monocytes rather than T cells. The interaction of IL-2 with its high affinity receptor is an essential requirement for T cells to express transferrin receptor and to start proliferating [24]. That the high affinity IL-2R is potentially expressed at a later time point on stimulated T cells rather than monocytes has been reported [17]. Delayed expression of the high affinity IL-2R on T cells may explain the delayed upregulation of CD71 and may be a basis of delay in the proliferation of T cells when compared with monocytes in an inflammatory response. IL-1, IL-6 and TNF-a are involved in the induction of fever and the acute phase response [7]. In this study the IL-6 secretion, by LPS-stimulated monocytes, was compared in adult and neonatal blood suggesting that the production of this cytokine in response to the stimuli was normal at birth. The data on the expression of IL-6 is in agreement with other reports [2, 10] but is in contrast to data in some other studies [9]. Schibler et al. [9] reported that only half as much IL-6 was produced from peripheral blood mononuclear cells (PBMC's) from five fullterm neonates, when compared to those from five adults. Schibler et al. [9] further reported that the peak production was even lower for monocytes from preterm infants. IL-1a and IL-1b were expressed by a significantly lower proportion of monocytes from cord blood at 4 h and 24 h following stimulation, when compared with expression by adult monocytes, although the kinetics of upregulation was similar to that for adults. These findings are consistent with those described elsewhere [2, 11, 12]. There have been conflicting reports in relation to the levels of expression of TNF-a for cord blood T cells [3, 7, 13]. In this study, a reduced proportion of stimulated monocytes and T cells from cord blood produced TNF-a when compared to the proportion for stimulated adult cells. The intensity of fluorescence, reflecting the quantity of cytokine produced per cell, was also significantly reduced. It is likely that the basis of the failure of neonates to mount a febrile response to infection is multifactorial with reduced IL1 and TNF-a production playing some part. For cord leucocytes, activation was observed but with a lower proportion of cells expressing the b chain of the IL-2R (CD122) and inflammatory cytokines IL-1a, IL-1b and TNF-a. A delayed expression of CD69 on NK cells was also observed. The data presented in this report provide a basis for further understanding of the immunological immaturity of leucocytes in newborn infants. R E F E RE N C E S
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