Summary
To evaluate the immunological development of preterm infants, especially in early infancy, we examined the serum cytokine levels and the expression of Th2 and Th1 chemokine receptors, CCR4 and CCR5, on days 0, 14 and 28 in 16 low birth weight infants (1720·38 ± 502·80 g) born at less than 37 (33·63 ± 3·29) weeks of gestation. Using an enzyme-linked immunosorbent assay (ELISA), serum interleukin (IL)-4 levels exhibited an increase on day 14, but decreased to the initial level on day 28 (P < 0·05). The significant elevation of serum transforming growth factor (TGF)-β levels was confirmed on day 14 (P < 0·05) but decreased to the initial level on day 28 (P < 0·05). The expression of CCR4 and CCR5 were examined using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. The RT-PCR confirmed the expression of CCR5-mRNA soon after birth, while there was no expression of CCR4-mRNA. Thereafter, the expression of CCR4-mRNA increased significantly and reached the level of CCR5-mRNA expression on day 28 (P < 0·05). Flow cytometric analysis, however, revealed that the expression levels of both CCR4 and CCR5 were low at birth. Thus, CCR4+ CD4+ cells were significantly increased from days 0–28 (P < 0·05), while CCR5+ CD4+ cells were not. Increased IL-4 and TGF-β synthesis as well as increased CCR4+ CD4+ cells suggest that, under extra-maternal circumstances, there is a shift in bias toward Th2 responses even in preterm infants soon after delivery, while they may be capable of developing Th1 mediated responses soon after birth.
Introduction
Infants can be barraged by all kinds of antigens soon after delivery. Dynamic changes are expected in the newborn's immunological system immediately after birth. It is thought that T cells removed from newborn infants are immunologically premature, and that the cytokine production is limited by cord-blood T cells stimulated with soluble anti-CD3 or anti-CD2 antibodies when compared to adult T cells in vitro[1]. However, newborn infants are capable of producing an adult level of interleukin (IL)-2 when stimulated with T cell receptor (TCR)-associated CD3 molecules, suggesting that cell-mediated immunological reactions mature with the help of certain stimuli [2]. Meanwhile, under extra-maternal circumstances, Th2-cell development is essential to produce immune globulins to newly encountered antigens in early infancy. Recently, Prescott et al. proved that initially T cells develop towards the Th2 cytokine profile, which may facilitate immune globulin synthesis including IgA [3,4]. However, there have been only a few thorough immunological analyses in preterm infants, especially in early infancy.
Th1 and Th2 cells are characterized by their cytokine synthesis patterns. Th1 cells, working mainly for cell-mediated immunological reactions, produce IL-2, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-12, while Th2 cells, working for antibodies synthesis, produce IL-4, IL-5, IL-6 and IL-13. Recently, chemokine receptors have been well characterized: cells expressing CCR5 and/or CXCR3 molecules are considered to be Th1 cells while those expressing CCR4 and/or CCR8 molecules are considered to be Th2 cells [5,6]. Examining the expression of these chemokine receptors enables us to analyse Th1 and Th2-cell development in preterm infants.
In this study, we investigate the immunological development in early infancy reflected in the serum cytokine levels and the expression of chemokine receptors, with particular emphasis on preterm infants.
Materials and methods
Subjects
Sixteen preterm infants admitted to our NICU (4 boys and 12 girls; mean body weight, 1720·38 ± 502·80 g; mean gestational week, 33·63 ± 3·29 weeks) were selected for this study. Infants with complications such as chromosome anomaly, congenital anomaly, those who experienced infection and those who underwent surgery were excluded from this study. Mothers or infants treated with corticosteroids were also excluded. Blood samples were collected from peripheral arterial vessels on days 0, 14 and 28 for further study. Soon after the blood samples were taken, peripheral blood mononuclear cells (PBMCs) were separated using a density gradient for reverse transcriptase-polymerase chain reaction (RT-PCR) and a flow cytometric analysis. Our local Ethics Committee approved this study protocol, and informed consent for participation was obtained from all the infants’ parents.
Cytokine concentration
To determine serum cytokine levels in preterm–low birth weight infants, the relevant enzyme-linked immunosorbent assay (ELISA) kits, such as human IL-4 (R&D Systems Europe Ltd, Oxon, UK), IL-5 (R&D Systems Europe Ltd), IL-6 (Fujirebio Inc., Tokyo, Japan), IFN-γ (Biosource International Inc., Camarillo, CA, USA), TNF-α (Japan Immunoresearch Laboratories Co., Ltd, Gunma, Japan) and transforming growth factor (TGF)-β (R&D Systems Europe Ltd) were used according to the manufacturer's instructions.
Chemokine receptor mRNA analysis
Total RNA was extracted from PBMC using a monophasic solution of phenol and guanidine isothiocyanate (RNA STAT 60 Reagent, Tel-Test, Inc., Friendswood, TX, USA) and chloroform, followed by isopropanol precipitation. cDNA was synthesized using RT at 42°C for 30 min from 0·5 µg total cellular RNA using the GeneAmp RNA PCR kit (Applied Biosystems, Branchburg, NJ, USA) according to the manufacturer's protocol. PCR primers for human CCR4 were designed as 5′-CTCCCTTTTTGGGGCTACTA-3′ (sense) and 5′-GTCGTGGAGTTGAGAGAGTA-3′ (antisense), resulting in a 297 base pairs (bp) PCR product. PCR primers for human CCR5 were designed as 5′-CTGACATCTACCT GCTCAAC-3′ (sense) and 5′-CTGCAGGTGTAATGAA GACC-3′ (antisense), resulting in a 323 bp PCR product. PCR primers for human CCR8 were designed as 5′-CACT TGACCTCAGTGTGACA-3′ (sense) and 5′-CACACTCAT GAGGGTGATGA-3′ (antisense), resulting in a 357 bp PCR product. PCR primers for human CXCR3 were designed as 5′-GGAGCTGCTCAGAGTAAATC-3′ (sense) and 5′-CAC GAGTCACTCTCGTTTTC-3′ (antisense), resulting in a 180 bp PCR product [7]. PCR primers for human β-actin, used as a housekeeping gene, were designed as 5′-AGAGATGGCCACGGCTGC-3′ (sense) and 5′-CTTCTG CATCCTGTCGGCA-3′ (antisense), which produce a 271 bp PCR product. The thermal cycle was programmed with a hot start at 95°C for 5 min followed by 30 cycles at 95°C for 1 min and annealing at 62°C for 1 min, followed by an extension at 72°C for 1 min, as described previously [8]. The RT-PCR products were electrophoresed in 1% agarose gel stained with ethidium bromide and visualized through a UV light. A PCR mixture with distilled H2O instead of RNA was used as a negative control. Band intensities were semiquantified by densitometry and the intensity ratio of each reading compared to β-actin as well as the intensity ratio of CCR4/CCR5 were also calculated.
Lymphocyte subpopulation analysis
Immunofluorescence staining was performed on T cells to examine their subpopulations with FITC/PE/Cy-Chrome conjugated monoclonal mouse antihuman CCR4, CCR5, CD4, and CD8 antibodies (PharMingen, San Diego, CA, USA). These antibodies were used at recommended dilutions according to the manufacturer's instructions. Three-colour flow cytometric analysis was performed using a FACScan (Becton Dickinson, San Jose, CA, USA). Lymphocytes were gated on light-scatter characteristics, and the percentage of positive cells was calculated in comparison to a negative control consisting of cells stained with FITC, PE and CyCr-conjugated antimouse IgG alone.
Statistical analysis
All data were analysed by two-way, paired Student's t-tests. A P < 0·05 was considered statistically significant.
Results
Serum cytokine concentration in early infancy
Serum IL-4, IL-5, IL-6, IFN-γ, TNF-α and TGF-β levels were measured using ELISA. Only serum IL-4 and TGF-β levels were detectable during this study. Serum IL-4 level tended to increase on day 14 but decreased to an initial level on day 28 (P < 0·05), while the significant elevation of serum TGF-β levels was confirmed on day 14 (P < 0·05) but decreased to an initial level on day 28 (P < 0·05) (Fig. 1).
Serum IL-4 and TGF-β levels after birth. Serum IL-4 and TGF-β levels were measured using ELISA. The serum IL-4 level tended to increase on day 14 but decreased to the initial level on day 28 (P < 0·05), while significant elevation of serum TGF-β levels was confirmed on day 14 (P < 0·05) but this decreased to an initial level on day 28 (*P < 0·05).
Expression of the chemokine receptors, CCR4, CCR8, CCR5, CXCR3 and β-actin, analysed by RT-PCR
To examine the development of CCR4, CCR5, CCR8, CXCR3 and β-actin-mRNA expression, RT-PCR was performed. CCR4 and CCR8 were used as Th2 cell markers, CCR5 and CXCR3 were used as Th1 cell markers. The expressions of both CCR5 and CXCR3-mRNA were stronger than those of CCR4 and CCR8-mRNA on day 0 (Fig. 2). Thus, the expression of CCR4-mRNA increased gradually and, as a result, the CCR4/CCR5 mRNA ratio was significantly increased (close to 1) on day 28 compared with that on day 0 (P < 0·05) (Fig. 3).
Expression of CCR mRNA on day 0. The expression of the chemokine receptors, CCR4 and CCR8 (Th2), and those of CCR5 and CXCR3 (Th1), was analysed using RT-PCR. The expression of both CCR5 and CXCR3 mRNA was stronger than that of CCR4 and CCR8-mRNA on day 0. β-actin was used as a housekeeping gene. Representative data from each infant are presented.
CCR4/CCR5-mRNA ratio. The CCR4 mRNA level increased gradually and almost reached that of CCR5 mRNA on day 28. The CCR4/CCR5 mRNA ratio had significantly increased to 1 on day 28 (P < 0·05).
Expression of the chemokine receptors, CCR4 and CCR5, analysed by FACScan
To analyse the development of Th1 and Th2 cells the chemokine receptors, CCR4 and CCR5, were used as representatives of Th2 and Th1 cells, respectively. Lymphocytes were gated with CD4 or CD8 positive cells and the expression of their chemokine receptors was then examined using FACScan. There were very few CCR4+ and CCR5+ cells at birth but the number of CCR4+ cells was increased markedly on day 28, while the increase in CCR5+ cells was not as large (Fig. 4). As a result, CCR4+ CD4+ cells were increased significantly from day 0 to day 28 (P < 0·05) (Fig. 5) and there were no significant changes in the numbers of CCR5+ CD4+, CCR4+ CD8+ and CCR5+ CD8+ cells during this period (data not shown).
The expression of chemokine receptors using FACScan. Lymphocytes were gated for CD4 and CD8 positive cells first and then the expression of the chemokine receptors CCR4 (Th2) and CCR5 (Th1) were analysed using FACScan. FACScan analysis revealed that the expression levels of both CCR4 and CCR5 were low at birth. Thus, the number of CCR4+CD4+ cells had gradually increased by day 28. Representative data from each infant are presented.
CCR4+CD4+ and CCR5+CD4+ cells in early infancy. FACScan analysis revealed that the expression of CCR4+CD4+ cells (closed square) was significantly increased from day 0 to day 28 (*P < 0·05), while the expression of CCR5+CD4+ cells (closed diamond) was not.
Discussion
The present study examined the expression of chemokine receptors using RT-PCR and flow cytometric analyses, which enabled us to understand better Th1/Th2 cell development at the nuclear (RT-PCR) and protein level (FACScan). Although there was a large difference in age, ranging from 30 to 36 weeks of gestation, there was no significant difference in immunological developmental manner individually.
Th1 cells are considered important for the recognition of and protection against infectious pathogens. Although it has been considered that T cells taken from newborn infants are immunologically premature, recent studies suggested that infants naturally infected or vaccinated with Bordetella pertussis develop preferentially pertussis-specific Th1 responses [9,10]. Moreover, in utero exposure to mycobacterial antigens has been reported to result in adult-level Th1 responses [11]. These findings suggested that newborn humans might be capable of developing efficient Th1-mediated responses. In the present study, detecting CCR5-mRNA expression in PBMC on day 0 infants revealed that even preterm infants might be capable of developing Th1-mediated responses soon after delivery. Although the FACScan analysis could detect very few CCR5+ cells in this study, this might be due to the lack of bacterial infections in these infants.
Meanwhile, CCR4+ CD4+ cells were increased significantly from days 0 to 28, suggesting that sufficient Th2 cell responses can also be introduced in preterm infants. Indeed, increased expression of CCR4-mRNA on day 28 suggested that Th2 cells developed gradually after birth. Prescott et al. suggested that the initial T cell development is towards the Th2 cytokine profile [3,4]. In this study, there was a relatively increased production of IL-4 and TGF-β levels in preterm infants on day 14, suggesting that extra-maternal environmental factors may possibly stimulate and introduce a Th2 response in these infants soon after birth. These cytokines assisted in enhancing the expression of CCR4-mRNA, and as a result CCR4+ CD4+ cells were increased significantly on day 28. Thus, there was a time lag in shifting T cell maturation towards Th2 response after cytokine stimulation, suggesting that immunological changes occurs gradually in vivo and not as fast we expected to see in vitro.
In the present study, increased production of TGF-β was observed in preterm infants in early infancy. TGF-β is an important cytokine in preventing inappropriate immunological reactions in infants, as TGF-β has a broad spectrum of activities in mucosal regulation, including induction of oral tolerance, potent anti-inflammatory effects, mucosal IgA expression and effects on epithelial cell proliferation and differentiation [12]. Continuous production of a certain amount of serum TGF-β and IL-4 and an increased number of CCR4+ CD4+ cells in these infants support the idea that natural antigen exposure is suitable to stimulate immunological development biased towards Th2 responses even in early infancy. These changes are considered convenient for early infancy, mainly for the production of IgA against natural antigens. In fact, early elevation of serum TGF-β and IL-4 levels on day 14 might possibly be an important signal for future Th2 development in preterm infants.
These findings indicate clearly that, under extra-maternal circumstances, there is a shift in bias toward Th2 mediated immunological responses even in preterm infants after delivery, while they may be capable of developing Th1-mediated responses soon after birth.
Acknowledgements
This study was supported by grants from the Japan Society for the Promotion of Science, Japan.
