Main

Neonates are vulnerable to infections because of their immature immunity (1). In particular, they are highly susceptible to specific bacterial, viral, and parasitic intracellular pathogens, including Listeria monocytogenes, Toxoplasma gondi, herpes simplex virus, and cytomegalovirus (2). Their susceptibility to these intracellular pathogens is secondary mainly to the immaturity and dysfunction of specific T-cell–mediated immunity (3, 4). The impairment of DC function in neonates could contribute to the dysfunction of T cells and their susceptibility to infection. The levels of intercellular adhesion molecule-1 (ICAM-1, CD54) and MHC class I and class II antigens in CB DC are significantly lower than those in adult blood DC, and IL-12p35 production from CB DC is impaired (5–8). In our previous studies, we not only demonstrated immaturity of T cells and monocytes in neonates, but also decreased phenotypic expression and function of CB immature monocyte-derived DC (4, 9, 10). The ability to improve the function of CB DC will have impact on the initiation of CB naïve T-cell response.

The maturation process is central to the function of DC, enabling DC to perform highly specialized yet different functions sequentially. There are many stimuli that initiate this maturation process, including pro-inflammatory cytokines such as TNF-α and IL-1, and bacterial products such as lipopolysaccharide (LPS) (11). IGF-I, as a lymphohematopoietic cytokine, has profound positive effects on immune function (12). It has been found that IGF-I enhances phagocytosis of human polymorphonuclear leukocyte and natural killer (NK) cell activity and induces TNF-α production from monocytes (13–15). IGF-I could also stimulate human B-cell proliferation and antibody secretion (16–18). Moreover, it has potential to augment lectin or anti-CD3 stimulated T-cell proliferation (4, 19). Our previous observations show that IGF-I not only promotes CB T-cell maturation in interferon γ production and CD45RA/RO transformation, but also maintains survival and inhibits apoptosis of CB T cells (4, 20, 21). We hypothesized that IGF-I may also modulate the maturation and survival of CB monocyte–derived DC.

The functions of IGF-I are mediated by the IGF-I receptor (IGF-IR), a highly specific membrane receptor associated with intracellular tyrosine kinase, homologous to the insulin receptor (22). Studies on signaling by the IGF-I and IGF-IR in different cell types have revealed two primary pathways by which these signals might be transmitted (23). Two primary substrates of the activated IGF-IR are IRS-1 and Shc. Phosphorylated tyrosines on IRS-1 serve as docking sites for multiple proteins containing SH-2 domains (24, 25). Among these proteins, Grb-2 has been shown to bind to IRS-1 and activate Ras, in turn activating the Raf-MAP kinase pathway (26). The second pathway is the PI 3-kinase pathway. Association of IRS-1 with p85/p110 PI 3-kinase results in its activation (24). Because the involvement of MEK and PI 3-kinase has been implicated in previous studies (27, 28), specific MEK inhibitor (PD98059) and PI 3-kinase inhibitor (LY294002) were used to study the intracellular signaling pathways involved. Cytokine-neutralizing antibodies were also used to determine whether the effects of IGF-I are mediated through the up-regulation of cytokine production. IGF-I is a survival factor for many cell types (27–31). To the best of our knowledge, this is the first report on the effects of IGF-I on CB DC.

METHODS

Isolation of monocytes.

Human umbilical CB was obtained from the placentae of normal, full-term infants, after the placentae were delivered and separated from the infants, with prior written informed consent of their mothers. The protocol was approved by the Ethics Committee of the University of Hong Kong. All samples were collected in heparinized flasks. Cord blood mononuclear cells (CBMC) were isolated from whole blood by centrifugation, using Ficoll-Hypaque gradients (Pharmacia Biotech, Uppsala, Sweden). CBMC at the interface were collected, washed, and resuspended at 1 × 108/mL in pH 7.2 Hanks/HEPES buffer. Monocytes were isolated from mononuclear cells by positive selection using anti-CD14 conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell viability, as measured by trypan blue exclusion, was more than 95%. The purity of monocytes was measured by flow cytometry with 85–95% of the cells being CD14 positive.

Generation of immature DC in vitro.

Isolated CD14+ monocytes were cultured at a density of 1 × 106 cells/mL in RPMI 1640 (Sigma Chemical Co., St. Louis, MO, U.S.A.) plus 10% FCS supplemented with 50 IU/mL penicillin and 50 μg/mL streptomycin (Invitrogen, Carlsbad, CA, U.S.A.). Monocytes were derived into immature DC in the presence of IL-4 (10 ng/mL; R & D Systems, Minneapolis, MN, U.S.A.) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL; R & D Systems) at 37°C in a humidified atmosphere containing 5% CO2, as in our previous study (10). The cultures were fed with fresh medium and cytokines after 3 d and cell differentiation was monitored by light microscopy. After 5 d culture, cells were harvested and washed three times with serum-free, hormone-free, and insulin-free Dulbecco's Modified Eagle Medium (DMEM)-F12 medium (Sigma Chemical Co.). Cells were then resuspended in DMEM-F12 plus IL-4 and GM-CSF with or without IGF-I (100 ng/mL, R & D Systems) for 2 more days. The concentration of IGF-I used has been titrated in previous studies (4, 20, 21).

Immunofluorescence staining.

After 7 d culture as described above, cells were collected, mixed with fluorochrome-conjugated antibodies at concentration of 4 μg/106 cells, washed, and resuspended in 300 μL of wash solution for flow cytometric analysis. Isotype controls (FITC, PE, and PC5 conjugated) and CD14-FITC, CD83-FITC, CD86-FITC, CD40-FITC, MHC class II-FITC, and MR-PE were all purchased from BD PharMingen (San Diego, CA, U.S.A.).

Apoptosis assay with AV/PI.

The percentage of cells undergoing apoptosis was determined by using the AV apoptosis kit (Immunotech-Coulter, Marseille, France) for the detection of membrane phosphatidylserine imbalance. According to the manufacturer's instructions, cells were resuspended in 300 μL of ice-cold binding buffer. Five microliters of AV-FITC and 5 μL of PI (1 mg/mL) were added to the cell suspension. The cell suspension was kept on ice and incubated for 10 min in the dark before being analyzed by flow cytometry.

Signal analysis.

Flow cytometric analysis was performed with a Coulter Epics Elite Flow Cytometer (Beckman Coulter, Inc., Fullerton, CA, U.S.A.). The machine was optimized daily using standard FlowCheck fluorescence beads (Beckman Coulter, Inc.) and linearity checked by Quantum25 beads (Flow Cytometry Standards Corp, San Juan, PR, U.S.A.). Ten thousand events per sample were collected into listmode files and analyzed by the WinMDI 2.8 analysis software. The DC analysis was gated to the standard forward scatter and side-scatter profile for large cells.

Mixed lymphocyte reaction (MLR).

In vitro–generated DC were washed with PBS, counted, resuspened, and treated with mitomycin C (10 μg/mL, Sigma Chemical Co.). Allogeneic CB CD3+ T cells (1 × 105 responder cells/well) were cultured in triplicate in 96-well flat-bottom microplates with different numbers of stimulator DC (DC to T cell ratios were 1:1000, 1:100, 1:10). Cultures were maintained in a humidified atmosphere at 37°C and 5% CO2. Twenty microliters of BrdU (100 μM) was added into the wells 16 h before the end of 5 d culture and its incorporation was then measured by Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Molecular Biochemicals, Indianapolis, IN, U.S.A.).

TNF- αproduction by DC.

The cell culture supernatants of non-IGF-I–treated and IGF-I–treated DC were harvested on d 7 and stored at −20°C before analysis. The levels of TNF-α were measured by Quantikine ELISA kits (R & D Systems). The detection range of TNF-α was 7.8–500 pg/mL.

Inhibition of signaling pathways.

On d 5, CB monocyte-derived DC were preincubated for 30 min with MEK inhibitor PD98059 (50 μM/mL, Calbiochem, San Diego, CA, U.S.A.) or PI 3-kinase inhibitor LY294002 (25 μM/mL, Calbiochem) before the addition of IGF-I (100 ng/mL). The concentration of inhibitors used was based on previous literature (27) and preliminary titration experiments. The inhibitors were left in the medium throughout the whole period of IGF-I stimulation. Phenotypic expression, apoptosis analysis, and cytokine assays were performed on d 7. The viability of cells on d 7 was within the range of 65–85% as determined by trypan blue exclusion.

Neutralization of TNF-α.

Anti-TNF-α neutralizing antibodies (10 μg/mL, R & D Systems) were added into d 5 CB monocyte-derived DC together with IGF-I (100 ng/mL) for a further 2 d in culture to inhibit the action of TNF-α on the cultured cells. The concentration used was based on the level of TNF-α assayed in previous culture and the suggested neutralization dose provided by the manufacturer.

Data presentation and statistical analysis.

In most cases, data were expressed as mean ± SEM. To facilitate the presentation of results, the mean fluorescence level (MFL) of test samples was normalized to the respective controls and expressed as relative expression level according to the equation:

All samples were paired and differences between groups analyzed by paired t test or the nonparametric equivalents.

RESULTS

IGF-I promoted CB monocyte-derived DC maturation.

The maturation of DC is marked by the up-regulation of co-stimulatory molecules and down-regulating the expression of MR (11). IGF-I significantly up-regulated the expression of CD83, CD86, CD40, and MHC class II molecules with down-regulated MR expression. To facilitate the presentation of data, the expression levels of IGF-I–treated DC were normalized to that of the non-IGF-I–treated DC (Fig. 1).

Figure 1
figure 1

The expression of maturation markers on IGF-I–treated CB DC. (A) IGF-I–treated CB DC (thick line) expressed higher levels of maturation markers than non-IGF–I treated CB DC (thin line). One representative experiment of more than five on different samples. (B) To consolidate our findings, the expression levels of DC markers in IGF-I–treated CB DC were normalized to that in non-IGF-I–treated CB DC (CD83: 33 ± 9 MFL; CD86: 336 ± 54 MFL; CD40: 111 ± 37 MFL; MHC class II: 1274 ± 168 MFL; MR: 198 ± 99 MFL). IGF-I significantly up-regulated the expression of CD83 (n = 9), CD86 (n = 11), CD40 (n = 5), and MHC class II molecules (n = 10) and down-regulated the expression of MR (n = 11). Results shown as mean ± SEM.

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IGF-I inhibited apoptosis in CB monocyte-derived DC.

To determine whether IGF-I is a survival factor for CB DC, apoptosis was studied in serum-free culture with or without IGF-I by flow cytometry (Fig. 2A). The percentage of AV+/PI− early apoptotic cells was decreased significantly in the IGF-I–treated DC (n = 7), but there was no significant difference in the percentage of AV+/PI+ late apoptotic cells between the two groups (Fig. 2B).

Figure 2
figure 2

The early and late apoptosis in IGF-I–treated CB DC. (A) Early and late apoptosis was determined by AV labeling for membrane phosphatidylserine imbalance. One representative experiment of seven different samples. (B) IGF-I reduced the percentage of early apoptotic cells (AV+/PI−) but had no effect on the percentage of late apoptotic cells (AV+/PI+). Results shown as mean ± SEM (n = 7).

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IGF-I–treated CB monocyte-derived DC stimulated allogeneic lymphocyte proliferation.

In vitro–generated DC were investigated for their ability to stimulate the proliferation of allogeneic CB CD3+ T cells in MLR. Background BrdU uptake by either CD3+ T cells (1 × 105) or mitomycin-treated DC was undetectable. Both non-IGF-treated and IGF-treated CB DC showed similar ability to stimulate CD3+ T cells at DC to T ratios of 1:1000; 1:100, and 1:10 (Fig. 3).

Figure 3
figure 3

Proliferative response of CB CD3+ T cell cultured with allogeneic non-IGF–treated or IGF–treated CB DC at different ratios. The abilities of IGF-treated and non-IGF-treated DC to stimulate the proliferation of CD3+ T cells were similar (n = 3). Results shown as mean ± SEM.

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IGF-I induced TNF-α production by CB monocyte-derived DC.

IGF-I alone induced TNF-α production from monocytes (15). We showed that IGF-I significantly induced TNF-α production by CB DC (Fig. 4). TNF-α production induced by IGF-I was completely blocked by PD98059 or LY294002.

Figure 4
figure 4

TNF-α production from IGF-I-treated and kinase inhibitor–treated CB DC. IGF-I induced significantly higher levels of TNF-α than in non-IGF-treated controls (34 ± 7 pg/mL vs 108 ± 36 pg/mL; n = 9; p = 0.008). The addition of PD98059 (50 μM/mL) or LY294002 (25 μM/mL) in cell culture for 30 min before the addition of IGF-I blocked the IGF-I induced TNF-α production. The mean ± SEM was 30 ± 2 pg/mL (n = 7, p = 0.016) and 30 ± 8 pg/mL (n = 3) for PD98059 and LY294002, respectively.

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MEK inhibitor PD98059 and PI 3-kinase inhibitor LY294002 blocked the effects of IGF-I on CB monocyte-derived DC maturation.

The intracellular pathways triggered after IGF-I binding with IGF-I receptor have been identified mainly as the MEK and PI 3-kinase pathways (24, 25, 28). To delineate the intracellular pathways of IGF-I effects on the CB DC maturation, PD98059 and LY294002 were used to inhibit the MEK and PI 3-kinase pathways, respectively. PD98059 completely blocked the effects of IGF-I on the expression of CD83, CD86, CD40, and MR in CB DC, whereas the expression of MHC class II molecules was only partially blocked (Fig. 5A). LY294002 had similar blocking effects for the expression of CD86 and CD40. Unexpectedly, it also down-regulated CD83 expression and up-regulated MR and MHC class II expression (Fig. 5B).

Figure 5
figure 5

The expression of maturation markers on kinase inhibitors-treated CB DC. PD98059 (50 μM/mL) or LY294002 (25 μM/mL) were added in cell culture for 30 min before the addition of IGF-I. (A) PD98059 completely blocked the effects of IGF-I on the up-regulation of CD83, CD86, and CD40, and down-regulation of MR (n = 5). The IGF-I–induced up-regulation of MHC class II molecules was only partially blocked (n = 4). (B) Similarly, LY294002 blocked the effects of IGF-I on up-regulation of CD83, CD86, and CD40, and down-regulation of MR (n = 3). However, LY294002 down-regulated CD83 expression and up-regulated MR and MHC class II molecules expression (n = 3). Results shown as mean ± SEM.

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MEK inhibitor PD98059 and PI 3-kinase inhibitor LY294002 blocked the effects of IGF-I on CB monocyte-derived DC apoptosis.

MEK and PI 3-kinase pathways play very important roles in DC survival and apoptosis induced by LPS (32, 33). We investigated whether the effects of IGF-I on CB DC survival and apoptosis were also mediated through these two pathways. We showed that MEK inhibitor PD98059 inhibited the effects of IGF-I on CB DC apoptosis (Fig. 6A). Similar but not entirely identical results were obtained for PI 3-kinase inhibitor LY294002 (Fig. 6B). In contrast to PD98059, which had no effects on the percentage of AV+/PI+ late apoptotic cells, LY294002 could significantly augment the percentage of AV+/PI+ late apoptotic cells in CB DC.

Figure 6
figure 6

The early and late apoptosis in kinase inhibitor–treated CB DC. PD98059 (50 μM/mL) or LY294002 (25 μM/mL) were added in cell culture for 30 min before the addition of IGF-I. MEK inhibitor PD98059 (A) and PI 3-kinase inhibitor LY294002 (B) completely blocked the effects of IGF-I on early apoptosis of CB DC. Moreover, LY294002 significantly increased the late apoptotic cells in CB DC. Results shown as mean ± SEM (n = 7).

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Anti-TNF-α could not neutralize the effects of IGF-I on the maturation and apoptosis of CB monocyte-derived DC.

Because TNF-α is a pivotal factor for DC maturation (34), we hypothesized that the effects of IGF-I on CB DC maturation and survival were mediated through TNF-α. However, even high concentration of anti-TNF-α neutralizing antibody did not neutralize the effects of IGF-I on CB DC maturation (Fig. 7A). Anti-TNF-α even induced higher expression levels of maturation markers, in particular CD83 and CD40, on CB DC. The addition of anti-TNF-α did not have any effect on the IGF-I induced reduction of apoptosis in CB DC (Fig. 7B).

Figure 7
figure 7

The expression of maturation markers and apoptosis in anti-TNF-α neutralizing antibody–treated CB DC. The addition of anti-TNF-α neutralizing antibody (10 μg/mL) did not neutralize the effects of IGF-I on CB DC maturation (A) and apoptosis (B). Unexpectedly, anti-TNF-α induced higher expression of maturation markers, in particular CD83 and CD40. Results shown as mean ± SEM (n = 7).

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DISCUSSION

Regulation of inflammation and immunity requires a vast network of interacting cells and cytokines. The endocrine system participates in various ways in immune regulation, through the actions of GH and IGF-I (35). In our previous studies on mixed populations of CBMC, we have shown that IGF-I could promote CB naïve T cell maturation from CD45RA to CD45RO (21), induce IL-6 production and stimulate the phytohemagglutinin-induced interferon γ secretion to adult level (4). IGF-I also maintains the survival and inhibits apoptosis of CB T cells (21) and increases the telomerase of CB T cells (20). In the present study, we further demonstrated that IGF-I promoted the CB monocyte-derived DC maturation with up-regulation of CD83, CD86, CD40, and MHC class II molecules expression and down-regulation of MR expression (Fig. 1). Furthermore, IGF-I inhibited the apoptosis of CB DC and induced TNF-α production (Figs. 2 and 4). The effects of IGF-I were blocked by MEK inhibitor PD98059 and PI 3-kinase inhibitor LY294002 (Figs. 4–6) but not by anti-TNF-α neutralizing antibodies (Fig. 7). Our results indicate that IGF-I induced CB DC maturation is regulated by activation of both intracellular signaling routes but not through up-regulation of TNF-α production.

The number of DC in the circulation is scarce and the in vitro generation of monocyte-derived DC serves as an important tool for the study of DC biology. Maturation of DC is characterized by morphologic, phenotypic, and functional changes (36). We demonstrated clearly that IGF-I induced CB DC maturation with up-regulation of co-stimulatory and MHC class II molecule expression and down-regulation of MR expression. Even though an up-regulation of antigen presentation capacity was not detected in IGF-I–treated DC (Fig. 3), the increase in expression levels of co-stimulatory molecules was statistically significant (Fig. 1). In previous studies, the effect of IGF-I may have been amplified by limiting the amount of cytokines, such as IL-4, in the cultures. This experimental design is difficult to achieve in the study of monocyte-derived DC, as they require IL-4 and granulocyte-macrophage colony-stimulating factor to maintain their stable differentiated stage (37).

The IGF-I induced maturation is mediated by the MEK pathway as shown after the addition of MEK inhibitor PD98059 (Fig. 5A). The involvement of the PI 3-kinase is, however, more complex. The addition of PI 3-kinase inhibitor LY294002 only blocked the IGF-I–induced up-regulation of CD86 and CD40. Interestingly, blocking the PI 3-kinase pathway led to reduced expression of CD83 and enhanced expression of MHC class II molecules and MR (Fig. 5B). It is possible that there are inhibitory factors in the system that are mediated through the PI 3-kinase pathway in down-regulating MHC class II molecule expression. Hence, blocking of inhibitory signals led to up-regulation of the molecules. Alternatively, there may be other maturation factors that share the PI 3-kinase pathway with IGF-I. Therefore, blocking of the pathway led to down-regulation of CD83 and up-regulation of MR. The role played by PI 3-kinase inhibitor alone and its interaction with molecules of the IGF-I signaling pathway will need to be investigated further.

It has been reported that IL-4 and IL-13 enhance fluid phase pinocytosis and MR-mediated uptake in human macrophages via the activation of PI 3-kinase (38). In rat liver endothelial cells, blocking PI 3-kinase pathway can suppress endocytosis that is due to both a reduction of the number of surface receptors and a reduction in the rate of receptor-ligand internalization (39). In this study, we did not investigate the pinocytosis or endocytosis function of CB DC, but our results showed that blocking the PI 3-kinase pathway significantly up-regulated MR expression, which may lead to increased MR-mediated endocytosis. Our results suggested that pinocytosis or endocytosis may be differentially regulated in different cell types and the mechanisms involved are worthy of further investigation.

IGF-I prevents apoptosis in a number of cell types, and acts as a survival factor in the absence of other factors (27–31). Specifically, IGF-I inhibits apoptosis of several IL-3–dependent cell lines when IL-3 is removed (30, 40). IGF-I also inhibits spontaneous apoptosis in granulocytes (41). Recent studies demonstrated that PI 3-kinase is a critical cellular protein that prevents apoptotic cell death in many cell types, including hemopoietic progenitor cells and T lymphocytes (42). IGF-I can protect myeloid progenitor cells from apoptosis by activating PI 3-kinase. Previous studies have shown that MEK and PI 3-kinase are involved in the survival, whereas p38 stress-activated protein kinase (p38 SAPK) and nuclear factor (NF)-κB family are involved in the maturation of human monocyte-derived DC induced by LPS (32, 33). In concordance with data published by Parrizas et al. (27) on rat pheochromocytoma cells, we demonstrated that IGF-I is capable of preventing apoptosis by activation of multiple signal transduction pathways. The fact that PI 3-kinase inhibitor LY294002 significantly increased the percentage of late apoptosis cells may be due to the blockade of signals from other cytokines, such as IL-4, which share the PI 3-kinase pathway with IGF-I (30).

TNF-α is one of the critical factors for DC maturation (34). Several TNF family members contribute to DC survival in addition to TNF-α itself. TNF-related activation-induced cytokine (TRANCE) is a DC-specific survival factor that regulates the expression of the anti-apoptotic molecule Bcl-XL(43, 44). We showed that IGF-I could significantly increase TNF-α production from CB DC and either PD98059 or LY294002 completely blocked this effect (Fig. 4). Our results on CB DC agreed with the increased in vitro production of TNF-α by IGF-I reported on monocytes, macrophages, and granulocytes (15, 41). The TNF-α released was not responsible for the IGF-I–induced maturation and apoptosis in CB DC, as high concentrations of anti-TNF-α neutralizing antibody did not neutralize the effects of IGF-I on CB DC. On the contrary, neutralizing TNF-α could potentiate the effects of IGF-I in the up-regulation of maturation markers of CB DC, in particular CD83 and CD40 (Fig. 7). This effect of TNF-α–neutralizing antibodies is similar to that of MEK inhibitors on TNF-α–stimulated cells reported recently by Puig-Kröger et al. (45). They demonstrated enhanced acquisition of maturation markers on DC when the TNF-α signaling pathway is blocked. The fact that blockade of the MEK pathway led to different cellular response in IGF-I– and TNF-α–stimulated monocyte-derived DC implicated the specificity and complexity of the signal transduction pathways.

In cerebellar granule neurons, picogram amounts of TNF-α can potently inhibit both the IRS-2 phosphorylation and PI 3-kinase activation that play a pivotal role in IGF-I signaling (46). In this study, PI 3-kinase was also involved in IGF-I–induced CB DC maturation. Hence, neutralizing TNF-α might decrease the inhibition of TNF-α on PI 3-kinase activity induced by IGF-I, leading to the additive effect of anti-TNF-α with IGF-I. The cross-talk by which vastly different receptors interact through sharing intracellular substrates such as PI 3-kinase needs to be studied further.

CONCLUSION

In conclusion, IGF-I not only promoted the maturation of CB monocyte-derived DC but also maintained the survival and inhibited the apoptosis of CB DC. In addition, IGF-I significantly induced TNF-α production. The maturation and survival of CB DC induced by IGF-I were mediated through both MEK and PI 3-kinase pathways. The effects of IGF-I on CB DC were not mediated by TNF-α inasmuch as anti-TNF-α antibody could not neutralize these effects. With the knowledge that IGF-I has positive effects on both CB T cells and DC, the use of IGF-I in designing a new strategy for ex vivo expansion of CB immunologic cells for immunotherapy and transplantation should be further explored.