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Impaired responses to toll-like receptor 4 and toll-like receptor 3 ligands in human cord blood
Journal of Autoimmunity 21 (2003) 277–281 www.elsevier.com/locate/issn/08968411
Impaired responses to toll-like receptor 4 and toll-like receptor 3 ligands in human cord blood Dominique De Wit, Sandrine Tonon, Ve´ronique Olislagers, Stanislas Goriely, Michae¨l Boutriaux, Michel Goldman, Fabienne Willems* Laboratory of Experimental Immunology, Universite´ Libre de Bruxelles, 808, Route de Lennik, B-1070 Brussels, Belgium Received 4 July 2003; revised 11 August 2003; accepted 12 August 2003
Abstract Toll-like receptor (TLR)-4 signaling pathway plays an essential role in host defense against gram-negative bacteria while TLR-3-mediated signaling is critically involved in anti-viral immunity. To gain insight into the defects responsible for impaired Th1 responses in human newborns, we investigated the responses of human cord blood cells to lipopolysaccharide, LPS, and to polyinosinic–polycytidylic acid, Poly (I:C), ligands of TLR-4 and TLR-3, respectively. Measurement of cytokine levels revealed a profound defect in IL-12 (p70) synthesis and an increased release of IL-10 in cord blood exposed to LPS or Poly (I:C), as compared to adult blood. Moreover, Poly (I:C)-induced IFN- production was found to be significantly impaired in cord blood. Phenotypic maturation of myeloid DC in response to LPS or Poly (I:C) was next compared in cord and adult blood. We observed that neonatal myeloid DC displayed decreased upregulation of CD40, CD80 whereas CD86 and HLA-DR upregulation did not differ significantly between adults and neonates. Taken together, these findings might be relevant to the increased vulnerability of human newborns to intracellular pathogens and to their inability to develop efficient Th1-type responses. 2003 Elsevier Ltd. All rights reserved. Keywords: LPS; Poly (I:C); Interleukin-12; Interferon-; Dendritic cell
1. Introduction Early life is characterized by an increased susceptibility to intracellular pathogens. This reflects the reduced ability of human newborns to mount protective T helper (Th) 1 immune responses [1]. While intrinsic defects of T cell functions contribute to impaired Th1 responses in neonates [2], dampening of neonatal immune responses may also be due to properties of antigen-presenting cells (APC). APC play a crucial role in the innate immune response against microbial products, which in turn leads to activation of the adaptative immune system [3]. Through their expression of Toll-like receptors (TLR), monocytes, macrophages and dendritic cells (DC) recognize evolutionary conserved molecular patterns derived from microbial pathogens [4,5]. Lipopolysaccharide (LPS), a major integral component from the outer * Corresponding author. Tel.: +32-2-555-39-25; fax: +32-2-555-69-14 E-mail address: [email protected] (F. Willems). 0896-8411/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.08.003
membrane of gram-negative bacteria and Poly (I:C), a viral double-stranded RNA (dsRNA), are two prototypical pathogen-associated molecular patterns using TLR-4 and TLR-3, respectively [6,7]. TLR-4 and TLR-3 signaling induces the release of an array of proinflammatory cytokines by APC. Furthermore, it leads to the maturation of myeloid DC, resulting in an increased expression of MHC class II and costimulatory molecules and increased production of IL-12, a critical factor for generation of Th1 type responses [8–11]. TLR-3 triggering on myeloid DC also induces the production of type I interferons (IFNs) that represent essential anti-viral components [12]. This indicates that TLR-4 and TLR-3-signaling pathways are critically involved in the host defense against gram-negative bacteria and virus, respectively. In order to characterize TLR-4 and TLR-3-mediated neonatal immune responses, we decided to explore the responses of human cord blood cells to LPS and Poly (I:C), a synthetic dsRNA. To mimic as much as possible the in vivo
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environment, we measured cytokine levels in cord and adult blood exposed to LPS or Poly (I:C) and compared the phenotypic maturation of neonatal and adult circulating blood myeloid DC in the same experimental settings.
2. Materials and methods 2.1. Blood samples Adult peripheral fresh blood was obtained from healthy volunteers and umbilical cord blood samples were collected from normal full-term deliveries at the department of obstetrics of the Erasme Hospital (Brussels, Belgium). All procedures were approved by the ethical committee of the faculty of medicine at the Universite´ Libre de Bruxelles. 2.2. Culture media and reagents Culture medium consisted of RPMI 1640 (Bio Whittaker, Verviers, Belgium) supplemented with 2 mM -glutamine (Bio Whittaker), gentamicin (20 µg/ml), 50 µM 2-mercaptoethanol (Bio Whittaker), 1% non-essential amino-acids (Bio Whittaker) and 10% fetal bovine serum (FBS) (HyClone, Erembodegem, Belgium). Lipopolysaccharide (LPS) from Escherichia coli (0128:B12) was purchased from Sigma Chemicals (Bornem, Belgium) and Poly (I:C) from Roche Diagnostics (Brussels, Belgium). 2.3. Whole blood and cell mononuclear cultures Heparinized whole blood samples (1 ml) were incubated at 37 (C with 10 ng/ml LPS or 20 µg/ml Poly (I:C). After an overnight incubation, blood cells were harvested for flow cytometry analysis and plasma collected for determination of cytokine levels by ELISA. In some experiments, mononuclear cells were separated by Ficoll-Hypaque Nycomed (Oslo, Norway) centrifugation and then stimulated overnight with Poly (I:C) (20 µg/ml) followed by determination of IFN- levels in culture supernatants by ELISA. 2.4. Cytokine determination IL-12(p40), TNF-, IL-10 concentrations in plasma were measured by two-site sandwich ELISA systems using antibodies (Flexia kits) from Biosource Europe (Nivelles, Belgium). The detection limit for these assays was 20 pg/ml. IFN- levels in plasma and culture supernatants were determined by a commercial ELISA kit with a detection threshold of 40 pg/ml (Biosource Europe). For the detection of IL-12 (p70), we used a
high sensitivity kit with a detection limit of 0.4 pg/ml (R&D Systems, Abingdon, UK).
2.5. Phenotype of myeloid DC Whole blood (1 ml) was incubated at 37 (C with 10 ng/ml LPS or 20 µg/ml Poly (I:C). After overnight incubation, phenotype of myeloid DC was analysed by four-colour flow cytometry as previously described [13]. Briefly, whole blood cells were incubated for 30 min at 4 (C with 7 µl of a cocktail of FITC-conjugated mAb specific for CD3, CD14, CD16, CD19, CD20, CD34 and CD56 receptors, 5 µl APC-conjugated anti-CD11c mAb, 5 µl PerCP-conjugated anti-HLADR mAb, and one of the following PE-conjugated mAb: anti-CD80 (Becton Dickinson, Mountain View, CA, USA), anti-CD86, anti-CD40 mAb (Biosource Europe, Nivelles, Europe). Expression of HLA-DR, CD40, CD80 and CD86 was quantified on lineage/HLADR+/CD11c+ DC after appropriate gating.
2.6. Statistical analysis Data were compared using two-tailed Mann– Whitney’s U test.
3. Results 3.1. Impaired IL-12 production by neonatal blood upon LPS and Poly (I:C) stimulation In a first set of experiments, we compared the effects of LPS (10 ng/ml) and Poly (I:C) (20 µg/ml) on cytokine production in whole blood from adult and cord samples (Table 1). LPS was able to induce the release of high levels of TNF- and IL-12 (p40) in adult whole blood whereas low levels of IL-10 were produced under the same conditions. Poly (I:C) was found to induce the release of TNF-, IL-12 (p40) and IL-10 by adult blood cells but at much lower levels as compared to LPS. We next observed that significant levels of bioactive IL-12 (p70) were induced by both stimuli in adult blood. In cord blood samples stimulated by LPS or Poly (I:C), TNF- was produced to adult levels, a trend toward the induction of higher TNF- levels being observed for LPS-stimulated cord blood, although the difference versus adult blood did not reach statistical significance. IL-12 (p40) was released to adult levels in response to both stimuli. In contrast, bioactive IL-12 (p70) was not detected in response to LPS and was strongly decreased under Poly (I:C) stimulation as compared to adult blood. Importantly, IL-10 production in cord blood
D. De Wit et al. / Journal of Autoimmunity 21 (2003) 277–281
279
Table 1 Cytokine production in whole blood cultures upon exposure to LPS and Poly I:C
Adult blood
Cord blood
Stimulusa
n
TNF-b
IL-12p40b
IL-12p70b
IL-10b
Medium
10
LPS
10
Poly I:C
8
40 (40–40)c 7174 (5184–11081) 113 (39–2208)
149 (116–195) 4037 (2944–6149) 814 (650–964)
0.4 (0.4–0.4) 16.0 (2.0–22.0) 34.0 (11.8–100.0)
5 (5–5) 90 (59–122) 34 (5–71)
Medium
10
LPS
10
Poly I:C
8
40 (40–40) 22521 (12987–30442) 123 (39–352)
625 (507–764) 2649 (2038–3679) 702 (596.5–882)
0.4 (0.4–0.4) 0.4*** (0.4–0.5) 3.2** (0.5–7.8)
5 (5–5) 1831* (1355–2346) 1360** (1115–1455)
a
Whole blood was cultured for 24 h with LPS (10 ng/ml), Poly I:C (20 µg/ml) or medium alone. Cytokine levels in plasma were assayed by ELISA and expressed in pg/ml. c Data represent median (25–75th quantiles) of 8–10 independent experiments on different donors. *** P<0.001 as compared to adult whole blood samples. * P<0.05 as compared to adult whole blood samples. ** P<0.01 as compared to adult whole blood samples. b
samples was significantly increased in response to both stimuli as compared to adult samples. 3.2. Poly (I:C)-induced IFN- secretion is deficient in neonatal blood Poly (I:C) is capable of inducing type I interferons from circulating DC [14]. We therefore compared IFN- release in adult and cord blood upon Poly (I:C) stimulation. We established that Poly (I:C) (20 µg/ml) was able to elicit IFN- secretion in adult whole blood (Fig. 1). In marked contrast, IFN- plasma levels were significantly lower in cord blood in the same condition. In order to exclude the presence of an inhibitory factor in cord blood, we next compared the effect of Poly (I:C) (20 µg/ml) on isolated mononuclear cells. The neonatal defect in IFN- production was still present in this setting as median (25–75th quantiles) IFN- levels produced by cord blood mononuclear cells (n=33) were 260 (39–738) pg/ml compared to 988 (263–1539) pg/ml produced by adult mononuclear cells (n=21) (P<0.01). 3.3. Incomplete maturation of neonatal myeloid DC exposed to LPS and Poly (I:C) In the next set of experiments, we determined the influence of LPS and Poly (I:C) on the phenotype of myeloid DC circulating in whole blood from adults and neonates. For this purpose, we used a four-colour flow cytometry method allowing the direct analysis of blood circulating myeloid DC (Lin, HLA-DR+, CD11c+). As shown in Fig. 2, incubation of whole blood with LPS or Poly (I:C) induced an upregulation of HLA-DR, CD40, CD80, CD86 expression on adult myeloid DC. In cord
Fig. 1. Induction of IFN- release in whole blood by Poly (I:C). Adult and cord blood samples (1 ml) were incubated in absence or presence of Poly (I:C) (20 µg/ml). After 20 h, plasma samples were collected and assayed by ELISA for detection of IFN- levels. The figure shows individual values and medians of 11 adult samples and 30 cord blood samples.
blood, neonatal myeloid DC also responded to LPS or Poly (I:C) as all markers of maturation were significantly upregulated as compared to resting DC (P<0.01 for each surface molecule). However, the intensity of the changes depended on the surface molecules considered.
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Fig. 2. Flow cytometry analysis of myeloid DC in whole blood. Cord blood (white histograms) and adult blood (dark histograms) samples (1 ml) were incubated overnight in absence or presence of 10 ng LPS or 20 µg Poly (I:C) and analysed by flow cytometry for surface expression of the indicated molecules on myeloid DC identified as Lin/CD11c+/ HLA-DR+ cells. Data represent the median (25–75th quantiles) of mean fluorescence intensity obtained from at least 5 independent experiments on diff rent donors.*, P<0.05; **, P<0.01, ***, P<0.001 as compared to adult blood samples.
While LPS and Poly (I:C)-induced HLA-DR and CD86 upregulation did not significantly differ between neonatal and adult DC, the magnitude of CD40, CD80 upregulation was significantly lower on cord blood myeloid DC (Fig. 2).
4. Discussion IL-12 is a key cytokine for the induction of Th1-type response [15]. The present study demonstrates that bioactive IL-12 (p70) synthesis is profoundly impaired in neonatal blood cells in response to LPS as well as to Poly (I:C), TLR-4 and TLR-3 ligands, respectively. Deficient IL-12 (p70) synthesis in cord blood is likely to be related to a defect in neonatal myeloid DC. In keeping with our data, the capacity of cord blood mononuclear cells to synthesize IL-12 (p70) in response to LPS or heat killed Staphylococcus aureus was found to be markedly impaired [16]. Moreover, deficient IL-12
(p70) release was also observed in cord blood cells when stimulated by another microbial product such as Bordetella pertussis toxin [13]. This impaired IL-12 (p70) synthesis in neonates could be partly attributed to concomitant increased IL-10 production as observed herein and in previous reports [13,16]. However, IL-10 is known to inhibit synthesis of both IL-12 (p35) and (p40) chains [17]. LPS- and poly (I:C)-induced IL-12(p40) levels were found to be comparable in adult and neonatal groups, thereby suggesting a selective inhibition of the p35 chain.These results are reminiscent of previous findings on LPS and Poly (I:C)-stimulated DC derived from cord blood monocytes [18,19]. Indeed, determination of IL-12 (p40) and IL-12 (p35) mRNA levels revealed that IL-12 (p35) gene expression was selectively repressed in LPS-stimulated neonatal DC whereas their IL-12 (p40) gene expression was not altered. We are currently evaluating IL-12 (p40) and (p35) gene expression by adult and neonatal blood upon LPS and Poly (I:C) stimulation. Preliminary data indicate decreased IL-12 (p35) mRNA levels in LPS-stimulated neonatal blood. Thus, impaired IL-12 (p35) gene expression by neonatal DC might constitute a critical control mechanism to prevent the development of potentially detrimental Th-1 immune response during fetal life. Type I IFNs play a crucial role in anti-viral innate immunity. However, due to their pleiotropic effects on various types of immune cells, they also appear to be key cytokines in the development of an effective adaptative immune response. Indeed, they are able to enhance cytotoxicity of NK cells and macrophages [20], to induce T cell activation [21], to maintain the survival of activated T cells [22], to promote DC maturation [23], and to stimulate IFN- production by human CD4+ T cells [24]. Here, we demonstrate that Poly (I:C)-induced IFN- release is profoundly impaired in cord blood cells as compared to adult cells. This finding is in line with previous studies that described decreased IFN- production by cord blood mononuclear cells exposed to virus [25,26]. Liu et al. demonstrated that CD11c+ DC is the only blood cell type that produces significant amounts of type I IFNs in response to Poly (I:C) [14]. Future work will determine whether deficient TLR-3-mediated IFN- production represents an intrinsic property of myeloid neonatal DC or results from the influence of immunomodulatory mechanisms during fetal life. The defects in IL-12 and IFN- synthesis observed in cord blood cells suggest that neonatal myeloid DC would inefficiently participate to natural resistance against intracellular pathogens. Moreover, we showed that cord blood myeloid DC are characterized by a reduced ability to acquire a complete mature adult phenotype in response to LPS and Poly (I:C). Taken together, the impaired neonatal TLR-4 and TLR-3mediated immune responses could participate to the decreased capacity of the human newborn to develop
D. De Wit et al. / Journal of Autoimmunity 21 (2003) 277–281
fully mature protective Th1 responses upon exposure to infectious agents.
Acknowledgements This work was supported by the Centre Interuniversitaire de Vaccinologie sponsored by GSK Biologicals and the Re´gion Wallone, the Neovac project of the European Commission and a Interuniversity Attraction Pole of the Belgian government
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Impaired responses to toll-like receptor 4 and toll-like receptor 3 ligands in human cord blood Dominique De Wit, Sandrine Tonon, Ve´ronique Olislagers, Stanislas Goriely, Michae¨l Boutriaux, Michel Goldman, Fabienne Willems* Laboratory of Experimental Immunology, Universite´ Libre de Bruxelles, 808, Route de Lennik, B-1070 Brussels, Belgium Received 4 July 2003; revised 11 August 2003; accepted 12 August 2003
Abstract Toll-like receptor (TLR)-4 signaling pathway plays an essential role in host defense against gram-negative bacteria while TLR-3-mediated signaling is critically involved in anti-viral immunity. To gain insight into the defects responsible for impaired Th1 responses in human newborns, we investigated the responses of human cord blood cells to lipopolysaccharide, LPS, and to polyinosinic–polycytidylic acid, Poly (I:C), ligands of TLR-4 and TLR-3, respectively. Measurement of cytokine levels revealed a profound defect in IL-12 (p70) synthesis and an increased release of IL-10 in cord blood exposed to LPS or Poly (I:C), as compared to adult blood. Moreover, Poly (I:C)-induced IFN- production was found to be significantly impaired in cord blood. Phenotypic maturation of myeloid DC in response to LPS or Poly (I:C) was next compared in cord and adult blood. We observed that neonatal myeloid DC displayed decreased upregulation of CD40, CD80 whereas CD86 and HLA-DR upregulation did not differ significantly between adults and neonates. Taken together, these findings might be relevant to the increased vulnerability of human newborns to intracellular pathogens and to their inability to develop efficient Th1-type responses. 2003 Elsevier Ltd. All rights reserved. Keywords: LPS; Poly (I:C); Interleukin-12; Interferon-; Dendritic cell
1. Introduction Early life is characterized by an increased susceptibility to intracellular pathogens. This reflects the reduced ability of human newborns to mount protective T helper (Th) 1 immune responses [1]. While intrinsic defects of T cell functions contribute to impaired Th1 responses in neonates [2], dampening of neonatal immune responses may also be due to properties of antigen-presenting cells (APC). APC play a crucial role in the innate immune response against microbial products, which in turn leads to activation of the adaptative immune system [3]. Through their expression of Toll-like receptors (TLR), monocytes, macrophages and dendritic cells (DC) recognize evolutionary conserved molecular patterns derived from microbial pathogens [4,5]. Lipopolysaccharide (LPS), a major integral component from the outer * Corresponding author. Tel.: +32-2-555-39-25; fax: +32-2-555-69-14 E-mail address: [email protected] (F. Willems). 0896-8411/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.08.003
membrane of gram-negative bacteria and Poly (I:C), a viral double-stranded RNA (dsRNA), are two prototypical pathogen-associated molecular patterns using TLR-4 and TLR-3, respectively [6,7]. TLR-4 and TLR-3 signaling induces the release of an array of proinflammatory cytokines by APC. Furthermore, it leads to the maturation of myeloid DC, resulting in an increased expression of MHC class II and costimulatory molecules and increased production of IL-12, a critical factor for generation of Th1 type responses [8–11]. TLR-3 triggering on myeloid DC also induces the production of type I interferons (IFNs) that represent essential anti-viral components [12]. This indicates that TLR-4 and TLR-3-signaling pathways are critically involved in the host defense against gram-negative bacteria and virus, respectively. In order to characterize TLR-4 and TLR-3-mediated neonatal immune responses, we decided to explore the responses of human cord blood cells to LPS and Poly (I:C), a synthetic dsRNA. To mimic as much as possible the in vivo
278
D. De Wit et al. / Journal of Autoimmunity 21 (2003) 277–281
environment, we measured cytokine levels in cord and adult blood exposed to LPS or Poly (I:C) and compared the phenotypic maturation of neonatal and adult circulating blood myeloid DC in the same experimental settings.
2. Materials and methods 2.1. Blood samples Adult peripheral fresh blood was obtained from healthy volunteers and umbilical cord blood samples were collected from normal full-term deliveries at the department of obstetrics of the Erasme Hospital (Brussels, Belgium). All procedures were approved by the ethical committee of the faculty of medicine at the Universite´ Libre de Bruxelles. 2.2. Culture media and reagents Culture medium consisted of RPMI 1640 (Bio Whittaker, Verviers, Belgium) supplemented with 2 mM -glutamine (Bio Whittaker), gentamicin (20 µg/ml), 50 µM 2-mercaptoethanol (Bio Whittaker), 1% non-essential amino-acids (Bio Whittaker) and 10% fetal bovine serum (FBS) (HyClone, Erembodegem, Belgium). Lipopolysaccharide (LPS) from Escherichia coli (0128:B12) was purchased from Sigma Chemicals (Bornem, Belgium) and Poly (I:C) from Roche Diagnostics (Brussels, Belgium). 2.3. Whole blood and cell mononuclear cultures Heparinized whole blood samples (1 ml) were incubated at 37 (C with 10 ng/ml LPS or 20 µg/ml Poly (I:C). After an overnight incubation, blood cells were harvested for flow cytometry analysis and plasma collected for determination of cytokine levels by ELISA. In some experiments, mononuclear cells were separated by Ficoll-Hypaque Nycomed (Oslo, Norway) centrifugation and then stimulated overnight with Poly (I:C) (20 µg/ml) followed by determination of IFN- levels in culture supernatants by ELISA. 2.4. Cytokine determination IL-12(p40), TNF-, IL-10 concentrations in plasma were measured by two-site sandwich ELISA systems using antibodies (Flexia kits) from Biosource Europe (Nivelles, Belgium). The detection limit for these assays was 20 pg/ml. IFN- levels in plasma and culture supernatants were determined by a commercial ELISA kit with a detection threshold of 40 pg/ml (Biosource Europe). For the detection of IL-12 (p70), we used a
high sensitivity kit with a detection limit of 0.4 pg/ml (R&D Systems, Abingdon, UK).
2.5. Phenotype of myeloid DC Whole blood (1 ml) was incubated at 37 (C with 10 ng/ml LPS or 20 µg/ml Poly (I:C). After overnight incubation, phenotype of myeloid DC was analysed by four-colour flow cytometry as previously described [13]. Briefly, whole blood cells were incubated for 30 min at 4 (C with 7 µl of a cocktail of FITC-conjugated mAb specific for CD3, CD14, CD16, CD19, CD20, CD34 and CD56 receptors, 5 µl APC-conjugated anti-CD11c mAb, 5 µl PerCP-conjugated anti-HLADR mAb, and one of the following PE-conjugated mAb: anti-CD80 (Becton Dickinson, Mountain View, CA, USA), anti-CD86, anti-CD40 mAb (Biosource Europe, Nivelles, Europe). Expression of HLA-DR, CD40, CD80 and CD86 was quantified on lineage/HLADR+/CD11c+ DC after appropriate gating.
2.6. Statistical analysis Data were compared using two-tailed Mann– Whitney’s U test.
3. Results 3.1. Impaired IL-12 production by neonatal blood upon LPS and Poly (I:C) stimulation In a first set of experiments, we compared the effects of LPS (10 ng/ml) and Poly (I:C) (20 µg/ml) on cytokine production in whole blood from adult and cord samples (Table 1). LPS was able to induce the release of high levels of TNF- and IL-12 (p40) in adult whole blood whereas low levels of IL-10 were produced under the same conditions. Poly (I:C) was found to induce the release of TNF-, IL-12 (p40) and IL-10 by adult blood cells but at much lower levels as compared to LPS. We next observed that significant levels of bioactive IL-12 (p70) were induced by both stimuli in adult blood. In cord blood samples stimulated by LPS or Poly (I:C), TNF- was produced to adult levels, a trend toward the induction of higher TNF- levels being observed for LPS-stimulated cord blood, although the difference versus adult blood did not reach statistical significance. IL-12 (p40) was released to adult levels in response to both stimuli. In contrast, bioactive IL-12 (p70) was not detected in response to LPS and was strongly decreased under Poly (I:C) stimulation as compared to adult blood. Importantly, IL-10 production in cord blood
D. De Wit et al. / Journal of Autoimmunity 21 (2003) 277–281
279
Table 1 Cytokine production in whole blood cultures upon exposure to LPS and Poly I:C
Adult blood
Cord blood
Stimulusa
n
TNF-b
IL-12p40b
IL-12p70b
IL-10b
Medium
10
LPS
10
Poly I:C
8
40 (40–40)c 7174 (5184–11081) 113 (39–2208)
149 (116–195) 4037 (2944–6149) 814 (650–964)
0.4 (0.4–0.4) 16.0 (2.0–22.0) 34.0 (11.8–100.0)
5 (5–5) 90 (59–122) 34 (5–71)
Medium
10
LPS
10
Poly I:C
8
40 (40–40) 22521 (12987–30442) 123 (39–352)
625 (507–764) 2649 (2038–3679) 702 (596.5–882)
0.4 (0.4–0.4) 0.4*** (0.4–0.5) 3.2** (0.5–7.8)
5 (5–5) 1831* (1355–2346) 1360** (1115–1455)
a
Whole blood was cultured for 24 h with LPS (10 ng/ml), Poly I:C (20 µg/ml) or medium alone. Cytokine levels in plasma were assayed by ELISA and expressed in pg/ml. c Data represent median (25–75th quantiles) of 8–10 independent experiments on different donors. *** P<0.001 as compared to adult whole blood samples. * P<0.05 as compared to adult whole blood samples. ** P<0.01 as compared to adult whole blood samples. b
samples was significantly increased in response to both stimuli as compared to adult samples. 3.2. Poly (I:C)-induced IFN- secretion is deficient in neonatal blood Poly (I:C) is capable of inducing type I interferons from circulating DC [14]. We therefore compared IFN- release in adult and cord blood upon Poly (I:C) stimulation. We established that Poly (I:C) (20 µg/ml) was able to elicit IFN- secretion in adult whole blood (Fig. 1). In marked contrast, IFN- plasma levels were significantly lower in cord blood in the same condition. In order to exclude the presence of an inhibitory factor in cord blood, we next compared the effect of Poly (I:C) (20 µg/ml) on isolated mononuclear cells. The neonatal defect in IFN- production was still present in this setting as median (25–75th quantiles) IFN- levels produced by cord blood mononuclear cells (n=33) were 260 (39–738) pg/ml compared to 988 (263–1539) pg/ml produced by adult mononuclear cells (n=21) (P<0.01). 3.3. Incomplete maturation of neonatal myeloid DC exposed to LPS and Poly (I:C) In the next set of experiments, we determined the influence of LPS and Poly (I:C) on the phenotype of myeloid DC circulating in whole blood from adults and neonates. For this purpose, we used a four-colour flow cytometry method allowing the direct analysis of blood circulating myeloid DC (Lin, HLA-DR+, CD11c+). As shown in Fig. 2, incubation of whole blood with LPS or Poly (I:C) induced an upregulation of HLA-DR, CD40, CD80, CD86 expression on adult myeloid DC. In cord
Fig. 1. Induction of IFN- release in whole blood by Poly (I:C). Adult and cord blood samples (1 ml) were incubated in absence or presence of Poly (I:C) (20 µg/ml). After 20 h, plasma samples were collected and assayed by ELISA for detection of IFN- levels. The figure shows individual values and medians of 11 adult samples and 30 cord blood samples.
blood, neonatal myeloid DC also responded to LPS or Poly (I:C) as all markers of maturation were significantly upregulated as compared to resting DC (P<0.01 for each surface molecule). However, the intensity of the changes depended on the surface molecules considered.
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Fig. 2. Flow cytometry analysis of myeloid DC in whole blood. Cord blood (white histograms) and adult blood (dark histograms) samples (1 ml) were incubated overnight in absence or presence of 10 ng LPS or 20 µg Poly (I:C) and analysed by flow cytometry for surface expression of the indicated molecules on myeloid DC identified as Lin/CD11c+/ HLA-DR+ cells. Data represent the median (25–75th quantiles) of mean fluorescence intensity obtained from at least 5 independent experiments on diff rent donors.*, P<0.05; **, P<0.01, ***, P<0.001 as compared to adult blood samples.
While LPS and Poly (I:C)-induced HLA-DR and CD86 upregulation did not significantly differ between neonatal and adult DC, the magnitude of CD40, CD80 upregulation was significantly lower on cord blood myeloid DC (Fig. 2).
4. Discussion IL-12 is a key cytokine for the induction of Th1-type response [15]. The present study demonstrates that bioactive IL-12 (p70) synthesis is profoundly impaired in neonatal blood cells in response to LPS as well as to Poly (I:C), TLR-4 and TLR-3 ligands, respectively. Deficient IL-12 (p70) synthesis in cord blood is likely to be related to a defect in neonatal myeloid DC. In keeping with our data, the capacity of cord blood mononuclear cells to synthesize IL-12 (p70) in response to LPS or heat killed Staphylococcus aureus was found to be markedly impaired [16]. Moreover, deficient IL-12
(p70) release was also observed in cord blood cells when stimulated by another microbial product such as Bordetella pertussis toxin [13]. This impaired IL-12 (p70) synthesis in neonates could be partly attributed to concomitant increased IL-10 production as observed herein and in previous reports [13,16]. However, IL-10 is known to inhibit synthesis of both IL-12 (p35) and (p40) chains [17]. LPS- and poly (I:C)-induced IL-12(p40) levels were found to be comparable in adult and neonatal groups, thereby suggesting a selective inhibition of the p35 chain.These results are reminiscent of previous findings on LPS and Poly (I:C)-stimulated DC derived from cord blood monocytes [18,19]. Indeed, determination of IL-12 (p40) and IL-12 (p35) mRNA levels revealed that IL-12 (p35) gene expression was selectively repressed in LPS-stimulated neonatal DC whereas their IL-12 (p40) gene expression was not altered. We are currently evaluating IL-12 (p40) and (p35) gene expression by adult and neonatal blood upon LPS and Poly (I:C) stimulation. Preliminary data indicate decreased IL-12 (p35) mRNA levels in LPS-stimulated neonatal blood. Thus, impaired IL-12 (p35) gene expression by neonatal DC might constitute a critical control mechanism to prevent the development of potentially detrimental Th-1 immune response during fetal life. Type I IFNs play a crucial role in anti-viral innate immunity. However, due to their pleiotropic effects on various types of immune cells, they also appear to be key cytokines in the development of an effective adaptative immune response. Indeed, they are able to enhance cytotoxicity of NK cells and macrophages [20], to induce T cell activation [21], to maintain the survival of activated T cells [22], to promote DC maturation [23], and to stimulate IFN- production by human CD4+ T cells [24]. Here, we demonstrate that Poly (I:C)-induced IFN- release is profoundly impaired in cord blood cells as compared to adult cells. This finding is in line with previous studies that described decreased IFN- production by cord blood mononuclear cells exposed to virus [25,26]. Liu et al. demonstrated that CD11c+ DC is the only blood cell type that produces significant amounts of type I IFNs in response to Poly (I:C) [14]. Future work will determine whether deficient TLR-3-mediated IFN- production represents an intrinsic property of myeloid neonatal DC or results from the influence of immunomodulatory mechanisms during fetal life. The defects in IL-12 and IFN- synthesis observed in cord blood cells suggest that neonatal myeloid DC would inefficiently participate to natural resistance against intracellular pathogens. Moreover, we showed that cord blood myeloid DC are characterized by a reduced ability to acquire a complete mature adult phenotype in response to LPS and Poly (I:C). Taken together, the impaired neonatal TLR-4 and TLR-3mediated immune responses could participate to the decreased capacity of the human newborn to develop
D. De Wit et al. / Journal of Autoimmunity 21 (2003) 277–281
fully mature protective Th1 responses upon exposure to infectious agents.
Acknowledgements This work was supported by the Centre Interuniversitaire de Vaccinologie sponsored by GSK Biologicals and the Re´gion Wallone, the Neovac project of the European Commission and a Interuniversity Attraction Pole of the Belgian government
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