SEARCH

SEARCH BY CITATION

Keywords:

  • neonatal immunity;
  • monocytes/macrophages;
  • signal transduction;
  • transcription factors;
  • FACS

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We reported earlier that neonatal monocyte-derived macrophages (MDM) could not be fully activated with IFN-γ, a finding that could not be attributed to lower expression of IFN-γ receptors on the neonatal cells. In this study we explored elements of IFN-γR-mediated signalling in cord monocytes and MDM. Intracellular expression of STAT-1 was analysed by flow cytometry. We have assessed phosphorylation of STAT-1 by using MoAbs that distinguish native and phosphorylated forms of STAT-1 on a discrete cell basis. Using MoAbs against the native form of STAT-1 revealed comparable expression of this protein in cord and adult cells (both monocytes and MDM). However, STAT-1 phosphorylation in response to IFN-γ was significantly decreased in neonatal monocytes (P < 0·05) and MDM (P < 0·01) compared to adult cells (n > 5 for each). These data suggest deficient cytokine-receptor signalling in neonatal mononuclear phagocytes exposed to IFN-γ. We propose that decreased STAT-1 phosphorylation and activation may represent developmental immaturity and may contribute to the unique susceptibility of neonates to infections by intracellular pathogens.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Neonatal macrophages cannot be fully activated with IFN-γ to kill viable microorganisms including Candida albicans and group B Streptococcus type III [1,2]. We found earlier that IFN-γ had a stimulatory effect on macrophage candidacidal function in both neonatal and adult macrophages, but its effect on neonatal cells was significantly weaker [1]. This finding is in agreement with the failure of IFN-γ to enhance superoxide anion generation and TNF secretion by cultured monocytes from neonatal blood [3]. We reported earlier that surface expression of IFN-γR1 and affinity of the receptor to its specific ligand were comparable in neonatal and adult macrophages [1]. In an attempt to gain more insight into mechanisms of macrophage activation in the newborn infant we have explored elements of IFN-γR-mediated signalling in cord monocytes and monocyte-derived macrophages (MDM).

IFN-γ binds to its cell-surface receptor consisting of two heterodimeric subunits, IFN-γR1 and IFN-γR2 which are associated with Janus kinases, JAK1 and JAK2, respectively, [4]. IFN-γ binding results in dimerization of the two receptor subunits and phosphorylation of JAK1 and JAK2. STAT-1 proteins are then in turn phosphorylated by JAK kinases allowing their dimerization and subsequent translocation into the nucleus, where they bind to activation sites of IFN-γ-inducible genes [5,6].

IFN-γ causes rapid serine/tyrosine phosphorylation of STAT-1 [5–7]. In this study, we have assessed STAT phosphorylation in cord and adult mononuclear phagocytes by using monoclonal antibodies that distinguish native and phosphorylated forms of STAT-1 on a discrete cell basis [8]. We report here profound deficiency of STAT-1 phosphorylation in neonatal monocytes and macrophages in response to activation with IFN-γ despite comparable expression of STAT-1 protein in resident monocytes and macrophages in newborns and adults.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Antibodies

Mouse antihuman STAT-1 cytoplasmic terminus MoAb (IgG2b) was obtained from Transduction Laboratories, Lexington, KY; mouse IgG2b (MOPC 141) was purchased from Sigma, St. Louis, MO; FITC-conjugated F(ab′)2 goat antimouse IgG was from Caltag Laboratories, Burlingame, CA. Rabbit antihuman phosphorylated STAT-1 directed against the phosphorylated tyrosine 701 of p91 STAT-1 [9] was obtained from New England Biolabs, Beverly, MA; normal rabbit IgG and FITC-conjugated F(ab′)2 goat antirabbit IgG were purchased from Caltag. Saturating concentrations of antibodies determined by flow cytometry were used.

Monocytes and macrophages

Studies on blood cells were approved by the Regional Ethics Committee of the Scientific Board of the University of Debrecen (DEOEC KEB No. MML-01–2001). Mixed mononuclear cells were isolated from heparinized (10 U/l) venous blood of 19 healthy adults and 18 cord blood of healthy term neonates with a gradient of Ficoll-PaqueTM Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden) [1,10]. The percentage of monocytes in fresh suspensions was between 18 and 33 as determined by Giemsa and esterase stainings.

The washed suspension of mononuclear cells was resuspended in X-VIVO 10 (Bio Whittaker, Walkersville, MD) medium supplemented with gentamycin and 1% heat-inactivated autologous serum [11]. Cells were plated on 6-well (35 mm) polystyrene plates (Corning Glass Works, Corning, NY) coated with 2% gelatine (Sigma) at a density of 5 × 106−107 cells per well. Nonadherent cells were removed by washing after 2 h incubation at 37°C and 5% CO2 and adherent cells were cultured for 3 days in fresh X-VIVO medium. Viability of cultured cells remained > 96% (trypan blue exclusion).

Treatment of mononuclear phagocytes with IFN-γ

Human rIFN-γ was obtained from R & D Systems. Monocytes or macrophages were prepared at 5 × 106 cells/ml in PBS containing 2% FCS and 100 μl aliquots of cell suspension were incubated for 10 min at 37°C without or with IFN-γ (10–1000 U/ml). Following incubation the cells were subjected to fixation and permeabilization before antibody addition (vide infra).

Flow cytometry

Expression of STAT-1 was analysed by indirect staining using 0·25 μg of the mouse antihuman anti-STAT-1 MoAb, or an isotype control IgG2b Ab, followed by FITC-conjugated goat antimouse IgG to detect bound Ab. Analysis of phosphorylated STAT-1 was performed by indirect staining using 0·1 μg of the rabbit antihuman antiphosphorylated STAT-1 IgG, or an isotype control IgG, followed by FITC-conjugated goat antirabbit IgG to detect bound Ab. Before analysis, cells were fixed and permeabilized as described [8]. In short, fixation reagent (100 μl Reagent A, Fix & Perm kit, Caltag) was added to untreated or IFN-γ-treated cells for 2–3 min at room temperature followed by incubation of cells with concentrated methanol at 4°C for 10 min. After washing in PBS, 100 μl permeabilization medium (Reagent B, Fix & Perm kit, Caltag) together with a primary antibody was added to the cells. After 30 min incubation at room temperature, the tubes were washed and incubated with the appropriate second antibody for 30 min at room temperature, and again washed before being resuspended in 200 μl of PBS for flow cytometry.

Samples were analysed on a modified Becton Dickinson FACStar Plus flow cytometer (Becton Dickinson, Mountain View, CA) equipped with an argon ion laser (Spectra-Physics Inc., Mountain View, CA). The 488/540 ± 10 nm (green) fluorescence intensity was detected for samples labelled with FITC-conjugated MoAb, while the 488/ > 580 nm (red) fluorescence intensity was measured in samples labelled with phycoerythrine conjugated MoAb. Monocytes or macrophages were identified on the basis of the small angle forward light scatter and side scatter intensities.

Electrophoresis and Western blotting

To determine specificity of the antihuman phosphorylated STAT-1 MoAb, SDS-PAGE was performed on 10% polyacrilamide gel [12]. Cultured monocytes (106) from adults were incubated with 1000 U/ml IFN-γ for 10 min at 37°C. Incubation was stopped by boiling the cells with sample buffer. Volumes of 25 μl were electrophoresed on 10% polyacrylamide gel. Proteins were transferred to nitrocellulose at 5°C for 2 h at 100 mA [13]. After blocking, the membranes were exposed to primary antibodies for 90 min at room temperature. For detection of primary antibodies on Western blots, peroxidase-coupled secondary antibodies were used with enhanced chemiluminescence reagents [13]. These control experiments revealed only single bands appropriate to the molecular weight of phosphorylated STAT-1 (91 kD) (data not shown).

Expression of data

Results are expressed as mean ± SEM. The symbol n refers to number of experiments, each done in duplicate or triplicate. Statistical significance was determined according to Student's t-test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dose-response and time course of STAT-1 phosphorylation in IFN-γ-stimulated mononuclear phagocytes

We studied concentration-dependent phosphorylation of STAT-1 in IFN-γ-exposed mononuclear phagocytes from newborns and adults. In Fig. 1, the extent of STAT-1 phosphorylation in monocytes and MDM as stimulated for 10 min is shown as a function of the concentration of IFN-γ. At baseline, fluorescence intensity was small in both monocytes and macrophages with no difference between adult and cord cells. Adult cells were more responsive to the activating effect of IFN-γ over the entire range of concentration tested (Fig. 1). Moreover, at all concentrations IFN-γ-induced STAT-1 phosphorylation was greater in macrophages than in monocytes (Fig. 1). Control experiments with monocytes exposed to 100 U/ml IFN-γ for varying time (1–30 min) were also performed. Maximal fluorescent intensity was detected after incubating of cells for 5–15 min (data not shown).

image

Figure 1. Concentration-dependent effect of IFN-γ on STAT-1 phosphorylation in cord (□) and adult (▪□) mononuclear phagocytes. Monocytes and macrophages were incubated for 10 min with medium alone of medium containing increasing concentrations of IFN-γ, as shown on the horizontal axis. Next, cells were fixed and permeabilized, and incubated with rabbit antihuman phosphorylated STAT-1 MoAb for 30 min. Ab binding was detected using FITC-conjugated F(ab′)2 goat antirabbit IgG. The results shown are representative of three separate experiments performed in parallel with cord and adult cells.

Download figure to PowerPoint

Effect of IFN-γ on STAT-1 phosphorylation in cord and adult monocytes

Based on results of the control experiments we compared STAT-1 phosphorylation in cord and adult monocytes exposed to 100 U/ml IFN-γ for 10 min. Experiments with cord and adult cells were performed on the same days, in parallel, under the same experimental conditions. Figure 2 shows that addition of IFN-γ to monocytes elicited a significantly lower STAT-1 phosphorylation in cord cells compared to adult monocytes (P < 0·05; fluorescence intensity range, 27–64 and 49–81 for cord and adult cells, respectively). In control experiments, comparable phosphorylation of STAT-1 could be detected after fixation and plasma membrane permeabilization of monocytes (Fig. 2).

image

Figure 2. STAT-1 phosphorylation in cord (□) and adult (▪□) monocytes exposed to IFN-γ. Monocytes were incubated for 10 min with medium alone or medium containing 100 U/ml IFN-γ. Rabbit antihuman phosphorylated STAT-1 MoAb was applied as described for Fig. 1. In isotype control experiments monocytes were incubated with rabbit IgG. Ab binding was detected by using FITC-conjugated F(ab′)2 goat antirabbit IgG. The heights of the columns represent means and the bars SEM; n = 5; *P < 0·05. Each experiment was performed on blood cells separated from different individuals (19 adults, 18 neonates) with comparable results.

Download figure to PowerPoint

Effect of IFN-γ on STAT-1 phosphorylation in cord and adult macrophages

Figure 3 shows STAT-1 phosphorylation in cord and adult macrophages. We found that both cord and adult cells showed increases in antibody binding upon activation with 100 U/ml IFN-γ compared to controls. However, treatment of adult cells with IFN-γ resulted in a significantly higher extent of STAT-1 phosphorylation compared to cord macrophages (P < 0·01; fluorescence intensity range, 56–110 and 100–165 for cord and adult cells, respectively). As shown in Figs 2 and 3, phosphorylation of STAT-1 was about half in monocytes compared to that in macrophages in both neonates and adults.

image

Figure 3. STAT-1 phosphorylation in cord (□) and adult (▪□) macrophages. Incubation with 100 U/ml IFN-γ was for 10 min. Phosphorylated STAT-1 protein detection was performed as described for Fig. 1. Rabbit IgG served as isotype control. Data represent mean ± SEM of at least five experiments; **P < 0·01. Each experiment was performed on blood cells separated from different individuals (19 adults, 18 neonates) with comparable results.

Download figure to PowerPoint

Expression of STAT-1 in cord and adult macrophages

We have explored the possibility that deficient STAT-1 phosphorylation in IFN-γ-exposed mononuclear phagocytes from the cord might be due to lower expression of STAT-1 protein in newborn cells. To this end, we used monoclonal antibodies, which bind to the native (inactive, nonphosphorylated) STAT-1 protein. As shown in Table 1, there was equivalent fluorescence intensity of STAT-1 positive cord and adult macrophages. Experiments with monocytes gave similar results (n = 4; data not shown). These data indicated that differences in STAT-1 phosphorylation in cord and adult monocytes and MDM could not be attributed to lower expression of STAT-1 protein in neonatal cells.

Table 1.  Antibody binding to STAT-1 in cord and adult macrophages
ConditionFluorescence intensity of STAT-positive macrophages*
Cord cellsAdult cellsP
  1. * Flow cytometry was performed on unstimulated (0 U/ml IFN-γ) and IFN-γ-stimulated (100 U/ml IFN-γ) macrophages from cord and adults. Incubation of macrophages with IFN-γ was for 10 min. Next, cells were permeabilized and incubated with mouse antihuman STAT-1 MoAb for 30 min. In isotype control experiments macrophages were incubated with mouse IgG2b. Ab binding was detected using FITC-conjugated F(ab′)2 goat antimouse IgG. Mean ± SEM; n = 5. Each experiments were performed on blood cells separated from different individuals.

Isotype control6 ± 48 ± 4NS
0 U/ml IFN-γ141 ± 16164 ± 20NS
100 U/ml IFN-γ264 ± 36296 ± 34NS

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The increased susceptibility of newborns to infections by viruses, bacteria, and fungi may be related to defective cell-mediated immunity mediated primarily by monocytes/macrophages and T lymphocytes. IFN-γ and IL-12 are major regulators of mononuclear phagocytes and T cells, respectively, and IFN-γ/IL-12 responses occurring early after infection constitute the most efficient form of innate immunity to intracellular pathogens [14–16]. Macrophages produce IL-12 in response to microbial products or to direct infection and the subsequent induction, by IL-12, of IFN-γ production by T and NK cells [4,16]. Neonatal T lymphocytes exposed either to mitogens or bacterial antigens seems to be deficient in their ability to produce IFN-γ[17,18]. In addition, neonates are deficient in the production of macrophage-derived IL-12, a major enhancer of IFN-γ production [19,20].

The clearest results to come from our studies are the repeated demonstrations that neonatal mononuclear phagocytes are deficient in their ability to respond to stimulation with IFN-γ. This includes the observations that neonatal macrophages cannot be fully activated to kill viable microorganisms [1,2], and that IFN-γ-induced STAT-1 phosporylation is severely deficient in cord cells compared to that of adults. The data presented in this study extend our understanding of neonatal host defense mechanisms against intracellular pathogens in demonstrating that IFN-γR-mediated signalling is markedly deficient in both monocytes and macrophages from the cord. To our knowledge, there is no previous report explaining neonatal deficiency of mononuclear phagocytic cell functions at the level of transduction. The defect in the ability of neonatal cells in signalling through the IFN-γR was relative rather than absolute, and may represent a transient immaturity. The results presented here are consistent with the failure of IFN-γ to efficiently enhance microbicidal capacity, superoxide anion generation, and TNF secretion in cultured monocytes from human neonates [1–3].

STAT-1 activation requires phosphorylation of upstream components of the IFN-γ signal transduction pathway including IFN-γR1, JAK1, and JAK2 [4,5]. The deficiency of neonatal monocyte/macrophage responses to stimulation with IFN-γ could not be explained by decreased number or ligand-binding capacity of IFN-γ receptors [1]. However, we cannot rule out the possibility that reduced phosphorylation of STAT-1 was the result of reduced phosphorylation of upstream JAK kinases and IFN-γR1. Recently, a new family that can inhibit JAK–STAT signalling pathways has been identified [21,22]. Such suppressors of cytokine signalling might be overexpressed in neonatal monocytes/macrophages. Whatever its basis, deficient STAT-1 phosphorylation has broad implication if present in vivo, because STAT-1 is a convergent point for immunological stimuli in a macrophage proinflammatory response [4–7,9,20].

The lack of adequate generation of proinflammatory cytokines and decreased responses of mononuclear phagocytes to stimulation with INF-γ may be due to a neonatal immune bias to polarized expression of Th2-like cytokines. During pregnancy, the placenta may synthesize Th2 cytokines to antagonize Th1 responses that could otherwise be harmful to the fetus. The abundant release of Th2 cytokines that act to suppress synthesis of IFN-γ at the maternal–fetal interface support this hypothesis [23,24].

In conclusion, there appears to be multiple pathways by which Th1 responses may be insufficiently developed in human newborns. Inappropriate activation of STAT-1, which is primarily, if not entirely, important for IFN-γ-dependent signalling, may be a major factor responsible for immature cellular immunity in neonatal infants. Decreased STAT-1 phosphorylation and activation may represent developmental immaturity and may contribute to the unique susceptibility of neonates to infections by intracellular pathogens.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Steven M. Holland for helpful discussions and Michael Vail for technical advice. We thank Dr Rita Káposzta, Erzsébet Nagy and Szilvia Taskó for excellent technical advice and assistance. This work was supported by grants from OTKA (T25780), and from the SOROS Foundation, Budapest, to Dr Maródi.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Maródi L, Káposzta R, Campbell DE, Polin RA, Csongor J, Johnston RB Jr. Candidacidal mechanisms in the human neonate: impaired IFN-γ activation in newborn infants. J Immunol 1994; 153:56439.
  • 2
    Maródi L, Káposzta R, Nemes É . Survival of group B Streptococcus type III in mononuclear phagocytes: Differential regulation of bacterial killing in cord macrophages by human recombinant gamma interferon and granulocyte-macrophage colony-stimulating factor. Infect Immun 2000; 68:216770.
  • 3
    Burchett SK, Weaver WM, Westall JA, Larsen A, Kronheim S, Wilson CB. Regulation of tumor necrosis factor/cachetin and IL-1 secretion in human mononuclear phagocytes. J Immunol 1988; 140:347381.
  • 4
    Bach E, Aguet M, Schreiber RD. The IFNγ receptor: a paradigm for cytokine receptor signaling. Annu Rev Immunol 1997; 15:56391.
  • 5
    Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994; 264:141521.
  • 6
    O'Shea JJ. Jaks, STATs, cytokine signal transduction, and immunoregulation. Immunity 1997; 7:111.
  • 7
    Decker T & Kovarik P. Serine phosphorylation of STATs. Oncogene 2000; 15:262837.
  • 8
    Fleisher TA, Dorman SE, Anderson JA, Vail V, Brown MR, Holland SM. Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin Immunol 1999; 90:42530.DOI: 10.1006/clim.1998.4654
  • 9
    Ihle JN. Cytokine receptor signaling. Nature 1995; 377:5914.
  • 10
    Maródi L, Forehand JR, Johnston RB Jr. Mechanisms of host defense against candida species. II. Biochemical basis for the killing of candida by mononuclear phagocytes. J Immunol 1991; 146:27904.
  • 11
    Káposzta R, Maródi L, Hollinshead M, Gordon S, Da Silva RP. Rapid recruitment of late endosomes and lysosomes in mouse macrophages ingesting Candida albicans. J Cell Sci 1999; 112:323748.
  • 12
    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:6805.
  • 13
    Murányi A, Erdõdi F, Ito M, Gergely P, Hartshorne DJ. Identification and localization of myosin phosphatase in human platelets. Biochem J 1998; 330:22531.
  • 14
    Biron CA & Gazzileni RT. Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome. Curr Opin Immunol 1995; 7:48596.
  • 15
    Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of Th1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 1993; 260:5479.
  • 16
    Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995; 13:25176.
  • 17
    Bryson YJ, Winter HS, Gard SE, Fisher TJ, Stiehm ER. Deficiency of immune interferon production by leukocytes of normal newborns. Cell Immunol 1980; 55:191200.
  • 18
    Wilson CB & Westal J. Activation of neonatal and adult human macrophages by alpha, beta, and gamma interferon. Infect Immun 1985; 49:3516.
  • 19
    Joyner JL, Augustine NH, Taylor KA, La Pine TR, Hill HR. Effects of group B streptococci on cord and adult mononuclear cell interleukin-12 and interferon-γ mRNA accumulation and protein secretion. J Infect Dis 2000; 182:9747.
  • 20
    Lee SM, Suen Y, Chang L. Decreased interleukin-12 from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-γ, natural killer, and lymphokine-activated killer activity by IL-12 in cord blood mononuclear cells. Blood 1996., 1996; 88:9459.
  • 21
    Starr R, Willson TA, Viney EM et al. A family of cytokine-inducible inhibitors of signaling. Nature 1997; 387:91721.
  • 22
    Endo TA, Masuhara M, Yokouchi M et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 1997; 387:9214.
  • 23
    Wegmann TG, Lin H, Guilbert LJ, Mosman TR. Bidirectional cytokine interactions in the maternal–fetal relationship: is succesful pregnancy a Th2 phenomenon? Immunol Today 1993; 14:3536.
  • 24
    Guleria I & Pollard JW. The trophoblast is a component of the innate immune system during pregnancy. Nature Med 2000; 6:58993.
Footnotes
  1. Correspondence: Dr László Maródi, Department of Infectology and Paediatric Immunology, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, POB:32, Hungary.