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Keywords:

  • CCAAT/enhancer binding protein β;
  • IL-12p40;
  • MAP kinases;
  • microglia;
  • NF-κB;
  • TNF-α

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

The present study was undertaken to explore the role of interleukin-12 (IL-12) p40 in the expression of TNF-α in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-α and the expression of TNF-α mRNA in BV-2 microglial cells. In addition to BV-2 microglial cells, p70, p402 and p40 also induced the production of TNF-α in mouse primary microglia and peritoneal macrophages. As the activation of both NF-κB and CCAAT/enhancer binding protein β (C/EBPβ) is important for the expression of TNF-α in microglial cells, we investigated the effect of p40 on the activation of NF-κB as well as C/EBPβ. Activation of NF-κB as well as C/EBPβ by p40 and inhibition of p40-induced expression of TNF-α by Δp65, a dominant-negative mutant of p65, and ΔC/EBPβ, a dominant-negative mutant of C/EBPβ, suggests that p40 induces the expression of TNF-α through the activation of NF-κB and C/EBPβ. In addition, we show that p40 induced the activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Interestingly, PD98059, an inhibitor of ERK, inhibited p40-induced expression of TNF-α through the inhibition of C/EBPβ, but not that of NF-κB, whereas SB203580, an inhibitor of p38 MAPK, inhibited p40-induced expression of TNF-α through the inhibition of both NF-κB and C/EBPβ. This study delineates a novel biological function of p40 in inducing TNF-α in microglia and macrophages.

Abbreviations used
C/EBPβ

CCAAT/enhancer binding protein β

EAE

experimental allergic encephalomyelitis

ERK

extracellular signal-regulated kinase

IL-12

interleukin-12

iNOS

inducible nitric oxide synthase

LPS

lipopolysaccharides

MAPK

mitogen-activated protein kinase

MRI

magnetic resonance imaging

MS

multiple sclerosis

STAT

signal transducer and activator of transcription

TNF-α

tumor necrosis factor-α

Tumor necrosis factor-α (TNF-α), produced mainly by cells of the macrophage lineage including microglia in the CNS, is a potent inflammatory mediator. It is involved at multiple levels of immune regulation (Vassalli 1992) and has also been strongly implicated in the pathogenesis of several neuroinflammatory and demyelinating diseases (Tyor et al. 1993; Raine 1995). Although recent clinical trials shows that TNF-α antagonists worsens clinical symptoms in patients with multiple sclerosis (MS) (Wiendl and Hohlfeld 2002), analysis of CSF from MS patients has shown increased levels of TNF-α compared with normal control, and levels of TNF-α in the CSF of MS patients also correlate with disease severity (Sharief and Hentges 1991). Most relevant to a role in demyelination is increasing evidence that the TNF-α and its receptor system is involved in triggering oligodendrocyte death. Both the p55TNFR and the p75TNFR are selectively expressed on oligodendrocytes located at the edge of active MS lesions (Bonetti and Raine 1997) and several studies have shown that TNF-α alone can kill cultured oligodendrocytes (Raine 1995; D'Souza et al. 1996).

In contrast, interleukin-12 (IL-12) plays a critical role in the early inflammatory response to infection and in the generation of T-helper type 1 Th-1 cells, which favor cell-mediated immunity (Hsieh et al. 1993). Recently, it has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of a number of auto-immune inflammatory diseases (e.g. multiple sclerosis, arthritis, insulin-dependent diabetes) (Zipris et al. 1996; Gately et al. 1998). IL-12 consists of a heavy chain (p40) and a light chain (p35) linked covalently by disulfide bonds to give rise to a heterodimeric (p70) molecule (Gately et al. 1998). It is known that the heterodimeric p70 molecule is the bioactive IL-12 cytokine and both subunits must be co-expressed in the same cell to generate the bioactive form (Gately et al. 1998). However, the level of p40 is much higher than that of p35 in IL-12 producing cells (Gately et al. 1998). Again, several reports (Fassbender et al. 1998; Gately et al. 1998; Van Boxel-Dezaire et al. 1999) indicate that the level of p40 mRNA in the CNS of patients with MS is much higher than the CNS of control subjects, whereas the level of p35 mRNA is about the same or decreases compared with that of controls. Similarly, in mice with experimental allergic encephalomyelitis (EAE), an animal model of MS, expression of p40 mRNA, but not that of p35 mRNA, increases in brain and spinal cord (Bright et al. 1998). These observations suggest that p40 has a key function as a monomer or homodimer, not just as part of the p40 : p35 heterodimer forming functional IL-12. However, biological functions of p40 monomer or homodimer in the CNS of MS patients and EAE animals are poorly understood.

Herein, we report the first evidence that p40 monomer markedly induces the expression of TNF-α through the activation of NF-κB and CCAAT/enhancer binding protein β (C/EBPβ) in mouse microglia.

Reagents

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

Fetal bovine serum, Hank's balanced salt solution (HBSS) and Dulbecco's modified Eagle's medium (DMEM)/F-12 were from Mediatech, Valley Park, MO, USA. Recombinant mouse IL-12 p70 and p402 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA. 125I-labeled protein A and [α-32P]dCTP were obtained from DuPont–NEN, Boston, MA, USA. The dominant-negative mutant of C/EBPβ (ΔC/EBPβ) was kindly provided by Dr Steve Smale of the University of California at Los Angeles.

Isolation of mouse microglia

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

Microglial cells were isolated from mixed glial cultures according to the procedure of Giulian and Baker (1986). Briefly, on day 7–9 the mixed glial cultures were washed three times with DMEM/F-12 and subjected to a shake at 240 r.p.m. for 2 h at 37°C on a rotary shaker (Lab-Line, Melrose Park, IL, USA). The floating cells were washed and seeded on to plastic tissue culture flasks and incubated at 37°C for 2 h. The attached cells were removed by trypsinization and seeded on to new plates for further studies. Of this preparation, 90–95% was found to be positive for Mac-1 surface antigen. For the induction of TNF-α production, cells were stimulated with IL-12 p70, p402 and p40 in serum-free DMEM/F-12.

Mouse BV-2 microglial cells (a kind gift from Virginia Bocchini of University of Perugia) were also maintained and induced as indicated above.

Isolation of mouse peritoneal macrophages

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

Resident macrophages were obtained from mouse by peritoneal lavage with sterile Roswell Park Medical Institute (RPMI) 1640 medium containing 1% fetal bovine serum and 100 μg/mL gentamicin (Pahan et al. 1997). Cells were washed three times with RPMI 1640 at 4°C and were maintained at 37°C in a humidified incubator containing 5% CO2 in air. Macrophages at a concentration of 106/mL in RPMI 1640 medium containing l-glutamine and gentamicin were added in volumes of 0.5 mL to each well of 12-well plates. After 1 h, non-adherent cells were removed by washing and 0.5 mL of serum-free RPMI 1640 medium with various stimuli were added to the adherent cells.

RNA isolation and northern blot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

Cells were taken out of the culture dishes directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories Inc., Houston, TX, USA), and total RNA was isolated according to the manufacturer's protocol. For northern blot analyses, 20 μg of total RNA was electrophoresed on 1.2% denaturing formaldehyde-agarose gels, electrotransferred to Hybond nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and hybridized at 68°C with 32P-labeled cDNA probe using Express Hyb hybridization solution (CLONTECH, Palo Alto, CA, USA) as described by the manufacturer. The cDNA probe for TNF-α has been described earlier (Pahan et al. 1997; Jana et al. 2002). After hybridization, the filters were washed two or three times in solution I (2 × SSC, 0.05% SDS) for 1 h at room temperature followed by solution II (0.1 × SSC, 0.1% SDS) at 50°C for a further 1 hour. The membranes were then dried and exposed to X-ray films (Kodak). The same amount of RNA was hybridized with probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

Nuclear extracts were prepared using the method of Dignam et al. (1983) with slight modifications. Cells were harvested, washed twice with ice-cold PBS, and lysed in 400 μL of buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 2 mm MgCl2, 0.5 mm DTT, 1 mm phenylmethylsulfonyl fluoride (PMSF), 5 μg/mL aprotinin, 5 μg/mL pepstatin A, and 5 μg/mL leupeptin) containing 0.1% Nonidet P-40 for 15 min on ice, vortexed vigorously for 15 s, and centrifuged at 16 000 g for 30 s. The pelleted nuclei were resuspended in 40 μL of buffer B [20 mm HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm DTT, 1 mm PMSF, 5 μg/mL aprotinin, 5 μg/mL pepstatin A, and 5 μg/mL leupeptin]. After 30 min on ice, lysates were centrifuged at 16 000 gfor 10 min. Supernatants containing the nuclear proteins were diluted with 20 μL of modified buffer C [20 mm HEPES, pH 7.9, 20% (v/v) glycerol, 0.05 m KCl, 0.2 mm EDTA, 0.5 mm DTT, and 0.5 mm PMSF] and stored at 70°C until use. Nuclear extracts were used for EMSA using 32P-end-labeled double-stranded [NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Promega, Madison, WI, USA) and C/EBPβ, 5′-TGCAGATTGCGCAATCTGCA-3′ (Santa Cruz Biotechnology, Santa Cruz, CA, USA)] oligonucleotides as described earlier (Pahan et al. 1997, 2001). Double-stranded mutated [NF-κB, 5′-AGTTGAGGCGACTTTCCCAGGC-3′, and C/EBPβ, 5′-TGCAGAGACTAGTCTCTGCA-3′ (Santa Cruz Biotechnology)] oligonucleotides were used to verify the specificity of NF-κB and C/EBPβ binding to DNA.

Assay of transcriptional activities of NF-κB and C/EBPβ

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

To assay the transcriptional activities of NF-κB and C/EBPβ, cells at 50–60% confluence were transfected with either pBIIX-Luc, an NF-κB-dependent reporter construct or pC/EBPβ-Luc using the LipofectAMINE Plus method (Life Technologies) (Jana et al. 2001, 2002; Liu et al. 2002). All transfections included 50 ng/μg total DNA of pRL-TK (a plasmid encoding Renilla luciferase, used as transfection efficiency control; Promega). After 24 h of transfection, cells were treated with p40 for 6 h. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract according to standard instructions provided in the Dual Luciferase Kit (Promega) in a TD-20/20 Luminometer (Turner Designs). Relative luciferase activity of cell extracts was typically represented as (firefly luciferase value/Renilla luciferase value) × 10−3.

Assay of ERK and p38 MAPK

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

BV-2 microglial cells (70–80% confluent) were stimulated under serum-free condition with 10 ng/mL of p40. The ERK and p38 MAPK activities were measured using assay kits obtained from Cell Signaling Technology, Beverly, MA, USA. Briefly, cells were harvested under non-denaturing conditions at different time intervals and cell lysates were prepared. Activated form of ERK and p38 were pulled down from the cell lysate by immunoprecipitation using immobilized phospho-ERK and phospho-p38 monoclonal antibodies, respectively. The pellets were washed twice with kinase buffer and finally resuspended with 50 μL kinase buffer supplemented with 200 μm ATP and either ELK-1 fusion protein (for ERK) or ATF-2 fusion protein (for p38). Following incubation at 30°C for a period of 30 min, samples were analyzed by western blot using antibodies against phospho ELK-1 or phospho ATF-2.

IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

IL-12 is a potent regulator of cell-mediated immune responses (Gately et al. 1998). To understand the role of IL-12 in the induction of TNF-α, we examined the effect of IL-12 p70 on the production of TNF-α in mouse BV-2 microglial cells. Results in Fig. 1 clearly show that mouse IL-12 markedly induced the production of TNF-α. Although the production of TNF-α was negligible at 2 h of stimulation by p70, the induction of TNF-α production started at 4 h of stimulation and peaked at 6 h (Fig. 1a). Dose-dependent studies also showed that p70 was able to induce the production of TNF-α even at 1 or 2 ng/mL concentration (Fig. 1b). However, the maximum induction was observed at 5 or 10 ng/mL of p70.

image

Figure 1. Induction of TNF-α production by IL-12 p70, p402 and p40 in mouse BV-2 microglial cells, primary microglia and peritoneal macrophages. (a) BV-2 cells were stimulated with 10 ng/mL of p70, p402 or p40 under serum-free condition. At different times (h) of incubation, supernatants were used for TNF-α assay by ELISA as mentioned in Materials and methods. (b) BV-2 cells were stimulated with different concentrations of p70, p402 or p40 under serum-free condition. After 8 h of incubation, supernatants were used for TNF-α assay. Mouse primary microglia (c) and peritoneal macrophages (d) were stimulated with 10 ng/mL of p70, p402 or p40 under serum-free condition. At different times (h) of incubation, supernatants were used for TNF-α assay by ELISA. Data are expressed as the mean of two separate experiments and duplicates were run within the same experiment.

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It is known that biologically active IL-12 p70 is comprised of disulfide-bonded 35 kDa (p35) and 40 kDa (p40) subunits (Gately et al. 1998). However, the p40, but not the p35, mRNA is expressed in excessive amount in neural tissues of MS and EAE (Gately et al. 1998; Van Boxel-Dezaire et al. 1999). Again, under physiological conditions, IL-12 p40 exists as both monomer and dimer (Gately et al. 1998). Therefore, to understand the role of IL-12 p40 in the induction of TNF-α, we examined the effect of mouse p40 homodimer (p402) and human p40 monomer (p40) on the induction of TNF-α production in BV-2 microglial cells. Similar to p70, both p402 and p40 markedly induced the production of TNF-α in time- and dose-dependent manner (Figs 1a and b). Although at 2 h of stimulation, both p402 and p40 were ineffective in inducing the production of TNF-α, marked TNF-α production was observed at 4 h of stimulation with the maximum production observed at 6 h (Fig. 1a). It is evident from Fig. 1(b) that the maximum induction of TNF-α production in response to p402 and p40 was observed at 10 or 15 ng/mL. However, p70 was most efficient in inducing TNF-α in BV-2 microglial cells followed by p40 and p402 (Figs 1a and b).

To understand whether IL-12 p70, p402 and p40 are also able to induce the production of TNF-α in primary cells, we examined the effect of these molecules on the production of TNF-α in mouse primary microglia and peritoneal macrophages. Consistent to the induction of TNF-α in BV-2 microglial cells, IL-12 p70 and p402 and p40 markedly induced the production of TNF-α in mouse primary microglia (Fig. 1c) and peritoneal macrophages (Fig. 1d). Similar to BV-2 microglial cells, the induction of TNF-α production in response to p70, p402 and p40 was very low at 2 h of stimulation in both primary microglia and peritoneal macrophages. However, marked induction of TNF-α production was observed at 4 h of stimulation followed by the maximum induction at 6 h. Similar to BV-2 microglial cells, p70 was the most efficient in inducing TNF-α followed by p40 and p402 in primary microglia (Fig. 1c). In contrast, in peritoneal macrophages, p402 was the most efficient in inducing TNF-α followed by p40 and p70 (Fig. 1d).

Next, to understand the mechanism of induction of TNF-α production, we examined the effect of p70, p402 and p40 on the mRNA level of TNF-α. Northern blot analysis for TNF-α mRNA of p70-, p402- and p40-stimulated BV-2 microglial cells clearly showed that these molecules dose-dependently induced the expression of TNF-α mRNA (Figs 2a–c). The maximum induction of TNF-α mRNA was observed at 5 ng/mL in the case of p70 and at 10 ng/mL in the case of p402 or p40 (Figs 2a–c). The time-dependent analysis of TNF-α mRNA showed that the induction of TNF-α mRNA began at 2 h of stimulation of p40 and peaked at 4 h (Fig. 2d). Induction of TNF-α production within 4 h of p40 stimulation (Fig. 1), and that of TNF-α mRNA expression within 2 h of p40 stimulation (Fig. 2d), suggest that the effect of p40 on TNF-α expression is direct.

image

Figure 2. IL-12 p70, p402 and p40 induce the expression of TNF-α mRNA in BV-2 microglial cells. Cells were stimulated with different concentrations of p70 (a), p402 (b) or p40 (c) under serum-free condition. After 4 h of incubation, cells were taken out directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) to the plates for isolation of total RNA, and northern blot analysis for TNF-α mRNA was carried out as described in Materials and methods. (d) Cells were stimulated with 10 ng/mL of p40 under serum-free condition. At different time (h) of incubation, northern blot analysis for TNF-α mRNA was carried out.

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Bacterial lipopolysaccharides (LPS) is a very potent inducer of TNF-α in different cell types (Pahan et al. 1997, 1998). As IL-12 p70, p402 and p40 alone are able to induce the expression of TNF-α markedly in microglial cells, one might consider the possibility of LPS contamination in p70 or p40 preparations. However, mouse IL-12 p70 and p402 and human p40 monomer are Sf-21 (insect cell)-expressed recombinant proteins (R & D Systems). Therefore, these preparations should be almost devoid of LPS. To further confirm the induction of TNF-α by p40, we examined the effect of transient expression of mouse p40 cDNA (Pahan et al. 2001) on the production of TNF-α in BV-2 glial cells. Consistent to the induction of TNF-α by p402 and p40, expression of different amounts of p40 cDNA, but not that of empty vector, induced the production of TNF-α (data not shown).

The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

It has been shown that activation of both NF-κB and C/EBPβ are involved in the expression of TNF-α in macrophages and monocytes following LPS stimulation (Wedel et al. 1996; Yao et al. 1997). These findings prompted us to ask whether activation of NF-κB and C/EBPβ may be responsible for the TNF-α release following p40 stimulation of BV-2 microglial cells. As activation of both NF-κB and C/EBPβ plays an important role in the induction of TNF-α, to understand the basis of expression of TNF-α, we examined the effect of p40 on the activation of both NF-κB and C/EBPβ in BV-2 microglial cells. Activation of NF-κB and C/EBPβ was monitored by both DNA binding and transcriptional activities. DNA binding activities of NF-κB and C/EBPβ were evaluated by the formation of a distinct and specific complex in a gel shift DNA binding assay. Treatment of BV-2 glial cells with 10 ng/mL of p40 resulted in the induction of DNA binding activity of NF-κB at 60 and 90 min of stimulation (Fig. 3a). This gel shift assay detected a specific band in response to p40 that was not found in the case of mutated double-stranded oligonucleotide, suggesting that p40 induces the DNA-binding activity of NF-κB. We then tested the effect of p40 on NF-κB-dependent transcription of luciferase in BV-2 glial cells, using the expression of luciferase from a reporter construct, pBIIX-Luc, as an assay. Consistent with the effect of p40 on the DNA binding activity of NF-κB, p40 also induced NF-κB-dependent transcription of luciferase in a dose-dependent fashion with the maximum induction observed at 10 ng/mL (Fig. 3b). Similarly, p40 also induced the DNA-binding activity of C/EBPβ in different minutes of stimulation (Fig. 3c). The specific band in response to p40 (upper arrow) was observed only in case of the double-stranded wild type oligonucleotides but not in case of the mutated ones (Fig. 3c). It is evident from C/EBPβ-dependent luciferase assay in Fig. 3(d) that p40 dose-dependently induced the transcriptional activity of C/EBPβ.

image

Figure 3. Activation of NF-κB and C/EBPβ by p40 in BV-2 microglial cells. Cells were stimulated with 10 ng/mL of p40 under serum-free conditions. After different minutes of stimulation, cells were taken out to prepare nuclear extracts, and nuclear proteins were used for EMSA to determine DNA-binding activities of NF-κB (a) and C/EBPβ (c) as described in Materials and methods. The upper and lower arrows in (a) indicate the induced NF-κB band and the unbound probe, respectively. However, the upper, middle and lower arrows in (c) indicate the induced C/EBPβ band, non-specific band, and the unbound probe, respectively. Cells plated at 50–60% confluence in 12-well plates were co-transfected with 0.5 μg of either pBIIX-Luc (b) or pC/EBPβ-Luc (d) and 25 ng of pRL-TK. After 24 h of transfection, cells were stimulated with different concentrations of p40 for 6 h under serum-free condition. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract as described in Materials and methods. Data are mean ± SD of three different experiments.

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Next, we examined if activation of both NF-κB and C/EBPβ is important for the expression of TNF-α in cells stimulated with p40. Overexpression of dominant-negative molecules provides an effective tool with which to investigate the in vivo functions of different transcription factors or signaling molecules. NF-κB was inhibited by a dominant-negative mutant of p65 (Δp65) (Zhong et al. 1997). Similarly, we used the dominant-negative mutant of C/EBPβ (ΔC/EBPβ) (Descombes and Schibler 1991) to inhibit the activation of C/EBPβ. Earlier we have shown that these dominant-negative mutants inhibit their respective target molecule in BV-2 microglial cells stimulated by the combination of IFN-γ and anti-CD40 (Jana et al. 2002). Expression of Δp65 and ΔC/EBPβ, but not that of the empty vector, inhibited p40-induced production of TNF-α(Fig. 4a) by 33–37%. Even after combining both forms of dominant-negative mutants during transient transfection, we were unable to achieve more than 40% inhibition (data not shown), and the reason behind this lies in the lack of achieving sufficient transfection efficiency. Earlier, we have shown that during successful transient transfection experiments by Lipofectamine Plus, transfection efficiency varies from 33 to 44% (Pahan et al. 2000). Consistent to the inhibition of TNF-α production, both Δp65 and ΔC/EBPβ inhibited the expression of TNF-α mRNA (Fig. 4b) in cells stimulated with p40. These studies suggest that activation of both NF-κB and C/EBPβ is important for p40-induced expression of TNF-α.

image

Figure 4. Dominant-negative mutants of p65 (Δp65) and C/EBPβ (ΔC/EBPβ) inhibit the expression of TNF-α in p40-stimulated BV-2 microglial cells. (a) Cells were transfected with 0.5 μg of either Δp65 or ΔC/EBPβ using Lipofectamine-plus. After 24 h of transfection, cells were stimulated with 10 ng/mL p40 for 12 h under serum-free conditions, and supernatants were used for TNF-α assay. Data are mean ± SD of three different experiments. bp < 0.05 versus p40. (b) Similarly, after 24 h of transfection, cells were stimulated with 10 ng/mL p40 under serum-free condition. After 4 h of incubation, northern blot analysis for TNF-α mRNA was carried out.

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Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

In eukaryotic cells, an important group of signaling pathways is the mitogen-activated protein kinase (MAPK) signaling cascades. As the activation of MAPK pathways such as ERK and p38 MAPK (p38) by LPS and other stimuli represents a potential signaling mechanism for TNF-α production during the inflammatory response (Rutault et al. 2001; Ye et al. 2001), we investigated the role of ERK and p38 in p40-induced expression of TNF-α. However, it was not known whether p40 is able to activate ERK and p38 in any cell types. Therefore, at first, we examined the effect of p40 on the activation of these kinases in BV-2 microglial cells. Interestingly, p40 alone induced the activation of both ERK (Fig. 5a) and p38 (Fig. 5b). The activation of both the kinases began at 5 min of stimulation (Fig. 5). However, activation of ERK peaked at 30–45 min of stimulation whereas activation of p38 peaked at 15 min (Fig. 5). These results suggest that, similar to other pro-inflammatory cytokines as described elsewhere, p40 alone can also induce MAPK pathways.

image

Figure 5. Activation of ERK and p38 MAP kinases by p40 in BV-2 microglial cells. Cells were stimulated with 10 ng/mL p40 under serum-free condition. At different minutes of incubation, activities of ERK (a) and p38 MAP kinase (b) were assayed as described in Materials and methods.

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Next, we investigated the role of ERK and p38 in p40-induced expression of TNF-α using specific pharmacological inhibitors of MEK-ERK (PD98059) and p38 (SB203580). The cells were pre-treated with different concentrations of PD98059 or SB203580 for 1 h before stimulation by p40. Both PD98059 and SB203580 were dissolved in Me2SO. These drugs were added to the cell culture at a final Me2SO concentration of 0.02–0.06%. Me2SO (0.06%) was used as a control. Both PD98059 and SB203580 dose-dependently inhibited the production of TNF-α(Fig. 6a) and the expression of TNF-α mRNA (Fig. 6b) in p40-stimulated cells. However, SB203580 was a more potent inhibitor of p40-induced expression of TNF-α than PD98059 (Fig. 6). The inhibition by PD98059 and SB203580 was not from Me2SO because Me2SO alone did not exhibit any inhibitory effect at the highest concentration used in our study. These experiments suggest that p40 induces the expression of TNF-α through ERK and p38. Because p40 also involved NF-κB and C/EBPβ to induce TNF-α, we next examined the effect of PD98059 and SB203580 on p40-induced activation of NF-κB and C/EBPβ. Interestingly, PD98059 at different doses tested had no effect on p40-mediated activation of NF-κB (Fig. 7a). In sharp contrast, SB203580 markedly inhibited p40-induced activation of NF-κB (Fig. 7a). However, both PD98059 and SB203580 dose-dependently inhibited the activation of C/EBPβ (Fig. 7b). These experiments suggest that PD98059 inhibits p40-induced expression of TNF-α by inhibiting the activation of only C/EBPβ, but not that of NF-κB, whereby SB203580 inhibits the expression of TNF-α by inhibiting the activation of both NF-κB and C/EBPβ.

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Figure 6. PD98059 and SB203580 inhibit the expression of TNF-α in p40-stimulated BV-2 microglial cells. Cells pre-incubated with different concentrations of PD98059 and SB203580 for 1 h received 10 ng/mL p40 under serum-free condition. (a) After 12 h of stimulation, supernatants were used for TNF-α assay by ELISA. Data are mean ± SD of three different experiments. ap < 0.05 versus p40; bp < 0.005 versus p40; cp < 0.001 versus p40. (b) After 4 h of stimulation, northern blot analysis for TNF-α mRNA was carried out.

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image

Figure 7. Effect of PD98059 and SB203580 on the activation of NF-κB and C/EBPβ in p40-stimulated BV-2 microglial cells. Cells were co-transfected with 25 ng of pRL-TK and 0.5 μg of either pBIIX-Luc (a) or pC/EBPβ-Luc (b). After 24 h of transfection, cells were incubated with different concentrations of PD98059 and SB203580 for 1 h followed by the stimulation with 10 ng/mL of p40 for 6 h under serum-free condition. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract. Data are mean ± SD of three different experiments. ap < 0.001 versus control; bp < 0.005 versus p40; cp < 0.001 versus p40.

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IL-12 p70, p402 and p40 used under these experimental conditions had no effect on the viability of BV-2 microglial cells, measured by trypan blue exclusion. Therefore, the conclusions drawn in this study are not due to any change in viability of the cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References

Microglia are considered as CNS-resident professional macrophages. Activation of microglia has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease (AD), Creutzfeld-Jacob disease, HIV-dementia, and multiple sclerosis (MS) (Gonzalez-Scarano and Baltuch 1999). Upon activation, microglia produce and secrete potentially neurotoxic pro-inflammatory cytokines including TNF-α (Gonzalez-Scarano and Baltuch 1999) that play a role in demyelination of MS patients. However, the mechanism by which TNF-α is produced in MS brain is unclear. Although LPS is a potent inducer of TNF-α in microglia, it has not been demonstrated to have a physiological relevance in MS (Meda et al. 1995). IFN-γ can also induce TNF-α in microglia (Meda et al. 1995), however, microglia isolated from adult human brain tend to be a poor source of TNF-α in response to IFN-γ (Becher et al. 1996). Results presented in this manuscript clearly establish the conclusion that heterodimeric IL-12 p70, homodimeric p402 and monomeric p40 are potent inducers of TNF-α in mouse microglia and macrophages. Earlier we have demonstrated that IL-12 p70 and p40 are able to induce iNOS in microglia (Pahan et al. 2001). Therefore, p40 monomer or homodimer may participate in the pathophysiology of MS and EAE by inducing iNOS and pro-inflammatory cytokine-like TNF-α from microglia. Consistently, recent studies have shown that it is p40 but not p35, which is involved in the disease process of EAE (Becher et al. 2002).

The level of p40 is much higher (5- to 500-fold) than that of the heterodimeric p70 in IL-12-producing cells (Gately et al. 1998). This excess p40 produced either in vitro in activated cells or in vivo in serum of endotoxin-treated mice exists as both dimer (20–40%) and monomer (the remainder) (Gately et al. 1998). Recently, it has been shown that p40 can also partner with p19 and the resulting heterodimer (IL-23) plays an important role in CNS inflammation that perhaps supercedes the role of IL-12 (Cua et al. 2003). Although the biological role of the monomeric as well as the dimeric form of p40 is not known, it has been suggested that p402 may act as a physiologic regulator of bioactive IL-12 p70 as p402 possesses the IL-12 antagonist activity (Germann et al. 1995; Gately et al. 1998). Therefore, the induction of TNF-α by p402 suggests that p402 exhibits the IL-12 antagonist activity possibly through the induction of TNF-α. However, our observation that both p402, p40 (with no IL-12 antagonist activity) and the so-called bioactive IL-12 (heterodimeric p70) induce the expression of TNF-α preclude this possibility. If TNF-α mediates the IL-12 antagonist activity of p402, then IL-12 itself can antagonize its own function through the induction of TNF-α. Although here we demonstrate that p40 leads to the activation of microglia which in turn secrete TNF-α, a very recent report by Dalpke et al. (2002) suggests that bacterial DNA stimulates microglia to release both p40 and TNF-α simultaneously. Therefore, it is possible that these cytokines can act in parallel during neuroinflammation.

The signaling events in the induction of TNF-α are not completely established so far. The presence of a consensus sequence in the promoter region of TNF-α for the binding of NF-κB (Udalova et al. 1998) and the inhibition of TNF-α expression with the inhibition of NF-κB activation (Pahan et al. 1998; Heiss et al. 2001) establishes an essential role of NF-κB activation in the induction of TNF-α. Similarly, presence of C/EBPβ binding site in the promoter of TNF-α and the inhibition of TNF-α expression with the inhibition of C/EBPβ suggest a role for C/EBPβ in the expression of TNF-α (Wedel et al. 1996; Yao et al. 1997). Results presented in this manuscript clearly demonstrate that activation of both NF-κB and C/EBPβ is essential for p40-mediated induction of TNF-α in mouse microglial cells. Firstly, p40 alone induced activation of NF-κB and C/EBPβ. Secondly, overexpression of Δp65 and ΔC/EBPβ, dominant-negative mutants, inhibited the expression of TNF-α in BV-2 glial cells stimulated with p40.

At present, it is unclear how p40 activates NF-κB and C/EBPβ to induce TNF-α in microglial cells. IL-12 p402 has been shown to antagonize bioactive IL-12 p70 by binding to the IL-12 receptor complex (Gately et al. 1998). The high affinity IL-12 receptor is composed of a low affinity IL-12Rβ1 combined with a low affinity IL-12Rβ2 (Gately et al. 1998). It appears that p402 binds to IL-12Rβ2 rather than IL-12Rβ1 whereas bioactive IL-12 binds the receptor complex on T cells with high affinity (Gately et al. 1998). In contrast, p40 reportedly does not have any IL-12 p70-antagonizing activity and does not bind to the IL-12 receptor complex (Gately et al. 1998). However, it is possible that p40 similar to p402 may bind to IL-12Rβ2 to induce signaling events. MAPKs are Ser-Thr kinases that have been shown to activate a number of transcription factors in different cell types in response to various stimuli (Dong et al. 2002). We have found that p40 induces the activation of ERK and p38 within an interval of minutes. Interestingly, using pharmacological inhibitors, we have elucidated that p40-induced ERK couples to only C/EBPβ but not to NF-κB, whereas that p38 couples to both NF-κB and C/EBPβ. As activation of both NF-κB and C/EBPβ are essential for p40-induced expression of TNF-α, SB203580, an inhibitor of p38, inhibited p40-induced expression of TNF-α more potently than PD98059, an inhibitor of ERK (Fig. 6). Taken together, our studies suggest p40 activates ERK and p38, that ultimately couple to NF-κB and C/EBPβ to induce the expression of TNF-α.

IL-12 is known to induce the activation of signal transducer and activator of transcription (STAT)-4 in T cells through the IL-12Rβ1/IL-12Rβ2 complex (Gately et al. 1998; Verhagen et al. 2000). Verhagen et al. (2000) have recently reported that expression of IL-12Rβ2 alone was insufficient to induce STAT4 activation in response to IL-12. Interestingly, they have also shown that IL-12-induced IFN-γ production through IL-12Rβ2 (in IL-12Rβ1-deficient T cells) could be inhibited by SB203580, the p38 MAP kinase inhibitor, and U0126, the MAP kinase kinase (MEK) 1/2 inhibitor, suggesting the involvement of MAP kinases in STAT4-independent IL-12 signaling pathway. Therefore, it is possible that p40 induces MAP kinase-dependent signaling events for the expression of TNF-α through IL-12Rβ2. Further works are underway to delineate receptor(s) involved in p70-, p40- or p402-induced signaling events for the expression of TNF-α.

Although the release of p40 monomer/homodimer has not been directly demonstrated in human cells, Fassbender et al. 1998) have demonstrated an increased (up to 1000-fold) compartmentalized release of the p40 subunit, but not of the heterodimer p70, in MS patients. Release of p40 correlates with classic markers of CNS inflammation (CSF cell counts, immunoglobulin G index) and is significantly increased in patients with gadolinium-enhancing plaques on MRI. It is apparent that this huge pool of p40 contains not only the newly described heterodimer IL-23, but also p40 monomer and dimer. In addition, we have recently found that p40 monomer and homodimer stimulates the expression of iNOS in human primary astrocytes (unpublished observation). Therefore, our findings may not necessarily apply to the murine system; p40 monomer and homodimer may induce/potentiate the neural injury in the CNS of patients with neuroinflammatory disorders through the induction of TNF-α production.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Isolation of mouse microglia
  6. Isolation of mouse peritoneal macrophages
  7. Expression of mouse p40 cDNA in BV-2 microglial cells
  8. Assay for TNF-α synthesis
  9. RNA isolation and northern blot analysis
  10. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
  11. Assay of transcriptional activities of NF-κB and C/EBPβ
  12. Assay of ERK and p38 MAPK
  13. Results
  14. IL-12 p70, p402 and p40 induce the expression of TNF-α in BV-2 microglial cells
  15. The p40 monomer induces the expression of TNF-α through the activation of NF-κB and C/EBPβ in BV-2 microglial cells
  16. Role of ERK and p38 MAPK in p40-mediated expression of TNF-α in BV-2 microglial cells
  17. Discussion
  18. Acknowledgements
  19. References
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