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Keywords:

  • B cells: irradiated, LPS-stimulated;
  • CD80: up-regulation;
  • NF-κB: nuclear factors;
  • oxidative stress

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

We have previously demonstrated irradiation-induced up-regulation of CD80 expression in A20-HL B lymphoma cells by inducing expression of tumour necrosis factor-α (TNF-α) and CD154. In the present study, we investigated whether irradiation also up-regulates CD80 expression in mouse spleen B cells. Because freshly prepared spleen B cells are highly sensitive to irradiation, we employed spleen B cells stimulated with lipopolysaccharide (LPS-B cells). X-irradiation (8 Gy) followed by incubation (9–12 hr) highly and selectively up-regulated CD80 expression in LPS-B cells, whereas the same treatment slightly increased expression of CD54 and did not affect expression of CD86, major histocompatibility complex class II, CD11a or surface immunoglobulin M. The irradiation-induced up-regulation of CD80 expression resulted in enhanced APC function of LPS-B cells. Up-regulation of CD80 expression on LPS-B cells was accompanied by an increase in CD80 mRNA accumulation and nuclear factor (NF)-κB activation. Activation of NF-κB was shown to be critical for up-regulation of CD80 expression as pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, severely decreased the observed up-regulation. X-irradiation of LPS-B cells induced expression of TNF-α but not CD154. However, anti-TNF-α monoclonal antibody (mAb) with anti-CD154 mAb did not inhibit X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, whereas these mAbs almost completely inhibited this up-regulation in A20-HL cells and bone marrow-derived dendritic cells (DCs). In contrast, a thiol antioxidant, N-acetyl-l-cysteine, completely blocked X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, but not in A20-HL cells or in DCs. Based on these findings, we concluded that X-irradiation up-regulates CD80 expression not only in A20-HL cells and DCs but also in LPS-B cells, and that this up-regulation in LPS-B cells via NF-κB activation is dependent on the generation of reactive oxygen species, while that in A20-HL cells and DCs is not.


Abbreviations:
APC

antigen-presenting cell

DC

dendritic cell

EMSA

electrophoretic mobility shift assay

LPS

lipopolysaccharide

MFI

median fluorescent intensity

NAC

N-acetyl-l-cyteine

NF-κB

nuclear factor-κB

OVA

chicken ovalbumin

TNF-α

tumour necrosis factor-α

TNP

trinitrophenyl

TPCK

n-tosyl-l-phenylalanine chloromethyl ketone

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Optimal T-cell response requires at least two signals, antigen-specific and antigen-non-specific. The former is given through the T-cell antigen receptor (TCR) and the latter is provided by a costimulatory molecule, one of which is CD80 (B7-1).1–4 We have previously shown that X-irradiation of A20-HL B lymphoma cells and dendritic cells (DCs) preferentially enhances expression of CD80 by inducing expression of tumour necrosis factor-α (TNF-α) and CD154 (CD40L), which results in enhanced antigen-presenting ability.5,6 Investigators recently reported that irradiation-induced CD80 expression is not restricted to this lymphoma line, and is also observed in other tumour cell lines that have been maintained in vitro for a long period of time, such as MOPC315 plasmacytoma and P815 mastocytoma,7–9 and in myeloid leukaemic cells freshly isolated from patients with acute myeloid leukemia.10 Irradiation also enhances expression of CD80 genes transfected into fresh leukaemic cells from patients as a potential immunotherapy-based gene therapy.11

In our study, irradiation of A20-HL B lymphoma cells was found to induce expression of TNF-α and CD154, which act on these cells in an autocrine and/or paracrine manner, and to activate nuclear factor (NF)-κB nuclear factor, which results in CD80 mRNA transcription.6 In other B lymphoma cell types, however, irradiation-induced CD80 expression is dependent on oxidative stress,7 suggesting the possibility that there are at least two signalling pathways for irradiation-induced up-regulation of CD80 expression, and that the signalling pathway activated by irradiation depends on cell type. Partial inhibition of protein synthesis12 and treatment with l-phenylalanine mustard (melphalan)9,13 have been also shown to enhance CD80 expression in A20-HL B lymphoma cells and in plasmacytoma or mastocytoma cells, respectively. l-phenylalanine mustard-induced up-regulation of CD80 expression is also observed in normal splenic B cells.14 These findings suggest that irradiation-induced up-regulation of CD80 expression might not only occur in A20-HL B lymphoma cells but also in normal B cells in an oxidative stress-dependent manner or in a TNF-α + CD154 induction-dependent manner. In order to further investigate this possibility, we prepared lipopolysaccharide (LPS)-activated splenic B cells and tested for irradiation-induced CD80 expression and then examined the mechanisms for induction in comparison with those in A20-HL B lymphoma cells and DCs. The results showed that irradiation-induced up-regulation of CD80 expression occurred in LPS-stimulated splenic B cells in an oxidative stress-dependent manner, whereas that in A20-HL cells and DCs was independent of oxidative stress.

Reagents

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Murine CD80 cDNA was kindly provided by Drs T. Uede and M. Isobe, the Institute of Immunological Science, Hokkaido University, Sapporo, Japan.15 Human α-actin cDNA was a generous gift from Dr T. Yoshimoto from the Institute of Medical Science at the University of Tokyo.16 Monoclonal antibodies (mAb), anti-I-AdEd (M5/114, rat immunoglobulin G (IgG)2b17), anti-mouse CD86 (GL-1, rat IgG2a18), anti-mouse CD4 (GK1.5, rat IgG2b19), anti-CD54 (intercellular adhesion molecule-1; ICAM-1) (YN1/1.7.4, rat IgG2b20), and anti-CD11a (leucocyte function-associated antigen-1; FD441.8, rat IgG2b21), were obtained from the American Type Culture Collection (Rockville, MD), and were kindly made available by Dr H. Nariuchi from the Institute of Medical Science of the University of Tokyo. Anti-mouse CD80 mAb (16-10A1, hamster IgG22) was kindly provided by Dr H. Reiser of the Dana-Farber Cancer Institute (Boston, MA) and was conjugated with fluoroscein isothiocyanate (FITC) or biotin. Anti-mouse TNF-α mAb (G281-2626, rat IgG1), anti-mouse CD154 mAb (MR1, hamster IgG), and monoclonal rat IgGl (R3-34) and IgG2a (R35-95), were obtained from Pharmingen (San Diego, CA). Hamster IgG and FITC-conjugated anti-mouse IgM goat IgG were purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA), and the biotinylated F(ab)′2 fraction of anti-hamster IgG goat IgG antibody was from Cedarlane Laboratories, Inc. (Hornby, Canada). As a control IgG for anti-mouse IgM goat IgG, FITC-conjugated anti-hamster IgG goat IgG, which was absorbed with mouse and rat IgG, was obtained from Southern Biotechnology Associates, Inc. (Birmingham, AL). Biotinylated antirat κ light chain mAb (MARK-1) and FITC-conjugated streptoavidin were obtained from Zymed Laboratories (San Francisco, CA). PE-Cy5-conjugated streptoavidin and anti-mouse CD16/31 mAb (clone 93, rat IgG2a) were purchased from Caltag Laboratories, Burlingame, CA, and eBioscience, San Diego, CA, respectively. The reagents n-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N-acetyl-l-cysteine (NAC), and chicken ovalbumin (OVA) were purchased from Sigma Chemical Co. (St. Louis, MO). pyrrolidine dithiocarbamate (PDTC) was from Calbiochem-Novabiochem Corp., San Diego, CA. OVA323−339 peptide was synthesized on a polyethylene glycol polystyrene-graft copolymer support by the solid-phase method using a Millipore Pepsynthesizer (Millipore, Bedford, MA) and Fmoc chemistry, and was kindly provided by Dr S. Imajoh-Ohmi of the Institute of Medical Science at the University of Tokyo.

Cells and irradiation

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

BALB/c mice were purchased from Charles River Co. (Atsugi, Japan), and were cared for in accordance with the institution's guide for the care and use of laboratory animals. Splenic B cells were prepared from 8–12-week-old mice, as described previously.23 Briefly, spleen cells were depleted of T cells by treatment with anti-Thy-1 IgM mAb and complement, and were depleted of adherent cells by passage through Sephadex G-10 column. LPS-stimulated B cells were prepared by incubation for 60 hr in the presence of 10 µg/ml LPS (Sigma Chemical Co.), followed by extensive washing. Bone-marrow-derived DCs were prepared as described elsewhere.24 Briefly, bone marrow cells from BALB/c mice were incubated in the presence of 200 U/ml rm-granulocyte–macrophage colony-stimulating factor (GM-CSF; PeproTech EC, London, UK) and 100 U/ml rm-interleukin(IL)-4 (PeproTech). After 10 days, the cells were used as DCs. A20-HL mouse B lymphoma cells expressing trinitrophenyl (TNP)-specific IgM were generated by transfection of A20.2 J cells with the rearranged κ- and µ-chain genes derived from Sp6 hybridoma.25 These cells were incubated or maintained in RPMI-1640 (Sigma Chemical Co.) supplemented with 10% fetal calf serum (FCS; Summit Biotechnology, Greely, CO), 5 × 10−5 m 2-mercaptoethanol, and 100 µg/ml kanamycin at 37° in a humidified atmosphere of 5% CO2 in air.

LPS-stimulated B cells, DCs, and A20-HL cells were irradiated with 8, 32, or 100 Gy, respectively, using an X-irradiator (MBR-1520 A; Hitachi Koki Co., Ltd, Hitachinaka, Ibaraki, Japan). Irradiated cells were incubated for 12 or 24 hr, unless otherwise stated, and were analysed for expression of surface molecules using flow cytometry, as described below. For analysis of CD80, TNF-α or CD154 mRNA expression, cells were incubated for 4–6 hr.

Antigen-presentation assay

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

In order to examine the antigen-presenting ability of LPS-stimulated B cells, 2 × 105 cells were incubated with 2 × 104 42-6A cloned T cells26 for 18 hr in 250 µl culture medium on a 96-well culture plate in the presence of OVA or OVA323−339 peptide at the indicated doses. To assess IL-2 production, 50 µl of culture supernatant was then harvested from each well and added to CTLL-2 cells, an IL-2-dependent T-cell line, as previously described.5,6,12 CTLL-2 cells were pulsed with 0·25 mCi [3H]thymidine during the last 6 hr of a 20 hr-incubation. Incorporation of [3H]thymidine was measured on a Matrix 96 direct beta counter (Packard Instrument Co., Meriden, CT). Where indicated, LPS-stimulated B cells were fixed as described elsewhere.27 Briefly, after washing with phosphate-buffered saline (PBS), cells were incubated 0·5% paraformaldehyde in PBS for 15 min at 37°, washed and further incubated in the absence of paraformaldehyde for 1 hr at 37°. After washing, they were used as APC (4 × 105 cells/well).

Flow cytometric analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

In order to examine surface molecules, cells were incubated with an appropriate FITC-conjugated mAb, or with an appropriate mAb, followed by staining with a biotinylated second antibody and FITC- or phycoerythrin (PE)–Cy5-conjugated avidin or with an appropriate FITC-conjugated second antibody. These cells were analysed on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems USA, Mountain View, CA). For quantitative comparison of CD80 expression, the fluorescence intensity of each sample was expressed as median fluorescence intensity (MFI).

Northern blot analysis of CD80

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Total cellular RNA was extracted using the single-step guanidium–thiocyanate method, as described previously.6,28 RNA samples were electrophoresed on a formaldehyde−1% agarose gel and were transferred onto a Zeta-probe nylon membrane (Bio-Rad Laboratories). Northern blots were hybridized with a 32P-labelled cDNA probe for CD80, and then exposed to RX film (Fuji Photo Film Co., Ltd, Tokyo, Japan). Blots were analysed using an image analysis system (BAS2000; Fuji Photo Film Co., Ltd). The membrane was then stripped of the first probe and reprobed using a 32P-labelled β-actin probe.

Electrophoretic mobility shift assay (EMSA)

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Nuclear extraction was conducted as previously described.29 Briefly, LPS-stimulated B cells, irradiated or non-irradiated, were washed and resuspended in 200–400 µl hypotonic buffer containing 10 mm Hepes (pH 7·9), 0·1 mm ethylenediaminetetraacetic acid (EDTA), 0·1 mm egtazic acid (EGTA), 10 mm KCl, 1 mm dithiothreitol (DTT), 0·75 mm spermidine, 0·15 mm spermine, 20 mmp-nitrophenyl phosphate, 20 mmβ-glycerophosphate, 1 mm Na3VO4, and protease inhibitors (1 mm phenylmethylsulphonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml peptstatin). After 15 min on ice, 0·6% NP40 was added and the lysates were further incubated for an additional 5 min and were centrifuged at 13 000 g in a microcentrifuge at 4°. Nuclear pellets were resuspended in 20–50 µl nuclear extract buffer (20 mm Hepes pH 7·9, 1 mm EDTA, 1 mm EGTA, 0·75 mm spermidine, 0·15 mm spermine, 0·4 m NaCl. 1 mm DTT, protease and phosphatase inhibitors) and freeze–thawed three times. After centrifugation at 13 000 g for 15 min, the supernatants were used as nuclear extracts.

For determination of NF-κB activity in nuclear extract, an EMSA was performed as previously described.29 A synthetic oligonucleotide probe containing nucleotides 2969–2945 of the human CD80 enhancer (5′-GGGAAAGGGGTTTTCCCAGCAGTCA-3′), which includes the NF-κB binding site.28 The probe was annealed with a synthetic nucleotide 5′-TGACTGCTGGGAAAACCCCTTT-3′ to form double-stranded DNA. The probe, endo-labelled with [α-32P]ATP, was incubated with nuclear extract for 30 min at room temperature. For competition assay, the nuclear extract was first incubated with 100-fold excess of the unlabeled probe for 15 min at room temperature, and then incubated with the endo-labelled probe. The free and protein-bound oligonucleotide probes in reaction mixtures were separated by electrophoresis on 5% polyacrylamide gels, which were subjected to autoradiography overnight.

Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Naïve murine B cells from the spleen are quite sensitive to irradiation. We therefore decided to prepare LPS-stimulated B cells to investigate irradiation-induced up-regulation of CD80 expression. To this end, B cells were prepared from spleen cells of BALB/c mouse and incubated for 2 days in the presence of 10 µg/ml LPS. LPS-stimulated B cells were then X-irradiated with 8 Gy, incubated for up to 24 hr, and examined for CD80 expression using a flow cytometer. The results are expressed as a median fluorescent channel and are shown in Fig. 1(a). CD80 expression was enhanced in a time-dependent manner, and reached the maximum levels at 9–12 hr after irradiation. CD80 expression then declined gradually, but was still much higher at 24 hr after irradiation than in nonirradiated LPS-stimulated B cells. The flow cytometric profiles of CD80 expression in LPS-stimulated B cells after irradiation and 12 hr-incubation are shown in the upper left panel of Fig. 1(b). Based on these results, LPS-stimulated B cells were irradiated with 8 Gy and incubated for 12 hr before use in further analysis in the following experiments. Irradiation of LPS-stimulated B cells preferentially up-regulated CD80 expression and slightly increased CD54 expression, but did not affect expression of CD86, MHC class II, surface IgM, and CD11a (Fig. 1(b)).

image

Figure 1. Irradiation selectively up-regulated CD80 expression in LPS-stimulated B cells. LPS-stimulated B cells were X-irradiated with 8Gy and incubated for 0–24 hr (a) or 12 hr (b). (a) Then, the cells were incubated with anti-CD80 mAb (16-10A1, hamster IgG), or hamster IgG as a control, and stained with biotinylated F(ab′)2 fraction of anti-hamster goat IgG antibody and FITC-conjugated streptoavidin. They were analysed on a flow cytometer. The results were shown as MFI. (b) The irradiated or nonirradiated cells were incubated with an antibody against CD80, CD86, MHC class II, IgM, CD54, or CD11a. They were stained with an appropriate biotinylated antibody and FITC–streptoavidin, and analysed on a flow cytometer.

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Irradiation-induced up-regulation of CD80 expression enhanced the APC function of LPS-stimulated B cells. As shown in Fig. 2, when nonirradiated LPS-stimulated B cells were used as APC, IL-2 production by 42-6A T cells was detected at 4·44 µm OVA (Fig. 2a) or 400 nm OVA323−339 peptide (Fig. 2b) and reached the plateau at 17·78 µm OVA (Fig. 2a) or at 3200 nm OVA323−339 peptide (Fig. 2b). When irradiated LPS-stimulated B cells were used as APC, IL-2 production was maximal at 4·44 µm OVA (Fig. 2a) or 800 nm OVA323−339 peptide (Fig. 2b). Therefore, irradiation of LPS-stimulated B cells enhanced not only CD80 expression but also APC function. This irradiation-induced enhancement of APC activity was almost completely inhibited by addition of anti-CD80 mAb (Fig. 2c), suggesting that up-regulation of CD80 molecules by irradiation enhanced APC function of LPS-stimulated B cells. Taken together, these results indicate that irradiation-induced CD80 molecules were functional.

image

Figure 2. Irradiation of LPS-stimulated B cells enhanced their APC function. LPS-stimulated B cells were X-irradiated with 8 Gy and incubated for 12 hr. In the experiment shown in the panel (a), 2 × 105 irradiated LPS-stimulated B cells (closed circle) or those non-irradiated (open circle) were incubated with 2 × 104 42–6A T cells in the presence of OVA at indicated doses. In the experiment shown in the panel (b), irradiated (closed circle) and non-irradiated (open circle) LPS-stimulated B cells were fixed with paraformaldehyde, and 4 × 105 fixed cells were incubated with 2 × 104 42-6A T cells in the presence of OVA323−339 peptide at indicated doses. After 20 hr-incubation, 50 µl culture supernatant was harvested from each well and assayed for IL-2 activity using IL-2-dependent proliferation of CTLL-2 cells. (c) Similar experiment to that in (b) was carried out in the presence of 20 µg/ml anti-CD80 mAb or hamster IgG as a control. When anti-CD80 mAb (mAb) or hamster IgG (h-IgG) was not included in the culture of irradiated and fixed LPS-stimulated B cells and 42-6A T cells, CTLL-2 cell proliferations were 15 988 ± 897 counts with 400 nm OVA323−339 peptide and 17 557 ± 1452 counts/3 min with 800 nm OVA323−339 peptide. Those in fixed LPS-B cells and the T cells were 3777 ± 647 and 11 245 ± 1004 counts/3 min, respectively.

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Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Irradiation-induced CD80 expression was accompanied by enhanced accumulation of CD80 mRNA. As shown in Fig. 3(a), CD80 mRNA was undetectable in freshly prepared B cells under the conditions employed, but was clearly detectable in LPS-stimulated B cells. Accumulation of CD80 mRNA in LPS-stimulated B cells was strongly enhanced by irradiation, suggesting that irradiation-induced CD80 expression was the result of enhanced transcription.

image

Figure 3. Irradiation-induced up-regulation of CD80 expression was observed at transcription level and accompanied by NF-κB activation. (a) For Northern blot analysis of CD80 mRNA, RNA was prepared from untreated B cells, LPS-stimulated B cells, or LPS-stimulated, 8 Gy-irradiated and 12 hr-incubated B cells. The RNA preparations were electrophoresed and hybridized with 32P-labelled CD80 cDNA. As a control, the membranes were re-hybridized with β-actin cDNA. The membranes were analysed using autoradiography (I) and densitometry (II). (b) For EMSA assay, nuclear extracts were prepared from LPS-stimulated and 8 Gy-irradiated B cells after incubation for indicated hours, and incubated with the α-32P-ATP-labelled probe containing NF-κB-binding site in CD80 enhancer. For examining specificity of NF-B binding, an unlabeled probe as a competitor was first added 100 fold excess to the nuclear extract 15 min prior to the addition of the labelled probe. The mixtures were then electrophoresed and analysed autoradiographically. Radioactive portions were shown corresponding to slow-migrating NF-κB complexes. (c) LPS-stimulated B cells or LPS-stimulated, irradiated and 12-hr-incubated B cells were stained for CD80, as described in the legend for Fig. 1. To examining the role of NF-κB activation, 300 µmPDTC was added during irradiation and 12-h-incubation.

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The transcription of CD80 mRNA has been shown to depend on NF-κB activation.28 Therefore, we investigated whether irradiation-induced CD80 expression in LPS-stimulated B cells was accompanied by enhancement of NF-κB activation. Nuclear extracts of LPS-stimulated B cells at various time points after irradiation were thus analysed using EMSA and a DNA probe containing the NF-κB binding site of the human CD80 gene. As shown in Fig. 3(b), a protein–DNA complex was detected and this was increased by irradiation. The amount of protein-DNA complex peaked at 1 hr after irradiation and returned to normal levels at 4 hr after irradiation. The specificity of this protein–DNA complex was demonstrated by the fact that the competitors (unlabelled probe) almost completely inhibited complex formation.

In order to examine the importance of NF-κB activation in irradiation-induced CD80 expression, the effects of PDTC, which is an NF-κB inhibitor, were investigated. As shown in Fig. 3(c), when 300 µm PDTC was present during irradiation and 12-hr incubation, the irradiation-induced increase in CD80 expression was almost completely inhibited. We therefore concluded that irradiation of LPS-stimulated B cells induced NF-κB activation, which resulted in increased accumulation of CD80 mRNA and subsequent up-regulation of CD80 expression.

Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Previously, we have shown that irradiation of A20-HL B lymphoma cells induces expression of TNF-α and CD154, which stimulate the cells in an autocrine and/or paracrine manner to up-regulate CD80 expression.6 Therefore, we examined whether irradiation induced TNF-α and CD154 expression in LPS-stimulated B cells. As shown in Fig. 4(a), irradiation of LPS-stimulated B cells did not alter expression of CD154, but enhanced TNF-α expression. However, enhancement of TNF-α expression did not play a role in irradiation-induced up-regulation of CD80 expression in these cells. The CD80 expression profiles in irradiated LPS-stimulated B cells were almost identical in the presence or absence of anti-TNF-α and/or anti-CD154 mAbs (Fig. 4b). Thus, irradiation-induced up-regulation of CD80 expression in LPS-stimulated B cells was independent of TNF-α and CD154, although these play critical roles in up-regulation in A20-HL B lymphoma cells.6

image

Figure 4. Irradiation up-regulated TNF-α expression, but not CD154, in LPS-stimulated B cells. (a) LPS-stimulated B cells were 8 Gy-irradiated and incubated for 12 hr, and stained for TNF-α (I) or CD154 (II) with anti-TNF-α mAb or anti-CD154, an appropriate biotinylated 2nd antibody, and FITC–streptoavidin. (b) Role of TNF-α and CD154 was examined in irradiation-induced up-regulation of CD80 expression. LPS-stimulated B cells were irradiated and incubated for 12 h in the absence (I) or presence of 20 µg/ml anti-TNF-α mAb (II), 20 µg/ml anti-CD154 mAb (III), or both (IV). Then, the cells were stained for CD80 as shown in the legend for Fig. 1. Staining profiles of LPS-stimulated, but not irradiated, B cells were also shown for comparison (LPS-treated). In these experiments antimouse CD16/32 mAb was included as a blocking antibody.

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Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

Oxidative stress was recently shown to induce CD80 expression. We therefore examined whether the thiol antioxidant NAC was capable of inhibiting up-regulation of CD80 expression in LPS-stimulated B cells. NAC (5, 10 or 20 mm) was added during irradiation and incubation, and cells were analysed for CD80 expression. NAC inhibited up-regulation in a dose-dependent manner and up-regulation was almost completely inhibited at 20 mm, indicating that reactive oxygen species were involved in this up-regulation. In Fig. 5(a, II), the results obtained with 20 mm NAC are shown.

image

Figure 5. Role of reactive oxygen species, TNF-α, or CD154 in irradiation-induced up-regulation of CD80 expression in LPS-stimulated B cells. CD80 expression was examined on LPS-stimulated, 8 Gy-irradiated and 12-hr-incubated B cells (a), irradiated A20-HL cells (b), and bone marrow derived dendritic cells (c). Cells were irradiated and incubated in the presence of 20 µg/ml anti-TNF-α mAb and anti-CD154 mAb (I) or 20 mm NAC (II). Then the cells were stained for CD80 expression as described in the legend for Fig. 1. In these experiments anti-mouse CD16/32 mAb was included as blocking antibody.

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These findings prompted us to examine the role of oxidative stress in the irradiation induced up-regulation of CD80 expression in A20-HL cells and in DCs. As shown in Fig. 5(b), irradiation of A20-HL cells enhanced CD80 expression. This enhancement was completely inhibited by addition of anti-TNF-α and anti-CD154 mAbs (left panel), as previously reported,6 but was not affected by addition of 20 mm NAC (right panel), indicating that irradiation-induced CD80 expression in A20-HL cells is independent of oxidative stress.

Irradiation of DCs increased expression of CD80 (Fig. 5c), as reported previously.6 Expression was completely inhibited by addition of anti-TNF-α and anti-CD154 mAbs (left panel), but 20 mm NAC did not affect irradiation-induced CD80 expression (right panel). These findings indicate that irradiation of DCs induced CD80 expression that was independent of oxidative stress, but dependent on induction of TNF-α and/or CD154. The up-regulations of CD80 expression in DCs also resulted in the enhancement of their APC function. Culture supernatant of irradiated DCs and 42-6A T cells in the presence of 800 nm OVA323−339 peptide induced CTLL-2 cell proliferation, 11 991 ± 2010 counts/3 min (102 ± 48 counts/3 min in the absence of the peptide), whereas that of non-irradiated DCs induced 7415 ± 1708 counts/3 min (150 ± 55 in the absence of the peptide).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References

CD80 is expressed in DCs, but not in resting B cells.29 CD80 expression in B cells is induced by stimulation with LPS, CD154, cross-linked CD21/CD35 or CD19 and partial inhibition of protein synthesis.12,30,31 Recently, irradiation has been shown to up-regulate CD80 expression in various B-cell lines in long-term culture, in freshly isolated B lymphoma cells from patients, and in CD80 gene-transfected cells.5–11 In the present study, we investigated whether irradiation up-regulated CD80 expression in mouse spleen B cells, as observed in a mouse B-cell line in long-term culture and bone marrow-derived DCs.6 Naïve spleen B cells are quite sensitive to irradiation and are difficult to examine for irradiation-induced up-regulation of CD80 expression, and thus we employed LPS-stimulated B cells. The present results indicate that irradiation (8 Gy) followed by incubation (12 hr) of LPS-stimulated B cells up-regulated CD80 expression, which resulted from enhanced accumulation of CD80 mRNA. This up-regulation was observed for CD80 expression, but not for other surface molecules, such as CD86, MHC class II, sIgM, CD54, and CD11a. Enhanced expression of CD80 was functional and resulted in increased ability to present OVA antigen for T-cell IL-2 production. Thus, irradiation up-regulates CD80 expression not only in B lymphoma cells in long-term culture and DCs but also in normal B cells. It is presently unknown why expression of other NF-κB-dependent molecules, such as CD86 or CD54, was not enhanced.32,33 The dependency of these molecules on NF-κB activation for expression might be different, as previously suggested.34

The mechanisms for up-regulation of CD80 expression in LPS-stimulated B cells were investigated and compared with those in A20-HL B lymphoma cells and DCs. Irradiation induced up-regulation of CD80 expression in LPS-stimulated B cells is mediated by reactive oxygen production, and is not dependent on induction of TNF-α and CD154 expression, whereas that in A20-HL B lymphoma cells and DCs is dependent on induction of TNF-α and CD154 expression rather than on reactive oxygen production. Both stimulation via reactive oxygen species and that by TNF-α and CD154 resulted in enhanced NF-κB activation-dependent CD80 expression. These findings suggest that there are two pathways by which irradiation activate NF-κB and induce up-regulation of CD80 expression. This interpretation is based on the observations that irradiation-induced up-regulation in LPS-stimulated B cells was accompanied by activation of NF-κB, and was inhibited by the NF-κB inhibitor, PDTC. In addition, up-regulation in A20-HL B lymphoma cells and DCs was inhibited by the NF-κB inhibitor TPCK and anti-TNF-α with anti-CD154.6 Presently, the factors that determine which pathway is activated by irradiation remain to be elucidated. It is possible that the activated pathway varies with cell type. Alternatively, stimulation with LPS might have affected the response to irradiation in normal B cells, which is consistent with previous findings that LPS stimulation produces reactive oxygen species.35,36

Previously, TNF-α has been shown to stimulate production of reactive oxygen species,37 raising the possibility that irradiation of A20-HL cells stimulates TNF-α expression, which activates NF-κB via production of reactive oxygen species. However, this possibility is unlikely in the present study, since NAC, a thiol antioxidant, did not inhibit irradiation-induced enhancement of CD80 expression in A20-HL cells.

Irradiation of LPS-stimulated B cells induced expression of TNF-α, but a neutralizing anti-TNF-α mAb did not inhibit up-regulation of CD80 expression, indicating that TNF-α does not play a critical role in this up-regulation. In contrast, irradiation-induced expression of TNF-α in conjunction with CD154 expression in A20-HL cells or DCs is critical for up-regulation of CD80 expression. It is possible that TNF-α expression was insufficient to induce up-regulation of CD80 expression, because TNF-α alone is able to up-regulate CD80 expression.38,39 The absence of CD154 expression may also explain why irradiation-induced TNF-α expression did not play a critical role in up-regulation of CD80 expression in LPS-stimulated B cells, as suggested by our previous findings that TNF-α and CD154 synergistically up-regulate CD80 expression in irradiated A20 B lymphoma cells. Although we did not detect irradiation-induced CD154 expression in LPS-stimulated B cells, CD154 has been shown to be present in the cytoplasm of B cells and to be easily released as a functional molecule,40 raising the possibility that soluble form of CD154 may bind to CD40 in LPS-stimulated and irradiated B cells and stimulate CD80 expression. However, this is unlikely since anti-CD154 mAb with anti-TNF-α mAb did not inhibit irradiation-induced up-regulation of CD80 expression in LPS-stimulated B cells.

Two pathways have recently been shown to activate NF-κB; IKKβ-dependent and IKKβ-independent.41–44 The latter is dependent on IKKα kinase. TNF-α is able to activate NF-κB via the IKKβ kinase pathway.45 On the other hand, reactive oxygen species have been shown to activate NF-κB via the IKKα-dependent pathway.46 Thus, irradiation of LPS-stimulated B cells induced generation of reactive oxygen species, which might have activated NF-κB via the IKKα-dependent pathway. Irradiation A20-HL cells induces expression of TNF-α and CD154, which might have activated NF-κB via the IKKβ-dependent pathway. Thus, irradiation could activate NF-κB via the IKKβ-dependent canonical pathway or the IKKβ-independent but IKKα-dependent non-canonical pathway. These possibilities are now under the investigation.

l-phenylalanine mustard has been also shown to preferentially up-regulate CD80 expression not only in B lymphoma cells in long-term culture but also in normal B cells.14 The mechanisms for irradiation to up-regulate CD80 in A20-HL B lymphoma cells and DCs were apparently different from those for l-phenylalanine mustard. The latter has been reported to increase CD80 expression via generation of reactive oxygen species and NF-κB activation.13,14 In contrast, irradiation of A20 B lymphoma cells up-regulates CD80 expression by inducing expression of TNF-α and CD154 and by activating NF-κB.6 As discussed above, the mechanisms for up-regulation of CD80 expression in LPS-stimulated B cells were similar to those for l-phenylalanine-induced CD80 expression, and up-regulation was dependent on generation of reactive oxygen species.

CD80 is a potent costimulatory molecule, which has led investigators to introduce this molecule into tumour cells to elicit or enhance antitumour immunity. The introduction of CD80 molecules activates both helper T cells and cytotoxic T cells against tumour cells.47,48 In the present study, we extended control of CD80 expression by irradiation of spleen B cells. Mild irradiation of LPS-stimulated B cells up-regulates CD80 expression. We also delineated the mechanism of this up-regulation, which is different from those in B lymphoma cells and bone-marrow-derived DCs. Irradiation-induced up-regulation of CD80 expression in chronic lymphatic leukaemia cells has recently been shown to correlate with chemosensitivity to purine analogues.49 Therefore, precise elucidation of the mechanisms for irradiation-induced up-regulation of CD80 expression will raise a possibility to develop strategies not only to potentiate immune responses via a simple procedure, such as mild irradiation, but also to improve chemotherapy for chronic lymphocytic leukaemia.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Cells and irradiation
  7. Antigen-presentation assay
  8. Flow cytometric analysis
  9. Northern blot analysis of CD80
  10. Electrophoretic mobility shift assay (EMSA)
  11. Statistical analysis
  12. Results
  13. Irradiation-induced enhancement of CD80 expression in LPS-stimulated B cells
  14. Irradiation-induced CD80 mRNA accumulation and NF-κB activation in LPS-stimulated B cells
  15. Irradiation-enhanced expression of TNF-α did not play a critical role in up-regulation of CD80 expression
  16. Irradiation-induced up-regulation of CD80 expression was inhibited by a thiol antioxidant, N-acetyl-l-cysteine (NAC)
  17. Discussion
  18. Acknowledgments
  19. References
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