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Abstract

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

Antigen presentation is a key rate-limiting step in the immune response. Dendritic cells (DCs) have been reported to be the most potent antigen-presenting cells for naïve T cells, but little is known about the biochemical pathways that regulate this function. We here demonstrate that mature murine DC can be infected with adenovirus at high efficiency (>95%) and that an adenovirus transferring the endogenous inhibitor IκBα blocks nuclear factor-kappa B (NF-κB) function in murine DC. This result indicates that antigen presentation in the mixed leucocyte reaction is NF-κB dependent, confirming data with human DC in vitro. However, the importance of this finding depends on verifying that this is true also in vivo. Using delayed type hypersensitivity with allogeneic cells, we show that NF-κB inhibition had a marked immunosuppressive effect in vivo. These results thus establish NF-κB as an effective target for blocking DC antigen presentation and hence inhibiting T-cell-dependent immune responses. This finding has potential implications for the development of therapeutic agents for use in various pathological conditions of the immune system, including allergy and autoimmunity, and also in transplantation.


Introduction

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

Dendritic cells (DC) are now recognized to be the most effective cells for presentation of antigen to naïve T cells in the primary immune response [1]. They are bone marrow-derived cells which were first described in the early 1970s by Steinman and Cohn [2] and subsequently studied by many groups [3–6]. The importance of antigen presentation in the generation of the immune response has been extensively confirmed in vivo by the demonstration that blocking antigen presentation downregulates both humoral and cell-mediated immune responses. This has been shown to be effective in treating animal models of disease and various antibodies that block antigen-presenting cell (APC) molecules such as antibody to murine major histocompatibility complex (MHC) class II has been used to treat experimental allergic encephalomyelitis [7]. Blocking the CD80/86 costimulatory molecules expressed on APCs with antibodies or CTLA4-Ig fusion protein (which binds to both CD80 and CD86) has been reported to be beneficial in transplants [8] or in animal models of autoimmunity such as murine collagen-induced arthritis or rat experimental allergic encephalomyelitis [9, 10]. This has led to a search for new ways of downregulating antigen presentation, which may be useful for therapy of human diseases or in transplantation. The success of this search would be enhanced by an understanding of the molecular regulation of antigen presentation not only in vitro but most importantly in vivo.

Nontransformed cells do not take up DNA by any of the classical transfection procedures. Hence we have sought to develop methods of augmenting the efficiency of adenoviral uptake into various normal cells, including human macrophages and human DC [11, 12]. This has enabled us to demonstrate, for e.g, that cytokine tumour necrosis factor-α (TNF-α) gene regulation is complex and stimulus dependent, even in a single cell type (macrophages), with different signals inducing TNF-α via different transcription factors [13]. In mature human DC, we found that antigen presentation is nuclear factor-kappa B (NF-κB) dependent. This new technique enabled us to ascertain that NF-κB coordinately regulated four aspects of antigen-presenting function, namely MHC expression, costimulatory molecule (CD80/CD86) expression and cytokine (e.g. interleukin-12 (IL-12)) production. Furthermore, the expression of molecules regulating the attachment of T cells to APCs (lymphocyte function-associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1) and ICAM-3) was also regulated by NF-κB, but in the opposite direction, suggesting that NF-κB also regulates the duration of APC-T-cell contact [12].

These results, although interesting, were entirely performed with human cells in vitro. To help determine whether these findings of the importance of NF-κB as a major regulator of APC function were of relevance in vivo, we first inhibited NF-κB in cultured murine DC, in vitro, using mixed lymphocyte reaction as a functional assay and confirmed the results from human cells. Subsequently, in vivo experiments were performed using the allogeneic delayed type hypersensitivity response. Here we show that mature murine DC can be infected with adenovirus at high efficiency (>95%) and that this procedure can be used to dissect out which pathways are essential for inducing dendritic antigen presentation to naïve T cells. The results indicate that antigen presentation in the mouse is dependent on NF-κB activation in both in vitro and in vivo models.

Materials and methods

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

Reagents.  Recombinant murine IL-4, granulocyte/macrophage-colony stimulating factor (GM-CSF) and TNF-α were purchased from R & D Systems (Minneapolis, MN, USA). Phorbol 12-myristate 7-acetate (PMA), lipopolysaccharide (LPS) and sterile 37% bovine serum albumin (BSA) solution were obtained from Sigma (St. Louis, MO, USA).

Mice.  For preparation of mouse DC, BALB/c mice were used (see below), and for the preparation of allogeneic T cells, C57/BL mice. Analysis of delayed type hypersensitivity was also performed in C57/BL mice. Mice (8–12 weeks) were obtained from Olac Ltd (Bicester, UK) and maintained in specific pathogen-free conditions.

Preparation of mouse dendritic cells.  Mouse DCs were prepared through an adaptation of the method previously described [14]. Mixed spleen and bone marrow from BALB/c mice was suspended in 1 mg/ml of Collagenase D (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37 °C. Debris and red cells were then removed, and low density cells collected by 37% BSA centrifugation (500 g, 10 min). After three washes, low density cells were cultured in RPMI-1640 supplemented with 5% fetal calf serum (FCS) and 50 µm of 2-mercaptoethanol, and with 20 ng/ml of murine IL-4 and murine GM-CSF for 6 days according to the procedure of Sallusto and Lanzavecchia [5]. Cytokines were freshly added throughout the culture period through exchange of medium.

Maturation of mouse dendritic cells.  On day 6, granulocytes were removed using a 50% FCS column. After two washes, cells were cultured in RPMI-1640 supplemented with 5% FCS and 50 µm of 2-mercaptoethanol, and with 10 ng/ml of murine TNF-α and 10 ng/ml of LPS for maturation. After 2 days, cells were harvested and resuspended for further experiments. It was observed that mouse DCs matured in this way had upregulation of I-A, CD86 and D11c, and some downregulation of CD16/32, as compared with immature mouse DCs (Fig. 1). All samples were analysed on a FACScan flow cytometer using the cellquest software (Becton Dickinson, San José, CA, USA). Analysis was carried out on a population of cells gated by forward and side scatter to exclude dead cells and debris. DC surface markers were studied using rat antimouse I-Ab, hamster antimouse CD11c, rat antimouse CD86 and rat antimouse CD16/CD32 (all R-PE conjugated, Pharmingen, San Diego, CA, USA).

image

Figure 1. Murine dendritic cell (DC)-surface markers were studied using rat antimouse I-Ab, hamster antimouse CD11c, rat antimouse CD86 and rat antimouse CD16/CD32 (all R-PE conjugated, Pharmingen, San Diego, CA, USA). Cell populations cultured with (red line) or without (green line) DC maturation using murine tumour necrosis factor-α and lipopolysaccharide were phenotyped with the monoclonal antibodies listed above and compared with isotype control (blue area) on FACScan (Becton Dickinson, San José, CA, USA).

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Adenoviral vectors.  Recombinant, replication-deficient adenoviral vectors encoding Escherichia coliβ-galactosidase (AdvβGal) or having no insert (Adv0) were provided by Drs A. Byrnes and M. Wood (Oxford, UK). An adenovirus- encoding porcine IκBα with a cytomegalovirus promoter and a nuclear localization sequence (AdvIκBα) was provided by Dr R. de Martin (Vienna, Austria) [15]. Viruses were propagated and titred as previously described [16, 17].

Analysis of infectibility.  Mature murine DCs were replated on 96-well flat-bottom plate at a density of 2 × 105 cells/well and were either left uninfected or infected for 2 h with a multiplicity of infection (m.o.i.) of from 40 : 1 to 500 : 1 of Adv0 or AdvβGal in serum-free RPMI-1640. Cells were then incubated in RPMI-1640 supplemented with 5% FCS. Cells were taken off the plates 48 h after infection, spun down, washed in fluorescence-activated cell sorter (FACS) staining solution and incubated at 37 °C for 10 min before 45 µl of a 2 mm solution of Fluorescein-di-(β-D)-galactopyranoside (Sigma) was added for 1 min [18]. Addition of excess (10×) ice-cold staining solution was used to stop the reaction, and cell fluorescence was analysed by FACS. Alternatively, infection was performed with 100 : 1, 200 : 1 and 500 : 1 of an adenovirus transferring the green fluorescent protein gene, and cells were photographed through a fluorescence microscope 1 and 2 days after infection.

Western blotting and electrophoretic mobility shift assay.  Batches of 10 × 106 DCs were either left uninfected, infected with 200 : 1 of Adv0 or infected with 200 : 1 of AdvIκBα. Two days after infection, cells were either left unstimulated or stimulated with LPS (50 ng/ml) for 60 min. Cytosolic and nuclear extracts were prepared as described [19] and proteins separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis on a 10% (w/v) polyacrylamide gel, followed by electrotransfer onto nitrocellulose membranes. IκBα and the p42/p44 mitogen-activated protein kinases (as a control) were detected using antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

About 20 µg of protein from the nuclear extracts prepared as described above were analysed for NF-κB activity by electrophoretic mobility shift assay as previously described [18]. In a separate experiment, competition analysis with excess cold NF-κB probe, and with an unrelated probe (AP-1), was performed to ensure specificity.

Analysis of cytokines.  In these experiments, mature murine DCs were either left uninfected or infected with a m.o.i. of 200 : 1 of Adv0 or AdvIκBα as described above. Cells were then incubated in RPMI-1640 supplemented with 5% FCS. Two days after infection, cells were replated at 2 × 105 cells per well on a 96-well plate and either left unstimulated or stimulated with PMA (10 nm) or LPS (50 ng/ml) for 24 h. Supernatants were taken off and analysed for murine TNF-α and IL-6 by enzyme-linked immunosorbent assay using kits purchased from Pharmingen.

Analysis of antigen presentation.  Mature DCs were left uninfected, infected with Adv0 or infected with AdvIκBα at m.o.i. of 200 : 1. DCs were then plated in graded doses for 105 purified, allogeneic T cells prepared from C57/BL mice, in triplicate in a 96-well round-bottom microtitre plate on day 1 after adenovirus infection. Proliferation was determined on day 6 using the tritiated thymidine uptake assay.

Analysis of delayed type hypersensitivity.  In these experiments, murine DCs were prepared from a total of 24 mice and matured as described above but for 3 days. Nine female C57/BL mice were immunized with these mature murine DCs by intraperitoneal injection of 106 cells/mouse in 100 µl medium. These mice were challenged with subcutaneous injection of 105 freshly matured murine DCs in 10 µl of medium, into the two hind paws. Three mice were injected with uninfected DCs, three with DCs that had 2 days previously been infected with 200 : 1 of Adv0 and three with DCs that had 2 days previously been infected with 200 : 1 of AdvIκBα as described above. Paw thickness of both injected paws was measured 24, 32 and 48 h after injection.

Statistical methods.  Statistical testing was performed using the two-sample, one-sided Student's t-test and the Wilcoxon rank sum test.

Results

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

Infectivity of murine dendritic cells by adenovirus and inhibition of NF-κB function after AdviκBα infection

Figure 1 shows the phenotype of the in vitro-generated mature murine DC, which was as expected from prior studies [14], namely upregulation of I-A, CD11c and CD86 but downregulation of CD16/CD32. The infectivity of these DCs with adenovirus was assessed using an adenovirus-encoding β-galactosidase (Advβgal) and was >95% at a multiplicity of infection of 200 : 1 (Fig. 2) but not at lower virus titres. This was reproduced using an alternative transgene, the green fluorescent protein (not shown). Infection of mature DCs with 200 : 1 was thus routinely performed with an adenovirus-encoding IκBα (AdvIκBα). This leads to abundant cytosolic overexpression of IκBα, whereas infection with an adenovirus with no insert had no effect on IκBα expression (Fig. 3A). An electrophoretic mobility shift assay demonstrated that AdvIκBα infection inhibits LPS-induced NF-κB activation (Fig. 3B). Competition experiments using excess cold NF-κB probe and using an unrelated probe were performed to ascertain that the shifted band seen really was active NF-κB (data not shown). These data were analogous to those obtained with the same adenoviruses in human DCs gd[12].

image

Figure 2. In excess of 90% of murine dendritic cells (DCs) can be infected with adenovirus. Mature murine DCs infected with Adv0 or AdvβGal as described in Materials and methods, and cell fluorescence from Adv0-infected (black area) and AdvβGal-infected (grey line) DCs was analysed by fluorescence-activated cell sorter.

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image

Figure 3. Murine dendritic cells were either left uninfected, infected with 200 : 1 of Adv0 or infected with 200 : 1 of AdvIκBα. Cytosolic and nuclear IκBα overexpression was determined by Western blotting, and the p42/p44 mitogen-activated protein kinases were used as a control (A), and nuclear extracts were analysed for NF-κB activity by electrophoretic mobility shift assay (B) as described in Materials and methods. The reason no immunoreactive IκBα protein is seen in the cytosol of uninfected or Adv0-infected cells is that the antibody used detects human and swine IκBα but not murine IκBα. LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase.

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Effect of AdvIκBα infection on the secretion of mouse dendritic cell cytokines

We studied the production of some key proinflammatory DC cytokines, which were produced by human DC, and observed that the production of IL-6 was significantly (P < 0.001) downregulated by AdvIκBα (Fig. 4), indicating its dependence on NF-κB. There was no increase in IL-6 when cells were stimulated with PMA or LPS, probably because of the fact that the cells had already been matured with LPS. The very low level of TNF-α (just 100–120 pg/ml) appeared to be unchanged by AdvIκBα infection (data not shown). The reason that the TNF-α production is as low as this is likely to be ‘exhaustion’ of DC, as recently described [20], after the long exposure to LPS during the maturation process. Similarly, the reason that AdvIκBα infection does not inhibit this low level of TNF-α may well be that NF-κB has become downregulated over time, after the long exposure to LPS. No IL-10 production was detectable from these DCs, whether unstimulated or treated with PMA or LPS (data not shown).

image

Figure 4. Mature murine dendritic cells were either left uninfected (white bars), or infected with a m.o.i. of 200 : 1 of Adv0 (grey bars) or AdvIκBα (black bars) as described above. Supernatants were analysed for murine tumour necrosis factor-α (TNF-α)(A) and interleukin-6 (IL-6) (B) by enzyme-linked immunosorbent assay. LPS, lipopolysaccharide; PMA, Phorbol 12-myristate 7-acetate.

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Reduction in antigen-presenting function by inhibiting NF-κB

We studied the antigen-presenting function of DCs in vitro by using the mixed lymphocyte reaction, as we have previously performed for human DCs [12]. DCs have long been acknowledged to be the most potent APCs for the mixed lymphocyte reaction [21]. There was no effect of infecting DCs with Adv0, but AdvIκBα infection reduced T-cell proliferation, as was consistently observed in four experiments using tritiated thymidine uptake to assay murine T-cell proliferation. A representative experiment is shown in Fig. 5. The number of APCs needed for the same proliferative response was about 10-fold higher with NF-κB-inhibited DCs.

image

Figure 5. Mature dendritic cells (DCs) were left uninfected (○), infected with Adv0 (▪) or infected with AdvIκBα (▴) at m.o.i. of 200 : 1. DCs were then plated in graded doses for 105 purified, allogeneic T cells in triplicate in a 96-well round-bottom microtitre plate on day 1 after adenovirus infection. Proliferation was determined on day 6 using the tritiated thymidine (3H-TdR) uptake assay. TdR.

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In vivo verification that antigen-presenting function depends on NF-κB

This was evaluated using the murine allogeneic delayed-type hypersensitivity assay, as described in Materials and methods. Paw thickness was measured at 24, 32 and 48 h after challenge with subcutaneous allogeneic DCs, for each injected paw in three mice in each treatment group (uninfected DCs, Adv0-infected DCs and AdvIκBα-infected DCs). There was no difference in paw thickness prior to injection and a good response with rapid increase in paw thickness increasing from 24 to 48 h (Fig. 6A–C). Using either the two-sample Student's t-test or the Wilcoxon rank sum test, there was significantly (P < 0.01) less increase in paw thickness in mice injected with AdvIκBα-infected DCs as compared with animals injected with Adv0-infected DCs at 24 and 32 h, and significantly (P < 0.05) less increase in paw thickness in mice injected with AdvIκBα-infected DCs as compared with animals injected with Adv0-infected DCs at 48 h (Fig. 6D). There was no statistically significant difference between Adv0-infected and -uninfected DCs.

image

Figure 6. Paw thickness in each paw of three mice that were injected with uninfected dendritic cells (DCs), three with DC that had 2 days previously been infected with 200 : 1 of Adv0 and three with DC that had 2 days previously been infected with 200 : 1 of AdvIκBα as described in Materials and methods, 24 (A), 32 (B) and 48 h (C) after injection. In (D), mean difference in paw thickness in mice challenged with uninfected (○), Adv0-infected (•) or AdvIκBα-infected (▴) DC is plotted over time.

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Discussion

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

The data presented here complete a series of human and murine studies that demonstrate unequivocally that NF-κB is a major regulator of the antigen-presenting function of mature DCs. This possibility had been suggested previously by the presence of activated NF-κB in the nucleus of mature DCs [22], studies with RelB knockouts, which have no DC of myeloid type [23], and studies involving relatively nonspecific small molecule inhibitors of NF-κB [24, 25]. Using adenoviral transfer of IκBα, which is a much more specific inhibitor than drugs available and acts as a direct test of the role of NF-κB, it was possible to confirm the importance of NF-κB in the APC function of mature DCs. This was done both in the mixed lymphocyte reaction [12] and in antigen-specific T-cell responses [26]. In the human system, we noted that the regulation of multiple aspects of DC function, namely antigen presentation through NF-κB, involves coordinate regulation of MHC, costimulatory molecule expression and cytokine production. All these three aspects of regulating DC function are essential: removing one (i.e. antibody to CD86) has a major effect, but adding back one in the absence of the other two is not sufficient [12]. Other authors have proposed that NF-κB is essential for DC differentiation and maturation [27, 28], and it may well be that after NF-κB downregulation, DCs revert to a more immature phenotype.

This study indicates that NF-κB is essential for DC antigen presentation in vivo. Using adenoviral infection of murine DC in vitro, it was demonstrated that adenoviral transfer of IκBα leads to inhibition of NF-κB activation (Fig. 3), inhibition of IL-6 production (Fig. 4) and most importantly, functional inhibition of DC antigen presentation (Fig. 5). It was then demonstrated, using the model of delayed-type hypersensitivity to allogeneic DCs, that this specific blocking of NF-κB function inhibited DC antigen presentation in vivo (Fig. 6).

These results agree well with the previous finding that in a model of rhesus monkey renal allografts, the nonspecific NF-κB inhibitor deoxyspergualin interacts with CD3 immunotoxin to achieve tolerance [29]. Another important finding is that in mouse bone marrow-derived DC, administration of NF-κB decoy oligodeoxyribonucleotides induces allogeneic donor-specific hyporeactiveness in mixed leucocyte cultures and inhibition of T helper 1 cytokine production. Importantly, infusion of these modified DCs with impaired NF-κB-function resulted in prolonged cardiac allograft survival in the absence of immunosuppression [30]. Our results suggest that adenoviral transfer of IκBα to APCs in the donor organ may well have a similar beneficial effect. Apart from the obvious context of transplantation, the demonstration of the central role of NF-κB in DC function has other important medical implications in the fields of allergy and autoimmunity. In terms of gene therapy, AdvIκBα or other viruses designed to inhibit DC antigen presentation may also have the beneficial effect of inhibiting the immune response to the adenovirus itself and thus may prolong the expression of other therapeutic transgenes. This prediction remains to be evaluated.

Acknowledgments

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

This work was supported by the Arthritis Research Campaign and by Suntory Ltd (salary of Mr S. Yoshimura). We thank Drs de Martin, Byrnes and Wood for their generous gift of reagents.

References

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