Direct Ni²⁺ antigen formation on cultured human dendritic cells

Abstract

The possible direct antigen formation of Ni²⁺ on antigen-presenting cells (APCs) was studied with cultured human dendritic cells (DCs) obtained from 10 subjects contact allergic to Ni²⁺ and six non-allergic control individuals. All contact allergic subjects showed a significantly increased peripheral blood mononuclear cell (PBMC) response in vitro to Ni²⁺. DCs were expanded from the plastic-adherent cell fraction of PBMCs by culturing with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days to obtain immature DCs, and with the addition of monocyte-conditioned medium for another 4 days, for DC maturation. The DCs were pulsed for 20 min with Ni²⁺ (50 μm) in protein-free Hank's balanced salt solution (HBSS) and added to freshly prepared autologous responder PBMCs. With five allergic subjects, immature DCs pulsed with Ni²⁺ demonstrated a significant capacity to activate Ni²⁺-reactive lymphocytes. With the remaining five patients and the six controls no difference in lymphocyte proliferation was observed between Ni²⁺-pulsed and non-pulsed immature DCs. In contrast, with mature Ni²⁺-pulsed DCs from both ‘positive responder’ (n = 4) and ‘non-responder’ (n = 4) patients, there was a significantly stimulated PBMC proliferation, whereas with the controls (n = 4) still no activation was observed. Our results indicate that direct formation of the antigenic determinant of Ni²⁺ on APCs is possible and that Ni²⁺ uptake and processing mechanisms may not play a major role. Differences in the ease of activation of Ni²⁺-reactive lymphocytes are discussed in terms of a possible heterogeneity in the availability of Ni²⁺-reactive groups presented on endogenous peptides bound in the antigen binding groove of human leucocyte antigen (HLA) class-II molecules.

INTRODUCTION

Ni²⁺ is the most frequent cause of contact allergy.¹ Yet, the antigenic structures formed by Ni²⁺ and the epitopes recognized by the metal ion-reactive T cells are essentially unknown. Ni²⁺-reactive T cells express CD3 and T-cell receptor (TCR)-αβ, are CD4⁺ or CD8⁺,² and are most often stimulated in a human leucocyte antigen (HLA)-DR-restricted fashion.³ Activation of T cells by Ni²⁺ leads to the release of cytokines with either a T helper 1 (Th1) (interferon-γ; IFN-γ), Th2 (interleukin-4; IL-4) or Th0 (IFN-γ+IL-4) pattern and is possibly associated with a limited number of TCR-Vβ elements.² There are indications for a genetic predisposition for the disease,⁴ possibly related to polymorphism of TAP genes.⁵ In contrast, it has not been possible to demonstrate an association of Ni²⁺ contact allergy and distribution of alleles expressed in HLA-DR regions.⁶

It is most probable that Ni²⁺ contact allergy is initiated by the formation of specific co-ordination complexes with electron-rich residues of proteins (or other biomolecules).⁷ In theory, the antigenic epitopes presented on HLA-class II molecules can originate from Ni²⁺ interaction with soluble proteins or from direct Ni²⁺ interaction with proteins of antigen-presenting cells (APCs), such as cell surface proteins, HLA-class II-associated proteins or intracellular self proteins. A possible direct antigenic metal ion–protein interaction on APCs has previously been suggested, but so far experimental evidence has been limited. For example, Ni²⁺ was found to inhibit unrelated peptide-specific T-cell reactions when the antigen contained histidine.⁸ But it still remains unclear whether those apparent interactions between Ni²⁺ and protein indeed form the antigenic epitopes recognized by Ni²⁺-reactive T cells. Stronger indications have been put forward by Weltzien and coworkers,⁹ who demonstrated that glutaraldehyde-fixed autologous Epstein–Barr virus-transformed B cells in a number of cases were capable of activating Ni²⁺-reactive T-cell clones. This indicates a possible non-processing-dependent pathway of Ni²⁺ antigen presentation, although the role of B cells in contact allergy responses may be questioned.

In this study, we investigated possible direct antigen formation of Ni²⁺ on cultured human dendritic cells (DCs). DCs highly express HLA-class II molecules and are the only APCs which are able to activate naive T cells.^10,11 In addition, DCs generated from human peripheral blood have been found to be far superior in stimulating antigen specific memory T cells in comparison with macrophages and B cells.^12,13 The hypothesis was studied with immature as well as mature DCs, because the functionality of DCs is known to be strongly dependent on the different stages of their development.^10,11

MATERIALS AND METHODS

Materials

NiCl₂·6H₂O and trypan blue were obtained from Merck KGaA, Darmstadt, Germany. Gentamicin, l-glutamine, HBSS, penicillin, RPMI-1640 medium and streptomycin were purchased from Gibco BRL, Life Technologies Ltd, Paisley, UK. 2-Mercaptoethanol was supplied by Sigma, St. Louis, MO, and phytohaemagglutinin (PHA) by Pharmacia Biotech., Uppsala, Sweden. [³H]Thymidine was purchased from Amersham International plc, Amersham Place, UK, and tuberculin purified protein (PPD) for in vitro use from Statens Seruminstitut, Copenhagen, Denmark. Granulocyte–macrophage colony-stimulating factor (GM-CSF) was supplied by Schering Plough, Kenilworth, NJ, and IL-4 by Genzyme Corporation, Cambridge, MA. Fetal calf serum (FCS) was purchased from Hyclone Laboratories Inc., Lagen, UT, and tetanus toxoid protein (TT) from SBL Vaccine Distribution, Stockholm, Sweden.

Media

RPMI-1640 supplemented with gentamicin (25 μg/ml), penicillin (100 IU/ml), streptomycin (100 μg/ml) and l-glutamine (2 mm), referred to as culture medium, was used. For the generation of DCs the culture medium was supplemented with 2-mercaptoethanol (50 μm) and heat-inactivated (56° for 30 min) FCS (10%; v/v). Conditioned medium, to maturate DCs, was prepared as previously described.^13,14 In short, 225 cm² culture flasks (Costar Corporation, Cambridge, MA) were coated with human immunoglobulin (11·25 ml, 10 mg/ml in phosphate-buffered saline (PBS); Endobulin, Immuno AG, Vienna, Austria) by incubation for 1 hr at room temperature. The flasks were washed three times with PBS. PBMCs from a buffy coat, obtained as described below, were added (6·3×10⁶ cells/ml; 22·5 ml/flask) and incubated in culture medium supplemented with FCS (10%; v/v) for 1 hr at 37° in an atmosphere of 5% CO₂ in air. The non-adherent cells were washed away with RPMI-1640 and the adherent cells were further cultured in fresh culture medium supplemented with 10% FCS (22·5 ml/flask) for 24 hr at 37°. The supernatant was taken, filter-sterilized (pore: 0·20 μm; Micro Filtration Systems, Dublin, CA) and stored at −20° until used.

The lymphocyte proliferation assays, i.e. allogeneic mixed lymphocyte reaction (MLR) and antigen presentation experiments, were performed in culture medium supplemented with autologous plasma (10%; v/v).

Subjects

One male and nine female subjects (range of age: 24–41 years, median: 33 years), with confirmed allergic contact dermatitis to materials containing nickel were recruited from the Department of Occupational and Environmental Dermatology, Karolinska Hospital. The patients had a positive patch test (++: erythaema, papules and infiltration or +++: erythaema, papules, infiltration and vesicles) to NiSO₄·7H₂O (5%) in petrolatum, within 3 years before the start of this study. In addition, six female control subjects (range of age: 25–56 years, median: 48 years), without a history of allergic contact dermatitis and with a negative patch test reaction to NiSO₄·7H₂O (5%) in petrolatum, participated in this study. All the subjects gave their informed consent to donate blood. The study was approved by the local ethics committee.

Generation of DCs from human peripheral blood

DCs were generated from human blood according to previously described procedures.^14–16 Briefly, freshly prepared PBMCs were resuspended to 3·5×10⁶ cells/ml in culture medium supplemented with 2-mercaptoethanol and 10% FCS and cultured for 1 hr in 75 cm² culture flasks (Costar Corporation) at 37° in an atmosphere of 5% CO₂ in air. The non-adherent cells were removed by washing three times with RPMI-1640 by gentle pipetting, and the adherent cells further cultured for 7 days in the presence of GM-CSF 50 ng/ml (550 U/ml) and IL-4 20 ng/ml (800 U/ml). At day 3, the cells were refed by removing 5 ml of the medium from the culture flasks and adding back 5 ml of fresh culture medium supplemented with FCS, 2-mercaptoethanol, cytokines (final concentration of 25 ng/ml GM-CSF and 400 U/ml IL-4). On day 7, the cells were either used as immature DCs or further cultured for 4 days in the presence of stimuli for maturation. The medium was removed from the culture flasks and fresh culture medium supplemented with 2-mercapthoethanol, 10% FCS, GM-CSF (50 ng/ml), IL-4 (800 U/ml) and conditioned medium (35% v/v; see above) was readded to the cells. Finally, the cells were harvested, washed two times with RPMI-1640 (unless otherwise stated) and used as mature DCs in experiments indicated below. The development and morphology of the DCs were examined by phase contrast light microscopy (Zeiss, Carl Zeiss Jena GmbH, Jena, Germany). Viability of the cells was verified by Trypan blue exclusion and routinely exceeded 95%.

Lymphocyte proliferation assays

The lymphocyte proliferation assays were performed in triplicates for 4·5 days in U-bottomed 96-microwell plates (Costar Corporation) at 37° in a humid atmosphere of 5% CO₂ in air. 1 μCi of [³H]thymidine per well (specific activity: 25 Ci/mmol) in RPMI-1640 was added for the last 18 hr. The cells were harvested on a Tomtec microplate harvester (Tomtec, Orange, CT) using glass-fibre filters. Filters were dried and assayed in a Wallac 1205 betaplate scintillation counter (Wallac Oy, Turku, Finland). Culture conditions were evaluated by the PHA (10 μg/ml)-induced cell proliferation. Results are expressed as mean c.p.m.×10³±SEM.

PBMCs were prepared from heparinized blood by Ficoll–Paque (Pharmacia-Biotech.) density gradient centrifugation. Cells from the interface were washed three times with RPMI-1640 and 2×10⁵ cells/well (200 μl/well) were used as responder cells in culture medium supplemented with 10% autologous plasma.

For the MLR, graded doses (50–5000 cells/well) of non-irradiated or 30 Gy γ-irradiated DCs were added as stimulator cells for freshly prepared allogeneic responder PBMCs. As a comparison, the allogeneic lymphocyte activating capacity of 30 Gy γ-irradiated PBMCs (10⁵ cells/well) was measured.

The capacity of DCs to present soluble antigenic proteins was determined with PPD or TT. DCs were pulsed with PPD or TT (25 μg/ml) for 2 hr in culture medium supplemented with 10% autologous plasma at 37° in an atmosphere of 5% CO₂ in air. The cells were washed two times with RPMI-1640 and added in graded doses (50–2500 cells/well) to freshly prepared autologous responder PBMCs (2×10⁵ cells/well).

The presentation of Ni²⁺ by DCs was determined as follows. The DCs were washed three times with protein-free HBSS to remove the exogenous protein content from the medium. Thereafter the DCs were incubated for 20 min with or without Ni²⁺ (50 μm) in HBSS at 37° in an atmosphere of 5% CO₂ in air. Finally, the DCs were spun down, washed two times with RPMI-1640 and added in graded doses (100–5000 cells/well) in culture medium supplemented with 10% autologous plasma to freshly prepared autologous responder PBMCs. In parallel the PBMCs alone were incubated with Ni²⁺ (10–50 μm) for 4·5 days.

The efficiency of exogenous protein removal from the culture medium supplemented with autologous plasma was evaluated by parallel washing procedures performed with HBSS under identical conditions as indicated above with the use of methyl-[¹⁴C] methylated protein molecular weight markers (a mixture of phosphorylase B, γ-globulins, bovine serum albumin, ovalbumin and cytochrome C with a molecular weight range of 12·3–97·4 kDa; NEN Life Science Products, Boston, MA). Before the first centrifugation of the DCs, the culture medium supplemented with autologous plasma was pulsed with methyl-[¹⁴C] methylated protein molecular weight markers (final concentration: 0·1 μCi/ml; specific activity: 30 μCi/mg) and the radioactivity of the subsequently collected supernatants was analysed in a Beckman LS5801 liquid scintillation counter (Beckman Instruments, Fullerton, CA). The washing procedure with HBSS efficiently removed 99·99±0·002% (mean±SEM; n = 6) of the exogenous protein from the culture medium supplemented with autologous plasma.

Immunocytochemistry

The cells obtained after the DC generation procedure were washed with PBS, air-dried on three-well (Ø int. 14 mm) microscope glass slides (2000 cells/well; Novakemi, Enskede, Sweden) and stored at −80° until used. Cells were fixed in acetone (50%; 4°) for 30 s followed by acetone (100%; 4°) for 5 min and allowed to react with normal rabbit serum (Dakopatts, Copenhagen, Denmark) for 10 min to reduce non-specific staining. Next, the cells were incubated for 60 min at room temperature with the following mouse monoclonal antibodies (mAbs): Leu-6 (CD1a), Leu-4 (CD3), Leu-3a (CD4), Leu-2a (CD8), Leu-M3 (CD14), Leu-12 (CD19), Leu-19 (CD56), anti-HLA-DR (all from Becton Dickinson, San Jose, CA); anti-CD40 (Serotec Ltd, Oxford, UK); anti-CD80 (Immunotech, Marseille, France); anti-CD83 (kindly provided by Dr T. Tedder, Duke University Medical Center, Durham, NC)¹⁷; anti-CD86 (Pharmingen, San Diego, CA) or Lag antibody (specific for Birbeck granule associated proteins, kindly provided by Dr K. Yoneda and Dr S. Imamura, Kyoto University, Japan)¹⁸ dissolved in bovine serum albumin (4% w/v; Sigma). The glass slides were washed with PBS, incubated with rabbit antimouse immunoglobulin (Dakopatts) for 30 min, washed with PBS, and incubated with preformed complexes of peroxidase–antiperoxidase (Dakopatts) for 30 min. The colour reaction was developed with 3-amino-9-ethylcarbazol (0·02% w/v; Aldrich-Chemie, Steinheim, Germany) dissolved in acetate buffered (pH = 5·5) hydrogen peroxide (0·01%) for 15 min and the cells were counterstained with Mayer's haematoxylin (Sigma) for 1 min. Specificity tests included omission of the primary mAbs or replacement with irrelevant mAbs. Staining was not observed in these tests. The mAbs gave positive staining on normal PBMCs or appropriate cryostat-sections from normal human skin or adenoid tissue stained in parallel. A minimum of 500 DCs were counted, omitting the lymphocytes, with a Leitz microscope with a ×40/0·70 PL Fluotar objective and a ×10 eyepiece (Laborlux K, Nürnberg, Germany). A positive cell was defined as one with a definite brownish-red staining and a visible nucleus. Results are expressed as the percentage of positive cells of the total number of counted cells.

Statistics

The statistical evaluation of the results was performed with the Mann–Whitney U-test. A P-value <0·05 was considered statistically significant.

RESULTS

Ni²⁺ reactivity of PBMCs

The PBMC cultures from the 10 contact allergic subjects showed a significantly increased lymphocyte proliferation induced by Ni²⁺ (10–50 μm) in comparison with the cultures from the six control individuals (Fig. 1). The optimal proliferative response was detected at a Ni²⁺ concentration of 50 μm, which was used in all additional experiments.

Figure 1.

Ni²⁺-induced lymphocyte proliferation in PBMC cultures (2×10⁵ cells/well) from patients contact allergic to Ni²⁺ (□n = 10) and healthy controls (◊n = 6). PBMCs were cultured in triplicates for 4·5 days in the presence of Ni²⁺ (10–50 μm). Cell proliferation was measured by [³H]-thymidine incorporation and expressed as mean c.p.m.×10³±SEM. *Significantly different from the controls, P < 0·05 or **P < 0·01.

Morphological and phenotypical characterization of the DCs

During the first 7 days of culture the plastic-adherent monocytes became non-adherent, rapidly increased in size and revealed an irregular shape. The following 4 days with the addition of monocyte-conditioned medium the DCs formed aggregates and displayed characteristic veils extending all over the cell body (Fig. 2). The morphological features were in accordance with previously presented data.,^15,16,18 From the starting number of PBMCs we obtained about 6±1% immature (n = 16) and 4±1% mature DCs (n = 12), with a purity of about 82±3% (n = 15) and 83±4% (n = 12), respectively, without any significant difference between patients and healthy controls. The majority of the non-DCs were CD19⁺ cells, 13±3% (n = 28, of the total cell population). There was no significant difference in this number between patients and controls or between immature and mature DCs populations.

Figure 2.

Phase contrast microscopic illustration of a mature DC after 11 days of culture. Plastic-adherent cells from human PBMCs were cultured for 7 days in the presence of GM-CSF (50 ng/ml) and IL-4 (800 U/ml), followed by 4 days in the presence of GM-CSF (50 ng/ml), IL-4 (800 U/ml) and monocyte-conditioned medium. Original magnification ×800.

Because of the limited number of DCs in each experiment immunoperoxidase staining on microscope slides was used for phenotypical characterization. All immature and mature DCs expressed HLA-DR and the costimulatory molecule CD40 (Table 1). The CD1a and CD4 expression did not differ significantly between the immature and the mature DCs (Table 1). CD80 was present on the majority of immature and all mature DCs, whereas the CD86 expression increased from around 50% in the immature cell population to almost 100% in the mature DC population. The CD83 expression was detectable on less than 10% of the immature DC population and on more than 95% of the DCs in the mature population, which corresponds to the general idea of CD83 being a marker for mature DCs (Table 1 ¹⁷). The Birbeck granules were detected in less than 1% of the DCs. The characteristic antigens for T cells (CD3, CD8), B cells (CD19) and natural killer (NK)-cells (CD56) were expressed in less than 1% of the DC populations. There was no significant difference between patients and healthy controls for the phenotypical markers investigated.

Phenotype of the generated immature and mature DCs

*DCs at day 7 of culture.

†DCs at day 11 of culture.

‡Positively stained DCs, as analysed by using peroxidase–antiperoxidase technique (mean percentage±SEM); a minimum of 500 cells were counted.

nd = not detectable.

Immunostimulating capacity of DCs

Immature DCs (250–5000 cells/well) strongly induced proliferation of allogeneic lymphocytes in a dose-dependent fashion (Fig. 3). However, per cell basis mature DCs (50–500 cells/well) were about 10-fold more efficient in lymphocyte activation (Fig. 3). No difference in the MLR response was seen between 30 Gy γ-irradiated and non-irradiated DCs (n = 7; data not shown). The allogeneic lymphocyte activating capacity of irradiated PBMCs tested in parallel was in the experiments with immature DCs 67±8 c.p.m.×10³ (n = 16) and with mature DCs 48±4 c.p.m.×10³ (n = 12). The PHA (10 μg/ml) induced proliferation for the PBMCs used in the experiments with immature and mature DCs was 93±8 c.p.m.×10³ (n = 16) and 85±10 c.p.m.×10³ (n = 12), respectively. The background proliferation of PBMCs in culture medium supplemented with autologous plasma only was 2·5±0·4 c.p.m.×10³ (n = 16) and 1±0·1 c.p.m.×10³ (n = 12), respectively. Background proliferation of DCs alone in culture medium never exceeded the background proliferation of the PBMCs. There was no significant difference in cell proliferation capacity in any of these assays between the patient group and the healthy controls.

Figure 3.

MLR in PBMC cultures (2×10⁵ cells/well), cultured for 4·5 days in the presence of graded doses of allogeneic immature DCs (◊n = 16) and allogeneic mature DCs (□n = 12). Cell proliferation was measured by [³H]thymidine incorporation and expressed as mean c.p.m.×10³±SEM.

The efficiency of soluble antigen uptake and presentation by the DCs was evaluated with the protein antigens PPD and TT. Immature DCs were very efficient in taking up and presenting PPD and TT to autologous PBMCs as was evident from the significantly enhanced lymphocyte proliferation induced by PPD- and TT-pulsed DCs (250–2500 cells/well; Fig. 4) in comparison with nonpulsed DCs (P < 0·01). In contrast, with mature DCs the efficiency of PPD presentation was significantly reduced compared to immature DCs (P < 0·05; Fig. 4a), and TT presentation was no longer apparent (Fig. 4b).

Figure 4.

Autologous lymphocyte proliferation in PBMC cultures (2×10⁵ cells/well), cultured for 4·5 days in the presence of graded doses of (a) immature DCs (◊n = 8) and mature DCs (□n = 9) pulsed with PPD (25 μg/ml), and (b) immature DCs (◊n = 7) and mature DCs (□n = 7) pulsed with TT (25 μg/ml). Cell proliferation was measured by [³H]thymidine incorporation and expressed as mean c.p.m.×10³±SEM. The background radioactivity incorporated into lymphocytes exposed to non-pulsed DCs (autologous MLR) was subtracted. *Significantly different from the antigen-pulsed mature DCs, P < 0·05.

Presentation of Ni²⁺ by DCs

Immature DCs pulsed with Ni²⁺ (50 μm) in protein free HBSS for 20 min demonstrated a significant capacity to activate Ni²⁺-reactive lymphocytes with five of the 10 allergic subjects (Fig. 5a). With the remaining five patients and the six controls a difference in lymphocyte activation was not observed between Ni²⁺-pulsed and non-pulsed DCs (Fig. 5a). The lack of lymphocyte activation was reproduced with two of the ‘non-responder’ patients (Fig. 5b). Higher concentrations of Ni²⁺ were not used since these resulted in a decreased viability of the DCs. Further kinetic studies with two ‘non-responders’ revealed that incubation periods of the DCs with Ni²⁺ up to 60 min did not affect the extent of immunostimulation (Fig. 5c). All patients PBMCs cultured in parallel in the presence of Ni²⁺ for 4·5 days did proliferate as expected (Fig. 1).

Figure 5.

Autologous lymphocyte proliferation in PBMC cultures, cultured for 4·5 days in the presence of graded doses of Ni²⁺ (50 μm)-pulsed (a) immature DCs from ‘positive-responder’ patients (□n = 5), from ‘non-responder’ patients (◊n = 5) and from healthy controls (○n = 6) (b) repeated experiments with two of the ‘nonresponder’ patients (♦, ▪) (c) prolonged incubation of immature DCs with Ni²⁺ up to 60 min in two of the ‘non-responder’ patients (♦, ▪) (d) mature DCs from ‘positive responder’ patients (□n = 4) from ‘non-responder’ patients (◊n = 4) and from healthy controls (○n = 4). Cell proliferation was measured by [³H]thymidine incorporation and the results are expressed as mean c.p.m.×10³±SEM for the indicated number of individuals in (a) and (d), and as mean c.p.m.×10³ of triplicates for each individual in (b) and (c). The background radioactivity incorporated into lymphocytes exposed to nonpulsed DCs (autologous MLR) was subtracted. *Significantly different from the non-pulsed DCs, P < 0·05or **P < 0·01.

The effect of DC maturation on the efficiency of Ni²⁺ antigen presentation was studied with four subjects from each ‘positive responder’, ‘nonresponder’ and control group. In contrast to the immature DCs (Fig. 5a), Ni²⁺-pulsed mature DCs of both the ‘positive responder’ and ‘non-responder’ subjects significantly stimulated PBMC proliferation in comparison with non-pulsed DCs, whereas with the controls still no activation was observed (Fig. 5d).

DISCUSSION

This study shows that Ni²⁺-pulsed immature DCs in HBSS activated Ni²⁺-reactive lymphocytes in 50% of the contact allergic subjects studied, whereas with the other 50% of the subjects no stimulation was found. In contrast, with mature Ni²⁺-pulsed DCs a homogenous pattern of lymphocyte activation was observed. These results indicate that direct Ni²⁺ antigen formation on APCs is possible and that antigen uptake and processing mechanisms may not play a major role in contact allergy to Ni²⁺.

It was decided to study the hypothesis of potential direct Ni²⁺ antigen formation on APCs with cultured human DCs, because these cells have been found to be superior in activating naive as well as memory T cells in comparison with monocytes or B cells.^10–13 However, in vitro as well as in vivo, DCs can exist in different stages of maturation, each of which corresponds with a different functionality. Immature DCs are specialized in capturing and processing protein antigens, whereas mature DCs are functionally characterized by an increased efficiency in stimulating T cells, accompanied with down-regulated antigen uptake and processing mechanisms.^10,11 The availability of both immature DCs and mature DCs was instrumental to investigate the molecular basis of Ni²⁺ antigen formation and the importance of antigen uptake/processing mechanisms in the activation of Ni²⁺-reactive lymphocytes.

DCs are only present as a trace population in normal blood and tissues.¹¹ However, various in vitro procedures have been developed to obtain substantial numbers of DCs from haematopoietic progenitor cells – in particular CD34⁺ cells – derived from peripheral blood,¹⁹ bone marrow²⁰ or neonatal cord blood.²¹ We successfully generated DCs from the plastic-adherent monocyte fraction from human blood according to recently published protocols,^14–16 and obtained DCs with a morphology (Fig. 2) and phenotype (Table 1) conforming with these described in earlier publications.^13,17 The costimulatory molecule CD86 and the DC line-specific marker CD83¹⁷ were both markedly upregulated in the mature DC population. There was no significant difference between patients and healthy controls regarding the DC characteristics.

In accordance with other functional studies, the immature DCs displayed, per cell, about a 100-fold higher efficiency than PBMCs to stimulate allogeneic lymphocytes,¹³ while with mature DCs the efficiency was further enhanced approximately another 10-fold (Fig. 3).^15,17 This increased capacity to activate lymphocytes upon DC maturation is probably related to an upregulated expression of HLA products^13,22 and adhesion/costimulatory molecules.¹³ Interesting to note is that the stimulation of autologous lymphocytes in the absence of exogenous antigen was also significantly enhanced by mature DCs in comparison with immature DCs. This phenomenon has been observed earlier,²³ and is possibly a result of presentation of FCS-derived peptide epitopes bound to the HLA-class II molecules, formed during the generation of DCs in FCS-containing culture medium. Alternatively, an upregulated presentation of DC-derived endogenous self antigens has been suggested, which in vivo is thought to be possibly related to the pathogenesis of autoimmune diseases.²⁴ The significantly reduced efficiency of lymphocyte activation by PPD- and TT-pulsed mature DCs in comparison with antigen-pulsed immature DCs (Fig. 4), is compatible with the known downregulated antigen uptake and processing mechanisms of mature DCs^10,11 and is in agreement with other studies.^13,15

Immature DCs pulsed with Ni²⁺ in protein-free HBSS displayed a dual behaviour with respect to activation of the Ni²⁺-reactive lymphocytes. With PBMC cultures from five of the 10 allergic subjects a significant stimulation of lymphocyte proliferation was induced by Ni²⁺-pulsed immature DCs (Fig. 5a). This suggests that with these five ‘positive responders’ the antigenic determinant of Ni²⁺ was not formed with exogenous proteins from the medium (only 0·01% was left after the washing procedure) and indicates that Ni²⁺ interacted directly with endogenous proteins expressed by the DCs themselves. In contrast, with the remaining five Ni²⁺ allergic subjects no significant activation of the Ni²⁺-reactive lymphocytes was induced by the Ni²⁺-pulsed immature DCs (Fig. 5a). Because there was no significant difference in severity of the clinical manifestation of contact allergy, PBMC reactivity to Ni²⁺ (Fig. 1) or DC characteristics between the five ‘positive responders’ and five ‘non-responders’, a possible heterogeneity of the mechanism of Ni²⁺ antigen formation/recognition was considered. In this respect, it is important to realize that from a chemical point of view Ni²⁺ is probably able to form various kinds of antigenic determinants with the abundantly available electron-rich residues of proteins or other biomolecules. Among the evidence supporting a heterogeneity of the Ni²⁺ response are for example the observations that Ni²⁺-reactive T cells can display differences in processing requirements of APCs,⁹ requirements of DC subsets,²⁵ TCR repertoires,²⁶ cytokine secretion patterns² and phenotype.^2,27

To evaluate the importance of antigen uptake and processing mechanisms on the activation of Ni²⁺-reactive lymphocytes, mature DCs were pulsed with Ni²⁺ in HBSS and the efficiency of immunostimulation was compared with that observed for the immature DCs described above. We decided not to work with chemically fixed DCs to manipulate antigen processing, since these procedures are liable to interfere with protein structures exposed on the cell surface of the DCs structures which could be essential for the interaction with Ni²⁺-ions. With Ni²⁺-pulsed mature DCs a difference between ‘positive responders’ and ‘nonresponders’ was no longer apparent (Fig. 5b). This observation gives further support to our hypothesis that the metal antigen can be directly formed on APCs themselves. Furthermore, it suggests that uptake and processing mechanisms of APCs do not play a major role in activating Ni²⁺-reactive lymphocytes, which implies that the Ni²⁺ ion itself probably plays a structural role in the antigenic determinant recognized by the TCR. In accordance with these deductions, it has been postulated that the Ni²⁺ antigen is formed directly on endogenous peptides bound in the antigen-binding groove of HLA-class II molecules of APCs.⁸ On the other hand, it is possible that Ni²⁺ alters HLA-class II molecules themselves which are then recognized by T cells as ‘altered self’, as has for example been suggested for contact allergy to gold.²⁸ However, since no HLA-class II association has been detectable in Ni²⁺ contact allergic subjects,⁶ it seems more likely that the antigenic determinants of Ni²⁺ are formed in the antigen binding groove; this is because HLA-class II molecules can bind and present a wide variety of endogenous peptides. Maturation of DCs leads to an upregulated total synthesis of HLA-class II molecules, an upregulated redistribution of these molecules from intracellular compartments to the cell surface and an increased half-life of HLA-class II molecules from about 10 hr to over 100 hr.²² Thus, DC maturation results in a rapid accumulation of a large number of long-lived peptide-loaded HLA-class II molecules on the cell surface of DCs, which will enhance the ease of antigenic Ni²⁺ interaction in comparison with immature DCs.

The finding of ‘non-responders’ with Ni²⁺-pulsed immature DCs (Fig. 5a) needs further explanation since immature DCs also express relatively large number of HLA-class II molecules. Apart from the finding that immature DCs were 10-fold less efficient in activating lymphocytes (Fig. 3), it should be kept in mind that just a minor fraction of the HLA-class II expression of immature DCs is present at the cell surface.²² Furthermore, in particular the outer domain of the peptide binding groove of HLA molecules displays considerable variation in the availability of chemical groups, e.g. histidinyl groups, directed to the TCR. As such, in accordance with earlier suggestions,^8,28,29 it is this heterogeneity in availability of reactive groups which can be responsible for differences in the ease of lymphocyte activation and the regulation of the immune response to Ni²⁺. Further indications of a heterogeneity in the ease of T-cell activation by Ni²⁺ have become apparent from findings that proliferation of some Ni²⁺-reactive T-cell clones required the permanent presence of Ni²⁺ whereas others started to proliferate when exposed to hapten-pulsed and washed APCs.⁹

In conclusion, direct Ni²⁺ antigen formation on APCs seems to be possible and uptake/processing mechanisms may not play a major role in contact allergy to Ni²⁺. This study gives support to earlier^8,9 indications that the antigenic determinant of Ni²⁺ is directly formed on endogenous peptides bound in the antigen binding groove of HLA-class II molecules of APCs.

Glossary

Abbreviations

DCs

dendritic cells

PPD

tuberculin purified protein

TT

tetanus toxoid protein