RESULTS

CpG ODN enhance CHS when applied at the site of DNFB sensitization

CHS to DNFB was performed by epicutaneous sensitization on the back skin at day 0 and challenge on the ear at day 5. CHS was tested in groups of mice, which received one subcutaneous (s.c.) injection of CpG ODN, control ODN, or PBS alone, on the back or on abdominal skin 1 d before DNFB sensitization. Two groups of mice were depleted in CD8+ T cells by in vivo treatment with anti-CD8 mAb.

When mice received CpG ODN at the same site as the DNFB sensitization, i.e., the back skin, the intensity of CHS to DNFB was enhanced compared with that observed in mice receiving control ODN or PBS alone (Figure 1). But the kinetics of the CHS reaction was similar in the three groups of mice with a maximal ear swelling at 24/48 h. Downregulation of the inflammation started at day 3 and complete recovery was achieved at days 10–15. In vivo treatment of mice before the sensitization phase with anti-CD8 mAb, but not control isotype mAb, depleted the CD8+ T cell subset (data not shown) and abrogated the CHS reaction, confirming that CD8+ T cells mediate the CHS reaction to DNFB. Alternatively, when mice received CpG ODN injections at a skin site distant from the sensitization site, i.e., the abdominal skin, CHS reaction was not affected by CpG ODN treatment. The magnitude of the CHS response was similar in both CpG ODN (abdomen)- and control ODN (back)-treated mice (Figure 1). These data show that CpG ODN function as a local, but not systemic, adjuvant for enhancement of contact hypersensitivity to DNFB in mice.

Figure 1.

Effect of cytidine – phosphate – guanosine – oligodeoxynucleotides (CpG ODN)-treatment on the contact hypersensitivity (CHS) reaction to 2,4-dinitrofluorobenzene (DNFB). CHS to DNFB was performed by sensitization on the back skin at day 0 and challenge on the ear at day 5. One day before DNFB sensitization, mice (5 mice per group) received either one subcutaneous (s.c.) injection of CpG ODN (), control ODN (), or PBS alone () on the back skin. Two groups of mice receiving CpG () and control ODN () were depleted in CD8+ T cells by in vivo treatment with anti-CD8 mAb. One group received CpG ODN treatment on the abdominal skin (abdomen ()). One group was left unsensitized and challenged at day 5 (). Results are expressed as the mean ear swellingSD at different time points after challenge. p (<0.05) indicates statistical significance between CpG- and control ODN-treated mice. Results show one representative experiment of four.

Full figure and legend (39K)

CpG ODN-treated mice develop enhanced recall T cell responses

We next analyzed the mechanisms of the local adjuvant effect of CpG ODN. First, we examined whether CpG ODN could increase the immunological memory to haptens. CpG ODN- and control ODN-treated mice which had developed a CHS response by hapten painting on the left ear on day 5 (as in Figure 1) were both re-challenged on day 28 on the right ear with DNFB. The recall CHS reaction was significantly enhanced in previously CpG ODN-treated mice compared with control ODN-treated mice (Figure 2), demonstrating the stimulatory effect of CpG ODN on T cell responses.

Figure 2.

Effect of cytidine – phosphate – guanosine – oligodeoxynucleotides (CpG-ODN) treatment on the recall contact hypersensitivity (CHS) response. Mice which developed the CHS response after treatment on the sensitization site with CpG ODN (A) or control ODN (B), or after treatment on a distant site with CpG ODN (C) (as in Figure 1) were re-challenged at day 28 with 2,4-dinitrofluorobenzene (DNFB) on the contra-lateral ear. One group of unsensitized naïve mice received DNFB on the ear at day 28 (D). Results are expressed as the mean ear swellingSD 48 h after the second challenge. p (<0.05) indicates statistical significance between CpG- and control ODN-treated mice. Results show one representative experiment of three.

Full figure and legend (13K)

CpG ODN treatment increases CD8+ T cell infiltration in challenged skin

Since CHS to DNFB is mediated by infiltration of IFN -producing CD8+ effector T cells in the epidermis at the challenge site (^{Akiba et al. 2002}), we next analyzed the contribution of CD8+ T cell recruitment to the enhanced CHS response observed in CpG ODN-treated mice. Ears from control ODN- and CpG ODN-treated mice were recovered 24 h after challenge at the peak of the CHS reaction. Histological analysis showed increased dermal edema and mononuclear cell infiltration in CpG ODN- compared with control ODN-treated mice (Figure 3a,b). Immunohistochemical analysis revealed that in control ODN-treated mice, CD8+ cells were mostly found in the upper dermis with a few CD8+ epidermal cells. Although there was a slight increase in CD8+ cell infiltration in the dermis of CpG ODN-treated mice compared with control ODN-treated mice, CpG ODN-treated mice exhibited much higher numbers of CD8+ cells infiltrating the epidermis (Figure 3c–e). That CD8+ epidermal cells were T cells was confirmed by additional experiments showing that they expressed CD3 but not major histocompatibility complex (MHC) class II molecules (data not shown).

Figure 3.

Histological analysis of the contact hypersensitivity (CHS) reaction to 2,4-dinitrofluorobenzene (DNFB) in cytidine–phosphate–guanosine–oligodeoxynucleotides (CpG ODN)-treated mice 24 h after challenge. (A, B): Hematoxylin and eosin (H&E) staining of the ear of control ODN-treated mice (A) and CpG ODN-treated mice (B). Arrowhead indicates a dilated capillary vessel. (C, D): Immunohistochemical staining of CD8+ T cells (CD8) in the skin of control ODN-treated (C) and CpG ODN-treated (D) mice. CD8+ T cells are found in the dermis in both groups of mice but are found in higher numbers in the epidermis of CpG ODN-treated mice (arrowheads). Dotted line represents the dermal–epidermal (D–E) junction. Bar=20 m. c, cartilage; e, epidermis; d, dermis. (E) Semi-quantitative analysis of CD8+ cells in the epidermis of control ODN and CpG ODN-treated mice at 24-h post-challenge. Results are expressed as the mean number of cells per mm of D–E junctionSD. p (<0.05) indicates statistical significance between CpG- and control ODN-treated mice. Results show one representative experiment of three.

Full figure and legend (80K)

We next examined the presence of CD8 and IFN- mRNA in the ears of mice treated with CpG ODN or control ODN 24 h after challenge and subjected to mRNA extraction and semi-quantitative RT-PCR analysis using HPRT mRNA as internal standard (Figure 4). CD8 and IFN- mRNA in the skin from CpG ODN-treated mice were 1.5–2 times higher than that of control ODN-treated mice.

Figure 4.

RT-PCR analysis of CD8 and IFN- mRNA expression in the skin during the contact hypersensitivity (CHS) response. Expression of CD8 and IFN- mRNA was determined by semi-quantitative RT-PCR analysis, 24 h after challenge with 2,4-dinitrofluorobenzene (DNFB) or vehicle, in the ears of cytidine – phosphate – guanosine – oligodeoxynucleotides (CpG ODN) or control ODN-treated mice (A). Histogram derived from the results presented in A showing the CD8 and IFN- mRNA relative quantities expressed as a ratio between optical densities (OD) obtained in experimental samples compared with HPRT mRNA as standard (B). Results are representative of four experiments.

Full figure and legend (45K)

Thus, increased CHS response in CpG ODN-treated mice is associated with enhanced infiltration of IFN--producing T cells in challenged skin.

CpG ODN induces in vivo activation of epidermal DC

Since CHS reaction is mediated by T cells which are primed in draining lymph nodes by hapten-bearing DC that migrate from the skin, we analyzed the effect of CpG ODN on epidermal DC phenotype and function. In order to test for the effect of CpG ODN on the expression of MHC and costimulatory molecules by skin DC, CpG ODN (or control ODN) was injected intradermally in the right ear of naïve mice 24 h before epicutaneous application of a sensitizing dose of 0.5% of DNFB onto the same ear. Ears were recovered 24 h after hapten application and analyzed for expression of DC surface molecules. We observed a more pronounced upregulation of MHC class II molecules and increased numbers of CD80+ and CD86+ cells in the epidermis of CpG ODN-injected skin, compared with control ODN-injected skin (Figure 5a–f) with a 2-fold increase in the number of CD80+ and CD86+ cells, respectively (Figure 5g).

Figure 5.

Immunohistochemical analysis of epidermal cells 24 h after 2,4-dinitrofluorobenzene (DNFB) sensitization at the site of cytidine–phosphate–guanosine (CpG) treatment. Mice received subcutaneous (s.c.) injections of control (A–C) or CpG ODN (D–F) on the ear skin at day 0, and DNFB painting at day 1. Skin was recovered 24 h after DNFB painting and cryostat sections were stained for major histocompatibility complex class II (A, D), CD80 (B, E), and CD86 (C, F) antigen expression using specific mAbs. Arrows in B, C, E, and F show positive epidermal cells. Bar=10 m. (G) Semi-quantitative analysis of CD80- and CD86-positive epidermal cells in control ODN- (white bars) and CpG ODN- (black bars) treated mice. Results are expressed as the mean number of cells per mm of dermal–epidermal (D–E) junctionSD. p (<0.05) indicates statistical significance in the results obtained in control versus CpG-treated mice. Results are representative of three experiments.

Full figure and legend (108K)

Next, we tested for the functions of CpG ODN-treated skin DC. DC recovered from the skin of normal mice were treated in vitro with CpG ODN, control ODN or left untreated, and then hapten derivatized by incubation with 2,4-dinitrobenzenesulfonic acid (DNBS) before s.c. transfer into naïve mice. Five days later, mice were ear-challenged with DNFB. As shown in Figure 6, control ODN-treated skin DC induced a CHS response upon transfer into naïve mice. Similar results were obtained with untreated, hapten-derivatized skin DC. More importantly, the CHS response induced by adoptive transfer of CpG ODN-treated skin DC was strongly increased compared with that induced by control ODN-treated skin DC, demonstrating that CpG ODN enhanced the immunostimulatory function of skin DC.

Figure 6.

Cytidine – phosphate – guanosine – oligodeoxynucleotides (CpG ODN) enhance the ability of cultured dendritic cells (DC) to transfer contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB). Skin DC recovered from naïve mice were treated in vitro with CpG ODN (A), control ODN (B) (1 M at 37°C for 12 h), or medium alone (C), and then haptenized with 2,4-dinitrobenzenesulfonic acid (DNBS) (4 mM DNBS at 37°C for 30 min). Cells were transferred to recipient naïve mice (subcutaneous back skin injection of 1 10⁵ DC per mouse). Five days later, recipient mice were challenged with DNFB (10 L of 0.2% DNFB on the ear) and CHS was determined by ear swelling at 24 h post-challenge. Controls included mice which received untreated/unhaptenized DC at day 0 and DNFB painting on the ear at day 5 (D). CpG ODN/DNBS-derivatized DC-transferred mice showed a significantly enhanced CHS response. Results are expressed as the mean ear swellingSD. p (<0.05) indicates statistical significance compared with mice which received the control ODN/DNBS-derivatized DC. The result is one representative experiment of three (consisting of 5 mice per group).

Full figure and legend (12K)

DISCUSSION

CHS reaction to DNFB is mediated by antigen-specific, MHC class I-restricted CD8+ T cells which are induced in lymphoid organs by presentation of haptenated peptides by skin DC (^{Krasteva et al. 1999a; Cavani et al. 2001}). Here we show that CpG ODN can enhance CHS responses by increasing the immunostimulatory properties of skin DC leading to optimal priming of T cells and increased recruitment of CD8+ T cells in the skin. This study extends to haptens previous results obtained with protein antigens and peptides indicating that CpG ODN-based vaccines can augment T cell responses to specific antigens (^{Sun et al. 1998}) and promote priming and differentiation of CD8+ T cells (^{Tascon et al. 2000; Vabulas et al. 2000}).

Our results are consistent with CpG motifs activating skin innate immunity (^{Kobayashi et al. 1999; Revillard, 2002}) thereby leading to a more robust effector T cell response. We show that CpG ODN-induced phenotypic and functional maturation of skin DC with in vivo upregulation of MHC class II, CD80, and CD86 surface molecules at sites of subcutaneous injection. Furthermore, immunization of naïve mice by CpG ODN-treated, hapten-derivatized skin DC resulted in enhanced CHS reaction. Finally, the adjuvant effect of CpG ODN was local but not systemic as: (i) activation of skin DC and upregulation of costimulatory molecules was observed at sites of CpG ODN injections but not at distant sites; (ii) enhanced CHS reaction was obtained when the hapten was administered at the CpG ODN-injected skin site but not when applied at sites distant from CpG ODN-injected skin. These data are in line with studies showing that skin DC are the main target of immunostimulatory DNA sequences (^{Vogel and Udey, 2000}) through interaction with toll-like receptor (TLR)-9 (^{Hemmi et al. 2000}) leading to migration and phenotypic and functional maturation of resident DC (^{Jakob et al. 1998, 1999; Ban et al. 2000}).

Differences in CpG reactivity between mouse and human models have been documented. Therefore, it cannot be concluded from these data that CpG ODN could worsen CHS in humans. Follow-up of side-effects during clinical studies on the therapeutic potential of CpG DNA as adjuvants for vaccination strategies in cancer, allergies, and infectious diseases have concluded that CpG motifs were safe (^{Weiner, 2000a, b}). Moreover, in patients receiving repeated immunizations with vaccine mixed with CpG, no significant local hypersensitivity reactions have been reported to date (^{Halperin et al. 2003}).

From this study, it is clear that CpG ODN could cause adverse cutaneous immune-mediated effects such as enhanced CHS reactions. This result was not unexpected since stimulation of TLR-9 is known to enhance CD8+ CTL responses, as does CpG (^{Schwarz et al. 2003}). Thus, the potential side-effects of CpG ODN are directly linked to their immune properties, i.e., adjuvanticity, with activation of skin innate immunity to increase the adaptive immune responses. Although the lack of systemic effect of CpG ODN observed in this study would appear to be encouraging, future clinical studies will have to rule out the possibility that CpG ODN could cause aggravation of skin diseases, especially those where antigen-specific T cells are continuously activated by antigen-bearing skin DC.

MATERIALS AND METHODS

Reagents

Phosphorothioate-stabilized oligodeoxynucleotides (ODN) were synthesized and purified under sterilized condition by Grainer Japan (Tokyo, Japan), and reconstituted in endotoxin-free water. The ODN sequences used in this study were as follows: CpG ODN (1668 ODN): 5'-TCCATGACGTTCCTGATGCT-3', and control ODN (1720 ODN): 5'-TCCATGAGCTTCCTGATGCT-3' (^{Krieg et al. 1995}). DNFB (Sigma, St Louis, Missouri) was diluted in acetone:olive oil (4:1) immediately before use. DNBS (Kanto Chemical, Tokyo, Japan) was used for in vitro pulsing of DC. Antibodies used in in vivo experiments comprised anti-CD8 mAb, produced by the hybridoma H 35.17.2 kindly provided by G. Milon (Institut Pasteur Paris) and an isotype control mAb, produced by the hybridoma GL113 (rat anti--galactosidase IgG) (ATCC, Manassas, Virginia). The culture medium used was RPMI 1640 (Gibco BRL, Grand Island, New York) supplemented with 10% fetal calf serum (FCS), 100 U per mL penicillin, 100 g per mL streptomycin (Gibco BRL), and 2.5 g per mL Fungizone (Gibco BRL). This was referred to as complete medium (CM).

Mice

Female BALB/C mice (5–8-wk old) were purchased from SLC (Shizuoka, Japan) and bred in a conventional animal facility. All animal experiments were performed following the guidelines of the Fukushima Medical University School of Medicine animal care and use committee (K01153).

ODN treatment of mice

Twenty micrograms of CpG ODN or of control ODN diluted in PBS were injected subcutaneously (s.c.) in the back or abdominal skin (total volume of 300 L) of groups of BALB/C mice 1 d before epicutaneous application of DNFB at the back skin. One group of mice received PBS alone and was used as control.

Induction of contact sensitivity

The procedure used is the "mouse ear swelling test" (MEST) which has been described in detail elsewhere (^{Garrigue et al. 1994}). Briefly, 25 L of 0.5% DNFB was applied to the shaved back skin. Five days later, mice were challenged by applying 10 L of 0.2% DNFB on both sides of one ear, and 10 L of vehicle on the contra-lateral ear. Some groups of mice were re-challenged, on the opposite ear, 28 d after the first challenge. Ear thickness was measured with calipers (Peacock, Tokyo, Japan) prior to challenge, and every day after challenge. Ear swelling was calculated by the following formula: Ear swelling=(ear thickness after challenge with DNFB-ear thickness before challenge with DNFB)-(ear thickness after challenge with vehicle-ear thickness before challenge with vehicle).

Antibody depletion of CD8+ T cells in vivo

Mice were given intraperitoneally (i.p.), injections of 200 L 1:10-diluted anti-CD8 mAb ascites (mAb concentration, 0.8 mg per mL) or i.p. injections of 200 L 1:10-diluted control mAb (mAb concentration, 1.2 mg per mL) on days -1, 0, +1, and +4 of skin sensitization. Cell depletion was assessed at days +1 and +4 by staining CD8 molecules on peripheral blood mononuclear cell recovered from retro-orbital plexus. In all cases, specific depletion exceeded 95% on both days.

Skin DC isolation

Skin DC were recovered from mouse skin as previously described (^{Ortner et al. 1996}). Briefly, murine ears were rinsed in 70% ethanol, and divided in half with forceps. Dorsal cartilage-free sides were cultured in 24-well tissue culture plates with dermal side down. The tissues were transferred onto culture wells with fresh medium every day. On day 3, all cells present in the culture medium were pooled. Analysis of the cell suspension showed that >80% of cells which have migrated out of the skin into the culture medium had a DC morphology and expressed a high level of MHC class II molecules.

Immunization of naïve mice with ODN-treated, hapten-derivatized skin DC

Skin DC were incubated in CM containing CpG ODN (1 M), control ODN (1 M) or left untreated for 12 h at 37°C. After extensive washing with PBS, the cells were haptenized with DNBS (4 mM, pH 8.0) for 30 min in serum-free RPMI, and then washed with PBS containing 10% FCS. 1 10⁵ ODN-treated and DNBS-derivatized DC were injected s.c. in 200 L of saline into the back skin of naïve BALB/C recipient mice. Five days later, the mice were challenged by epicutaneous application of 0.2% DNFB on the ear. One group of naïve mice received untreated and unhaptenized-DC injections at day 0 but were painted with DNFB on day 5 and served as controls. Ear swelling was evaluated as described above.

Immunohistochemical analysis of mouse skin

Skin samples were collected from sensitized mice at 24 h after challenge. In order to test for the effect of CpG ODN treatment on the phenotype of skin DC, 20 g of CpG ODN and control ODN (total volume of 50 L) was injected intradermally in the right ear of naïve mice 24 h before epicutaneous application of a sensitizing dose of 0.5% of DNFB onto the same ear. Ears were recovered 24 h after hapten application and analyzed for expression of DC surface molecules.

Ears of mice were cut in two parts, and one-half was fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin (H&E). The other half was frozen in OCT compound (MILES Inc., Torrance, California). Frozen sections were fixed for 10 min in 4°C acetone, air-dried, treated with 5% FCS in Tris-buffered saline. Primary antibodies included: anti-CD80 mAb (RMMP-1, Rat IgG2a) (Caltag Lab, Burlingame, California), anti-CD86 mAb (PO3.1, Rat IgG2a), anti-MHC class II mAb (M5/114.15.2, Rat IgG2b) (Pharmingen, San Diego, California), and anti-CD8 mAb (KT-15, Rat IgG2a) (Harlan Sera-Lab, Loughborough, UK). After washing, slides were incubated with biotinylated rabbit anti-rat IgG (H+L) secondary Ab (Vector Burlingame, California). Slides were next incubated with biotinylated alkaline phosphatase–streptavidin (Strept ABComplex/AP, Dako, Lavoratories, Glostrup, Denmark), and the reaction was developed using BCIP/NBT (Bio-Rad, Hercules, California), followed by counterstaining with nuclear fast red. Irrelevant rat IgG mAbs (Caltag Lab) were used as primary antibody to confirm the specificity of immunostaining. In some experiments, sections were further incubated with anti-CD3 mAb (145-2C11, Hamster IgG1) (Pharmingen), and then HRP-conjugated anti-hamster IgG (H+L) Ab (Santa Cruz Biotechnology, Santa Cruz, California). Specific binding was revealed with 3-amino-9-ethylcarbazole (AEC) (Dako). Semi-quantitative analysis of stained cells was done by counting the number of positive cells in epidermis per 5 mm of dermo-epidermal (D-E) junction of two different sections. Results represent the mean number of positive cells per mm of D-E junction.

RNA extraction and RT-PCR analysis of CD8 and IFN- mRNA

At 24 h after challenge, ear samples were collected from CpG ODN- or control ODN-treated and sensitized mice, and frozen in liquid nitrogen. Total RNA extracted using a SV Total RNA Isolation System (Promega, Madison, Wisconsin) was reverse transcribed using poly dT15 primers and Superscript II RT (Invitrogen, Carlsbad, California) (90 min 37°C). The amount of RNA to be used for the detection was normalized using the housekeeping gene HPRT (hypoxanthine phosphoribosyltransferase) as reference as previously described (^{Akiba et al. 2002}). The cDNA obtained was amplified using different sets of primers, for HPRT (5' primer: 5' GTA ATG ATC AGT CAA CGG GGG AC 3'-3' primer: 5' CCA GCA AGC TTG CAA CCT TAA CCA 3') for CD8 (5' primer: 5' AGG ATG CTC TTG GCT CTT CC 3'-3' primer: 5' TCA CAG GCG AAG TCC AAT CC 3') for IFN- (5' primer: 5' GCT CTG AGA CAA TGA ACG CT 3'-3' primer: 5' AAA GAG ATA ATC TGG CTC TGC 3'). The amplifications were carried out with 29 cycles for HPRT and 33 cycles for IFN- and CD8 (1 min at 94°C, 1 min 30 s at 60°C, 2 min at 72°C). The PCR products were subjected to gel electrophoresis using ethidium-containing 1.5% agarose gel, and visualized by UV light. To evaluate the mRNA expression, we measured the signal intensities of HPRT, CD8, and IFN- by densitometry and compared their arbitrary densitometric units. The results were expressed as ratios of optical densities (OD) to HPRT band. We confirmed that the mRNA levels could be compared in the linear range under the conditions mentioned above.

Statistical analysis

All experimental groups consisted of five mice, and all experiments were performed at least three times. The statistical significance of differences between mean values of groups was evaluated with the one-way factorial ANOVA (p<0.05).

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Acknowledgments

We thank Naoko Suzuki, Yukiko Horikoshi for performing the histological study and Ms Jenny Messenger for the English corrections. This work was supported by a grant from the Fukushima Medical Foundation and the Région Rhône Alpes (HHC03F)-contract 00 81 60 45.