The role of interleukin-16 in murine contact hypersensitivity

Abstract

Contact hypersensitivity (CHS) is a T-cell-mediated skin inflammatory response. It is controversial whether CD4⁺ T cells play an enhancing or regulatory role in the pathogenesis of CHS. Because interleukin (IL)-16 is a chemoattractant cytokine for CD4-expressing cells, we investigated the involvement of IL-16 in the CHS reaction. IL-16 production was induced in the epidermis and dermis during the elicitation phase of the CHS response with trinitrochlorobenzene. In the sensitization phase, the single application of haptens such as trinitrochlorobenzene and oxazolone also induced IL-16, whereas primary irritants or vehicle control did not. IL-16 was produced mainly by CD11c-negative cells in the epidermis during the elicitation phase. Furthermore, treatment of sensitized mice with anti-IL-16 neutralizing MoAb enhanced the ear swelling and reduced the number of infiltrating CD4⁺ T cells. These data indicate that IL-16 plays a role in CHS, whereby IL-16 induces CD4⁺ T cells and these CD4⁺ T cells subsequently exhibit down-regulating properties.

Introduction

Contact hypersensitivity (CHS) is a T cell-mediated immune reaction that occurs after epicutaneous sensitization and challenge with reactive haptens [1,2]. Traditionally, it is believed that CHS is representative of delayed-type hypersensitivity (DTH), which is mediated by CD4⁺ T cells and down-regulated by CD8⁺ T cells [3]. However, recent experiments indicate that CD4⁺ T helper type 1 (Th1) and CD8⁺ type 1 cytotoxic T cells (Tc1) are crucial effector cells in CHS, unlike DTH [4]. Alternatively, another study suggested that CD8⁺ Tc1 are effector cells whereas regulatory CD4⁺ T cells have down-regulating properties in CHS [5]. The nature of the cell type involved in hapten presentation to specific T cells during the elicitation phase of CHS remains to be determined.

Interleukin (IL)-16 was described originally as a lymphocyte chemoattractant factor that is generated from mitogen- or antigen-stimulated human peripheral blood mononuclear cells [6]. Subsequent studies found that IL-16 is produced by lymphocytes, mast cells, dendritic cells, epithelial cells, fibroblasts and eosinophils [7–9] and induces chemotaxis via direct interaction with CD4 molecule as its natural ligand in CD4-expressing cells such as T cells, eosinophils, mast cells, dendritic cells and monocytes [8,10,11]. In addition to being a chemoattractant factor, several immunomodulatory functions of IL-16 have been identified. IL-16 stimulates cytokine production in monocytes and up-regulates their expression of co-stimulatory molecules and MHC class II molecules [12,13]. In contrast, it reduces proliferative responses and IL-2 production in CD4⁺ T cells [14,15]. Although IL-16 was reported to play a role in DTH by chemoattracting CD4⁺ T cells [16], no information is available on the expression and role of IL-16 in CHS.

In the present study, we show that IL-16 is produced by epidermal cells, especially keratinocytes, and dermal cells during the sensitization and elicitation phases of hapten-induced CHS. We also show that treatment of sensitized mice with neutralizing monoclonal antibody against IL-16 before hapten challenge significantly suppresses the CHS response, suggesting that IL-16 plays a regulatory role in the elicitation phase.

Materials and methods

Animals

Male and female BALB/c mice, female C57BL6 mice and C3H/HeN mice aged 6–8 weeks were purchased from Shimizu Laboratory Supplies Co.Ltd (Kyoto, Japan). Each experimental group consisted of at least four mice.

Chemicals

The following chemicals were obtained commercially; trinitrochlorobenzene (TNCB) (Tokyo Kasei, Tokyo, Japan), oxazolone (Aldrich Chem., Milwaukee, WI, USA), sodium lauryl sulphate (Sigma, St Louis, MO, USA), benzalkonium (Takasugi Pharma., Fukuoka, Japan) and croton oil (Wako, Osaka, Japan).

Sensitization and challenge with haptens

The mice were sensitized with 50 µl of 3% TNCB in a 4 : 1 acetone–olive oil mixture on shaved abdomens. Five to 7 days later, 20 µl of 1% TNCB solution was applied to induce CHS in both ears or 100 µl on the trunk as described previously [17].

Detection of IL-16 by enzyme-linked immunosorbent assay (ELISA)

The detection of IL-16 was carried out as described previously [16]. The mice were killed and the ears were cut off at various times after sensitization or challenge with TNCB. The epidermis was then separated from the dermis by incubation in 0·5 m ammonium thiocyanate at 37°C for 20 min. Each piece of tissue minced in phosphate-buffered saline (PBS) (two ear lobes per ml) and incubated at 4°C for 1 h to release soluble materials. Afterward, the supernatant was collected and frozen at − 80°C until use. Sandwich ELISA for mouse IL-16 was carried out using anti-IL-16 (17·1, Endogen, Woburn, MA, USA) as the capture MoAb and biotinylated anti-IL-16 (14·1, Endogen) as the detection MoAb, and recombinant IL-16 (Endogen) as a standard.

Reverse transcription-polymerase chain reaction (RT-PCR) for IL-16

Using the same mice as in the ELISA experiments, truncal skin was cut at various times after the application of the hapten and incubated with 1% trypsin to obtain epidermal cells [18,19]. Total RNA was then isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed to cDNA using a first-strand cDNA Synthesis Kit™ (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. PCR was performed using Taq polymerase (Takara, Ohtsu, Japan) and the primers specific for IL-16: sense 5′-AACCGAGGACAGGAACCACT-3′, antisense, 5′-CTTGAGAGATTTGCCATTGA-3′, I-Ad; sense 5′-TAGGCATTCATTCCCACCC-3′, antisense 5′-CCAGTCGC CATTCCTAATAAGC-3′ (kind gift of Dr Y. Koyama, Nippi Research Institute of Biomatrix) and G3PDH (R&D Systems, Minneapolis, MN, USA). The PCR products were then analysed on 2% agarose gels. The signal strength of IL-16 was normalized to the G3PDH signal. The specificity of the amplified bands was validated by their predicted size [316 base pairs (bp), 262 bp and 983 bp for IL-16, I-Ad and G3PDH, respectively].

Cell purification of epidermal cell suspension

Twenty-four hours after the TNCB challenge, ear and truncal skin was removed and incubated with 1% trypsin to obtain an epidermal cell suspension. The cells were incubated with antimouse CD11c-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and selected on MACS™ separation columns (Miltenyi Biotec). The positively selected cells were regarded as Langerhans cells (LC) and the negatively cells were regarded as keratinocytes. These separated cells were used for isolating total RNA or the cells (1 × 10⁶ per ml) were cultured for 24 h at 37°C and the amount of IL-16 in each supernatant was measured by ELISA.

Neutralization of IL-16 in vivo with monoclonal antibodies

To neutralize endogenous IL-16, TNCB-sensitized mice were injected intraperitoneally with 500 µg of anti-IL-16 (14·1) or an equal volume of mIgG at 15 h and immediately before the elicitation, challenged with 1% TNCB on the ears.

Immunohistochemical examination

An ear lobe from each mouse treated with anti-IL-16 or with mIgG was removed 24 h after challenge and frozen with Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA) in liquid nitrogen. After fixation in acetone for 20 min, the tissue sections were incubated for 1 h at 37°C in PBS containing 5% normal rabbit serum and 5% skimmed milk to block non-specific IgG binding. They were then incubated with rat anti-CD4 (GK1·5, rat IgG2b, eBioscience, San Diego, CA, USA), anti-CD8 (53–6·7, rat IgG2a, eBioscience) or isotype control overnight at 4°C. Detection was performed with a streptavidin–horseradish peroxidase conjugate and diaminobenzidine substrate.

Statistical analysis

The data were expressed as means ± standard deviation (s.d.) unless indicated otherwise and analysed using the two-tailed Student's t-test on statview version 5 (SAS Institute Inc., NC, USA). A P-value of less than 0·05 was regarded as statistically significant.

Results

Release of IL-16 from the epidermis and dermis during the elicitation phase of the CHS response

During the elicitation phase of the classical DTH response, IL-16 can be detected in epidermal and infiltrating cells in the dermis of the footpads of sensitized mice [16]. We analysed kinetically IL-16 production of the ear skin during the elicitation phase of CHS by ELISA (Fig. 1a). No detectable levels of IL-16 were found under steady-state conditions before hapten challenge. Similar to DTH, IL-16 was detected 12 h after challenge in the epidermis and dermis, and IL-16 levels increased in a time-dependent manner for up to 72 h. No IL-16 was detected in the epidermis and dermis from the ears of sensitized mice challenged with vehicle alone (data not shown). Throughout the analysis period, higher values of IL-16 were detected in the dermal supernatant than in the epidermal supernatant.

Fig. 1.

Kinetic analysis of IL-16 production after TNCB application during the elicitation (a) and sensitization phase (b) of the CHS response. IL-16 production in mice of various ages or strains during the elicitation (c) and sensitization phase (d). IL-16 was analysed 24 h after the application of the hapten or the challenge. The data are shown as means ± s.d. for 16 mice. *P < 0·05 compared with the epidermis.

Single application of haptens onto non-sensitized mice skin causes the release of IL-16 from the epidermis and dermis

There has been no study concerning IL-16 production in the skin after single hapten application in non-sensitized mice. To examine this, we applied 1–3% TNCB onto the ears of non-sensitized mice and analysed IL-16 production by ELISA. We found that IL-16 was present in the epidermis 12 h after application (Fig. 1b). IL-16 was also detected in the dermis 12 h after application and IL-16 levels increased for up to 72 h. More IL-16 was found to originate from the dermis than from the epidermis. On the other hand, no IL-16 was detected in the epidermis and dermis from non-sensitized mice treated with vehicle (data not shown). Less IL-16 was observed during the induction phase than during the elicitation phase with significance. Next, we compared IL-16 production during the sensitization and elicitation phases between males and females, different ages of BALB/c mice, and also other strains, C57BL/6 and C3H/HeN (Fig. 1c,d). The production was almost the same between these mice and there was no significant difference between them.

IL-16 production was also observed after the single application of another hapten, oxazolone, in the epidermis and dermis, but primary irritants such as sodium lauryl sulphate, benzalkonium and croton oil did not induce IL-16 production (Fig. 2a). IL-16 was detected in TNCB-sensitized mice 24 h after the application of oxazolone, but its quantity was significantly smaller than that from TNCB-sensitized mice 24 h after the application of TNCB (Fig. 2b). These results suggest that production of IL-16 during the elicitation phase of the CHS response was affected not only by hapten application, but also antigen-specific reaction.

Fig. 2.

IL-16 production after the application of haptens or primary irritants to the epidermis and dermis. (a) Twenty-four hours after the application of haptens or primary irritants on the ear lobes of non-sensitized mice or TNCB sensitized mice. (b) Sensitization with 3% TNCB, oxazolone (Oxaz) and vehicle (Veh) and elicitation with 1% TNCB, oxazolone and vehicle were carried out. IL-16 values for the ear skin were determined 24 h after elicitation. The data are shown as means ± s.d. for 16 mice.

IL-16 mRNA expression in epidermal cells during the induction and elicitation phases

Next, we performed semiquantitative RT-PCR to examine the expression of IL-16 mRNA in epidermal cells. (Fig. 3a,b). The epidermal cells of untreated mice slightly expressed IL-16 mRNA. During the induction phase, IL-16 mRNA was increased until 12 h after the challenge, but decreased at 24 h after the challenge. In the elicitation phase mRNA was decreased once at 12 h, but increased again at 24 h after the application of the hapten (Fig. 3b). The IL-16 mRNA expression in epidermal cells of mice treated with vehicle alone was as low as that of untreated mice (data not shown).

Fig. 3.

RT-PCR analysis of IL-16 mRNA after TNCB application in epidermal cells. Total RNA was extracted from epidermal cells and RT-PCR was conducted (a). The relative amounts of the PCR products were determined by densitometry and the densitometric value of IL-16 was normalized to that of G3PDH (b). The histogram represents untreated mice (▴), the induction phase (▪), and elicitation phase (). Similar results were obtained from four experiments.

IL-16 is produced mainly by keratinocytes in the epidermis during CHS

In atopic dermatitis (AD), another prototypic allergic inflammatory skin disease, most epidermal cells are IL-16 mRNA-positive, suggesting that keratinocytes are the major source of IL-16 [20]. However, Reich et al. reported that LC produced mainly IL-16 in the epidermis of patients with atopic dermatitis [21]. Therefore, to examine whether keratinocytes or LC are the main producers of IL-16 during a CHS response, we separated epidermal cells into keratinocytes and LC using CD11c-coated magnetic beads and MACS™ separation columns, then performed PT-PCR and ELISA. IL-16 was observed in both CD11c positive (less than 5% of epidermal cells) and negative cells (over 95%), and IL-16 levels increased in a time-dependent manner (Fig. 4a). Moreover, we found that higher levels of IL-16 were detected in CD11c-negative cells than in the positive cells (Fig. 4a,b). These results suggest that IL-16 was produced mainly by keratinocytes in the epidermis during the elicitation phase of the CHS response.

Fig. 4.

Analysis of IL-16 in epidermal cells 24 h after the TNCB challenge. Epidermal cells were separated into CD11c⁺ LC and CD11c^– keratinocytes. IL-16 production in each culture supernatant was measured by ELISA (a). Total RNA was extracted from the epidermal cell suspension (lane 1) or CD11c^– (lane 2) or CD11c⁺ (lane 3) cells and analysed by RT-PCR (b). Similar results were obtained from four experiments.

Enhancement of the CHS response by anti-IL-16 treatment

During the elicitation phase of the classical DTH response, treatment of BSA-sensitized mice in vivo with anti-IL-16 neutralizing MoAb suppresses the DTH response, together with decreased infiltration of CD4⁺ and CD8⁺ T cells [16]. However, no information is available about the role of IL-16 in the recruitment of CD4⁺ and CD8⁺ T cells in CHS. To examine whether the IL-16 activity is involved in the CHS response, we treated TNCB-sensitized mice with neutralizing MoAb against IL-16, followed by challenge with 1% TNCB on the ears. The ear swelling observed for untreated and isotype-matched control-treated mice was enhanced by the treatment with anti-IL-16 MoAb (Fig. 5a).

Fig. 5.

Enhancement of the CHS response by anti-IL-16 treatment. (a) Time-course of the enhancing effect of anti-IL-16 on CHS. (b) CD4⁺ and CD8⁺ T cell infiltration of mice treated with anti-IL-16 or with mIgG 24 h after challenge. Positively stained cells were counted in randomly selected microscopic fields analysed at a magnification of ×200. The data are shown as means ± s.d. for four mice. *P < 0·05 compared with control. Similar results were obtained from four experiments.

Immunohistochemical analysis revealed that treatment with anti-IL-16 reduced the number of infiltrating CD4⁺ T cells but increased the number of CD8⁺ T cells (Fig. 5b). These results suggest that CD4⁺ T cells are recruited by IL-16, which down-regulates the CHS response.

Discussion

In this study, we found that a single application of hapten on non-sensitized murine skin and hapten-sensitized skin induces IL-16 production from the epidermis and the dermis. In addition, we discovered that IL-16 is produced mainly by keratinocytes and to a lesser extent by LC in epidermal cells. Furthermore, we found that the application of haptens and not of primary irritants or of control vehicle induces IL-16 production in the epidermis. In AD, Laberge et al. showed that keratinocytes are the main cells which express IL-16 mRNA among epidermal cells [20]. Epithelial cells in the bronchus and nose of patients with asthma and allergic rhinitis, respectively, also produce IL-16 [22,23]. During a DTH response, a considerable number of epidermal cells, most probably keratinocytes, express IL-16-immunoreactivity [16]. Keratinocytes are being recognized increasingly for their ability to modulate immune reactions in the skin partly through their capacity to synthesize and release mediators, including cytokines, growth factors and chemokines. IL-16 should thus be added to the list of immunomodulators released from keratinocytes.

Reich et al. reported that the main source of IL-16 in epidermal cells is LC in an atopy patch test site and lesional skin in AD [21]. This is in line with the observation that dendritic cells are a major source of IL-16 among immunocompetent cells [7]. Single hapten application induces keratinocyte production of cytokines such as tumour necrosis factor (TNF)-α and  IL-1β [24].  In  addition,  only  the  application of haptens and not primary irritants activates LC and induces IL-1β production [24,25]. Therefore, single hapten application may stimulate keratinocytes and LC directly to produce IL-16 in a similar fashion. Alternatively, IL-16 production by epidermal cells may be induced by TNF-α produced by these cells after hapten application in an autocrine and paracrine manner, because TNF-α induces IL-16 production by epithelial cells [10]. IL-16 produced by epidermal cells and resident cells in skin-associated lymphoid tissue chemoattracts CD4⁺ T cells and monocytes, which thus produce more IL-16. In our study, the level of IL-16 mRNA expression was increased at 6 h and 24 h after elicitation. First the expression increased at 6 h, induced possibly by the application of hapten, which directly stimulates epidermal cells; it then increased at 24 h, possibly induced by a cytokine such as TNF-α produced by epidermal cells and the cells infiltrating the dermis after the hapten was applied. We therefore suggest that skin may regulate immune reactions in preparation for subsequent antigen challenge after single hapten application by accumulating CD4⁺ inflammatory cells at the site through IL-16 release.

The role of CD4⁺ and CD8⁺ T cells during the elicitation phase of CHS is controversial. For years, CHS was considered to be mediated by CD4⁺ T cells, as in classical DTH [3]. However, recent studies indicate that interferon (IFN)-γ-producing CD8⁺ type 1 cytotoxic T cells and perhaps CD4⁺ Th1 cells as well are effector cells which contribute to the development of CHS, while CD4⁺ Th2 cells induce down-regulating effects during CHS by producing immunoregulatory cytokines such as IL-10 and IL-4 [4,26,27]. During the elicitation phase of CHS, CD8⁺ T cells are found in the skin as early as 6 h after challenge [26], whereas IL-16 was not detected in hapten-treated skin tissue in our study. At the same time, CD4⁺ T cells appear first in the challenged site 24 h afterward. The accumulation of CD4⁺ T cells is associated with the peak of the CHS response and the beginning of resolution of skin inflammation [26]. It is possible that IL-16 may contribute to the resolution of a CHS response by chemoattracting CD4⁺ T cells.

In this study, blocking IL-16 by neutralizing MoAb treatment did not block completely the infiltration of CD4⁺ cells. This was also the case in the experiment concerning the effect of neutralizing antibody against IL-16 on dermal cell infiltration during DTH. These results suggest that other chemotactic factors such as IL-8 and RANTES may act during the recruitment of T cells in the absence of IL-16. In addition to decreasing numbers of CD4⁺ T cells at the reaction site, we observed increasing numbers of CD8⁺ T cells in anti-IL-16 MoAb-treated mice. Although the precise mechanism by which the blocking of IL-16 results in increased CD8⁺ T cell accumulation in the dermis is not known, it is possible that a decreased number of CD4⁺ T cells is associated with decreased concentration of CD4 T cell-derived cytokines which inhibit T cell chemotaxis. One of these candidates is IL-4, because it inhibits only CD8⁺ T cell chemotaxis toward RANTES, IL-8 and IL-10 [28]. It was suggested recently that CHS is mediated by IFN-γ-producing CD8⁺ T cells and Th2 cells, while DTH is mediated by Th1 cells [27]. The difference in cytokine profiles of accumulating cells may explain the discrepancy of CD8⁺ T cells in the IL-16-blocking experiment between the CHS and DTH responses; the latter showed a decreased number of CD8⁺ cells in IL-16-blocked mice.

In addition to being a chemoattractant factor, several immunomodulatory functions of IL-16 have been identified. IL-16 stimulates cytokine production in monocytes and up-regulates their expression of co-stimulatory molecules and MHC class II molecules [12,13,29]. In contrast, preincubation of CD4⁺ T cells with IL-16 reduces their proliferative response and IL-2 production through stimulation with CD3/TCR and concanavalin A (Con A), respectively [14,15]. IL-16-transfected keratinocytes inhibit the activation of allogeneic lymphocytes stimulated with transfectant cells [30]. IL-16 also enhances the T cell expression of the receptor for IL-2, whose main function is recognized as promoting the development of T regulatory cells [31]. These observations implicate the immunosuppressive effect of IL-16 in T cell-mediated hypersensitivity.

Collectively, we obtained evidence that keratinocytes are important producers of IL-16 during the sensitization and elicitation phases of CHS. IL-16 may have a role in resolving the CHS response by recruiting CD4⁺ T cells.