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. Author manuscript; available in PMC: 2013 Feb 15.
Published in final edited form as: J Immunol. 2009 Jan 1;182(1):675–683. doi: 10.4049/jimmunol.182.1.675

Heat shock proteins HSP27 and HSP70 are present in the skin and are important mediators of allergic contact hypersensitivity

Nabiha Yusuf 1,3, Tahseen H Nasti 1, Chun-Ming Huang 1, Brad S Huber 1, Tarannum Jaleel 1, Hui-Yi Lin 1,2, Hui Xu 1,3, Craig A Elmets 1,3
PMCID: PMC3573708  NIHMSID: NIHMS79462  PMID: 19109201

Abstract

Proteomic analysis of murine skin has shown that a variety of heat shock proteins (HSPs) are constitutively expressed in the skin. Using murine allergic contact hypersensitivity as a model, we investigated the role of two heat shock proteins – HSP27 and HSP70 – in the induction of cutaneous cell-mediated immune responses. Immunohistochemical examination of skin specimens showed that HSP27 was present in the epidermis and HSP70 was present in both the epidermis and dermis. Inhibition of HSP27 and HSP70 produced a reduction in the DNFB contact hypersensitivity response and resulted in the induction of antigen specific unresponsiveness. Treatment of dendritic cell cultures with recombinant HSP27 caused in the upregulation of IL-1β, TNF-α, IL-6, IL-12p70 and IL-12p40 but not IL-23p19, which was inhibited when antibodies to HSP27 were added. DNFB conjugated dendritic cells that had been treated with HSP27 had an increased capacity to initiate contact hypersensitivity responses compared to control dendritic cells. This augmented capacity required TLR4 signaling because neither cytokine production by dendritic cells nor the increased induction of contact hypersensitivity responses occurred in TLR4 deficient C3H/HeJ mice. Our findings indicate that a cascade of events occurs following initial interaction of hapten with the skin that includes increased activity of heat shock proteins, their interaction with TLR4 and, in turn, increased production of cytokines that are known to enhance antigen presentation by T-cells. The results suggest that heat shock proteins form a link between adaptive and innate immunity during the early stages of contact hypersensitivity.

Keywords: HSP27; HSP70; Contact Hypersensitivity; 1-Fluoro-2,4-dinitrobenzene (DNFB); Toll-like receptor (TLR)

INTRODUCTION

Heat shock proteins (HSPs) are a highly conserved family of intracellular proteins that are constitutively expressed in virtually all nucleated cells. Their synthesis is enhanced by exposure to a variety of stressful conditions, including heat, toxins, oxidative stress and glucose deprivation (1). HSPs have been categorized into various families (Hsp110, Hsp90, Hsp70, Hsp60, Hsp27 and other small heat shock proteins) depending on their molecular weights and functional attributes (2, 3).

Increasing evidence suggests that HSPs may play important roles in both innate and adaptive immunity by acting as chaperones for immunologically important molecules such as MHC I, immunoglobulins, T-cell receptors and Toll like receptors. HSPs can also deliver chaperoned haptens and antigens from non-antigen presenting cells to MHC molecules of antigen presenting cells. As a result, HSPs have been found to be remarkably potent in their ability to elicit T-cell mediated immune responses to tumors, viruses and minor histocompatibility antigens (4). HSPs that make their way to the extracellular milieu also serve as cytokines to activate innate functions of antigen presenting cells. Depending upon the mode of tissue damage, the release of HSPs may also play an immunoregulatory role in vivo (5.

As in any other organ system, several HSPs are constitutively expressed in the skin and can be upregulated as a result of exposure to stresses such as heat, cold shock, prostaglandins, arsenite, and oxidative stress (2, 3). Using proteomic mapping, we have shown that six molecular chaperones - HSP27, HSP60, HSP70, HSP84, ER60, and GRP78 - are constitutively expressed in the skin of C3H/HeN and BALB/c mice. Of the multiple protein molecular chaperones identified in the skin proteome, only HSP27 was found at predominantly higher levels in the epidermis (6). The 27-kD HSP (HSP27, small heat shock protein was originally named as HSP25 in mice; in this manuscript the molecule will be termed HSP27) form large oligomers that can act as molecular chaperones and can protect cells from heat shock and oxidative stress in vitro. Also, expression of HSP27 is reduced or absent in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and other malignancies arising from keratinocytes, suggesting that the lack of HSP27 might be a marker of epidermal malignancy (7, 8). Another relevant HSP family is the 70-kD family. The 70-kD family is among the most abundant and most thoroughly investigated. HSP70 family members are expressed constitutively within keratinocytes and are elevated in both epidermis and dermis after skin samples are heat shocked in vivo and in vitro (9, 10). Stressors such as ultraviolet light and 815 nm diode laser treatment of skin may also elevate their levels in keratinocytes (11, 12).

Despite the fact that the skin is proficient at initiating and eliciting immune responses and that a variety of HSPs have important immunological activities, the role of HSPs in the immunopathology of the skin has not been investigated. Using murine allergic contact hypersensitivity as a model, the studies presented here were designed to examine the role of HSP27 and HSP70, two HSPs that are expressed in the skin, in the induction of cutaneous cell-mediated immune responses. We found that inhibition of HSP27 and HSP70 retarded the development of contact hypersensitivity to DNFB and led to the induction of tolerance to that hapten. We also observed that HSP27 treatment of dendritic cells led to an increase in the secretion of several cytokines that are known to be involved in antigen presentation to T-cells and an augmented capacity to initiate contact hypersensitivity responses. Finally, we found that the effect of HSPs on dendritic cells did not occur in TLR4 deficient mice, indicating that components of the innate immune system were required for HSP27 effects.

MATERIALS AND METHODS

Animals & Reagents

Adult female, 6–8wk old C3H/HeN mice were obtained from Charles River Laboratories (Wilmington, MA) and adult female, 6–8wk old C3H/HeJ mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Animals were maintained in accordance with institutional guidelines.

Normal goat IgG and goat polyclonal anti-HSP27 and anti-HSP70 IgG were purchased from Santa Cruz Biotechnology CA, USA. Alexa Fluor™ 488 conjugated donkey anti-goat IgG, Alexa Fluor™ 594 conjugated goat anti-rabbit IgG and Texas Red X-phalloidin and sheep anti-rat IgG dynabeads were from Invitrogen (Carlsbad, CA). Hybridoma lines GK1.5 (anti-CD4), Lyt-2 (anti-CD8) and HB-32 (anti-Iak) were acquired from ATCC (Manassas, VA). CD3e, CD45R/B220 and recombinant GM-CSF were obtained from Pharmingen (San Diego, CA). Recombinant HSP25 (The 27-kD HSP was originally named as HSP25 in mice; in this manuscript the molecule will be termed HSP27) was purchased from Calbiochem (San Diego, CA). DNFB, DNBS, LPS, polymyxin B and recombinant IL-4 and were purchased from Sigma Chemical Co. (St. Louis, MO).

Immunofluorescent staining

Staining for HSP27 and HSP70 was done as described in our previous studies (6). Briefly, fixed skin sections were incubated with anti-HSP27/anti-HSP70 primary antibody and were then treated with Alexa Fluor™ 488 conjugated donkey anti-goat IgG; tissues incubated with Texas Red X-phalloidin were subsequently treated with Alexa Fluor™ 594 goat anti-rabbit IgG. Following incubation with secondary antibody, all tissues were counterstained with 20 µg/ml Hoechst 33258 and mounted in polyvinyl alcohol mounting medium. Fluorescent emissions were detected with a Zeiss Axiovert 100 microscope with epifluorescent optics and analyzed with LSM 3.8 software (Carl Zeiss, Oberkochen, Germany). The dilution factor for anti-HSP27 and anti-HSP70 antibodies was 1:100.

Antibody application on skin

In order to assess the contribution of HSP27 and 70 in contact hypersensitivity, the abdominal skin of mice was prepared by removing hair with an electric trimmer in conjunction with gentle brushing of the skin with a soft-bristle toothbrush for 30 strokes. This was followed by epicutaneous application of 2µg of control antibody, anti-HSP27, anti-HSP70 antibody or a combination of anti-HSP27 and anti-HSP70 antibody in PBS for 2 h under occlusion with a bioocclusive dressing (Tegaderm®, 3M, Maplewood, MN). The site was examined for erythema and edema using a modified Draize scoring system of 0 to 3 (13). All animals employed for experiments had a Draize score of <1. Studies were also performed which showed that application of antibody to the shaved abdominal skin allowed antibody to penetrate into the epidermis and dermis (Fig. 2).

Figure 2.

Figure 2

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Antibody penetration into the skin. Goat IgG was applied on the shaved back and animals were sacrificed at the indicated times. Frozen sections were cut as 7um thick sections and fixed in methanol for 10 minutes and then rehydrated with PBS. After blocking with 3% H2O2 in rat serum, the sections were incubated with biotin labeled anti-goat IgG for 1h. The sections were incubated with streptavidin-HRP and then after washing with PBS were incubated with DAB for 10 min and then counter stained with hematoxylin and visualized under a light microscope. Goat IgG was applied on the skin and sections cut after various time intervals. Peroxidase staining with DAB was done as indicated in materials and methods. (i) control skin (ii) antibody applied skin after 15 min (iii) antibody applied skin after 2h (iv) antibody applied skin after 4h. There were two mice per group and each experiment was repeated twice with the same results.

Contact hypersensitivity to DNFB

Contact hypersensitivity to DNFB was performed as described previously (14). Briefly, the shaved abdominal skin of C3H/HeN mice was sensitized on day 0 with 25 µl of a 0.5% solution of DNFB in a 4:1 mixture of olive oil:acetone. Five days later, a challenge dose of 20 µl of 0.2% DNFB was painted on the ear after measuring baseline ear thickness. The increase in ear swelling was measured at 24h intervals to quantitate the magnitude of the contact hypersensitivity response as described previously (14, 15).

Generation of bone marrow-derived DC (BMDC)

Bone marrow-derived DCs were prepared from C3H/HeN and C3H/HeJ mice as described with some modifications (16). Briefly, bone marrow cells from femurs and tibias were incubated in RPMI 1640 medium with a cocktail of antibodies against Iak, CD45R/B220, Lyt-2 and GK1.5 (2 µg/106 cells) on ice for 1 h and washed once with HBSS after lysis of RBCs. Different cellular populations were removed from the cell suspension by antibody mediated depletion using sheep anti-rat IgG dynabeads according to the manufacturer’s instructions. Cells were washed once with HBSS and cultured in 10% FCS RPMI 1640 media supplemented with recombinant mouse GM-CSF (10 ng/ml) and IL-4 (10 ng/ml) in 6-well plates (5×105 cells/well). On day 5, half of the medium was replaced with fresh medium and cells were stimulated on the following day for the experiments.

Stimulation of DNFB primed lymph node cells with hapten-conjugated BMDC for cytokine production

In order to assess antigen specific cytokine production, bone marrow derived dendritic cells (BMDC) were used for in vitro stimulation of primed lymph node cells as described (14). Briefly, mice were pre-treated with anti-HSP27/70 or species matched control antibody as described earlier in this section and were then sensitized with DNFB in a 4:1 mixture of olive oil:acetone on day 0. On day +5 mice were sacrificed and lymph node cell suspensions were prepared by gentle pressure through a wire mesh screen. Ten million BMDC were suspended in 1 ml DNBS solution (5mM in PBS) for 15 min. The cells were then washed three times with RPMI containing 5% FCS and resuspended in the culture medium. DNFB primed lymph node cells (2 × 106/ml) were stimulated with DNBS labeled BMDC (2 × 105/ml). Cytokine concentrations in culture supernatants were measured 48 hours after cultures by cytokine-specific ELISA.

Assessment of Tolerance

Tolerance to DNFB was assessed by a modification from the protocol of Schwarz et al (17). On day -14, the abdominal skin of mice was treated as described above for antibody application on the skin. Two µg of control antibody, anti-HSP27, anti-HSP70 or a combination of anti-HSP27 and anti-HSP70 antibodies in PBS was applied to the skin for 2 h under occlusion with a bioocclusive dressing. Immediately thereafter, 25µl of 0.5% DNFB was applied topically to the antibody treated site. On day 0, mice were resensitized on the dorsal skin, a site that had not been treated with antibody, with 25µl of 0.5% DNFB. Five days later mice were challenged with 20µl of 0.2% DNFB on the ear. The increase in ear swelling was measured at 24h intervals as described above.

In order to evaluate antigenic specificity of tolerance, mice were treated in the same manner as above. One group of anti-HSP27 and anti-HSP70 antibody treated mice was sensitized with 25µl of 0.5% DNFB on day -14 but on day 0 they were sensitized with 50µl of 3% oxazolone on their shaved dorsal side and 5µl on each foot pad. On day +5, hapten sensitized and control mice were challenged with 20µl of 0.2% DNFB or 1% oxazolone on the ear. The increase in ear swelling was measured at 24h intervals as described above.

Adoptive transfer of regulatory cells

Mice were treated epicutaneously with 2 µg of control or anti-HSP27 and anti-HSP70 antibody on their shaved abdomen for 2h as described in the Methods section above on antibody application on the skin. Subsequently, 25µl of 0.5% DNFB was applied topically to the abdomen on the antibody treated site. On day +5 donor mice from the two groups were sacrificed and single cell lymphocyte suspensions were prepared from draining lymph nodes. Lymph node cells, 50×106 in 200µl PBS, were injected intravenously in the tail vein of naïve recipient mice. The recipients were contact sensitized immediately after adoptive transfer of the lymph node cells with 25µl of 0.5% DNFB on the shaved abdominal skin. On day +5 they were challenged with 20µl of 0.2% DNFB on the ear. The increase in ear swelling was measured at 24h intervals as described above.

In vitro stimulation of bone marrow derived dendritic cells with HSP27

Bone marrow derived dendritic cells (BMDC) were stimulated with 1µg/ml recombinant HSP27. LPS was used as a positive control for inducing DC maturation. Polymyxin B (10µg/ml) was added to the samples prior to stimulation to prevent LPS contamination. After 24h of culture at 37°C in the presence of 5% CO2, culture supernatants were collected and stored at −20°C until cytokine measurements were performed.

In vitro measurement of cytokine production

Cytokine concentrations in culture supernatants from stimulated BMDCs were measured using cytokine-specific enzyme linked immunosorbent assay (ELISA) kits from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions.

Assessment of CHS response after injection of hapten labeled BMDC

Bone marrow dendritic cells (naïve and HSP27 stimulated) from C3H/HeN and C3H/HeJ mice were labeled with DNBS according to previously established protocols with some modifications (14). Ten million cells were suspended in 1 ml DNBS solution (5mM in PBS) for 15 min. The cells were then washed three times with RPMI containing 5% FCS and resuspended in the culture medium. One million cells were injected subcutaneously on the right and left ventral side of each group of mice. Later, the mice were ear challenged with 20µl of 0.2% DNFB. The positive control groups were sensitized with 25µl of 0.5% DNFB and on day +5 they were ear challenged with 20µl of 0.2% DNFB. The increase in ear swelling was measured at 24h intervals as described above.

Statistical analysis

Data were analyzed by one-tailed Student’s t test, and the p values are indicated in the text and figure legends. The group difference was compared using ANOVA test followed by Tukey’s post hoc test for multiple comparison adjustment in one experiment. Differences were considered significant at p<0.05.

RESULTS

Localization of HSP27 and HSP70 in the murine skin

In previous studies, proteomic analysis of the skin of C3H/HeN and BALB/c mice revealed that there was constitutive expression of the heat shock proteins HSP27 and HSP70 (6). To identify where in the skin these molecules were expressed, sections of skin were subjected to immunohistochemical analysis. HSP27 was only detected in the superficial layers of epidermis whereas HSP70 was present throughout the entire epidermis and also to some extent in the dermis (Fig. 1).

Figure 1.

Figure 1

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HSP27 is expressed in the epidermis while HSP70 is expressed in both epidermis and dermis in C3H/HeN mice (A). HSP27 and HSP70 expression in naïve skin samples were collected from naïve mice and stained with HSP27 or HSP70 antibody. Left panel is naive skin incubated with control antibodies; middle panel is naive skin treated with HSP27 antibodies; the right panel is naive skin treated with HSP70 antibodies.

It has been demonstrated that under certain conditions, topically applied proteins and DNA can reach deeper levels of the skin (18–21). In order to determine whether the procedures we had employed allowed antibody to penetrate, antibody was applied to the skin under a biooclusive dressing as described in the Materials and Methods. By immunohistochemical analysis, the antibody could only be detected in the most superficial portion of the epidermis after 15 minutes. But with greater lengths of time up to 4 hours, the antibody had penetrated to the dermis and subcutaneous tissue (Fig. 2).

Heat shock proteins HSP27 and HSP70 play a role the in contact hypersensitivity response to DNFB

Having determined that antibodies could penetrate into the epidermis and dermis, experiments were performed to assess the functional relevance of HSP27 and HSP70 in cutaneous cell-mediated immune responses. To do this, C3H/HeN mice were contact sensitized to DNFB after anti-HSP27 and anti-HSP70 antibodies had been applied topically to abdominal skin that had been prepared in the manner described above. Inhibition of the induction of DNFB contact hypersensitivity could be achieved by pretreatment with both anti-HSP27 and anti-HSP70 antibodies (Fig. 3). When both anti-HSP27 and anti-HSP70 antibodies were applied simultaneously there was an additive effect leading to greater inhibition of contact hypersensitivity than with either alone (Fig. 3). When goat IgG was used as a species matched control, there was no inhibition, indicating that the effect of topical antibody treatment was specific for the anti-HSP antibodies employed (Fig. 3). Inhibition of the induction of DNFB contact hypersensitivity with anti-HSP27 was dose dependent with maximum inhibition occurring at 2 µg/mouse. Similar results were obtained with anti-HSP70 (data not shown).

Figure 3.

Figure 3

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Contact hypersensitivity to DNFB is inhibited by pretreatment of skin with antibodies to heat shock proteins 27 (HSP27) and 70 (HSP70). (A) C3H/HeN mice were treated with anti-HSP antibodies (2µg/mouse in 100 µl of PBS). After 2h, the mice were sensitized with DNFB on the antibody treated site. The mice were ear challenged 5 days later and auricular thickness was measured at 24 hours. The application of anti-HSP antibodies significantly inhibited the CHS response (*p<0.05). Results were expressed as change in auricular thickness ± SEM. There were four mice per group and each experiment was repeated twice with the same results. The results are from one representative experiment. The group difference was compared using ANOVA test followed by Tukey’s post hoc test for multiple comparison adjustment. B) Histologic sections were taken from representative mice at 24 hours after DNFB challenge, stained with hematoxylin and eosin, and photomicrographs were taken (original magnification ×10 for all sections).

Studies on the contact hypersensitivity response to DNFB have demonstrated that this response is mediated by IFN-γ and IL-17 in mice (14, 22). Experiments were therefore also performed in which hapten primed lymph node cells were isolated from the draining lymph nodes of DNFB sensitized mice and placed in culture with DNBS labeled BMDC. This served as an alternative method of assessing the immune response to topically applied hapten. Hapten labeled BMDC stimulated the production of IL-17 and IFN-γ by primed lymph node cells whereas they were not able to stimulate naïve lymph node cells (Fig. 4). Pretreatment of mice with anti-HSP27 and anti-HSP70 antibody, significantly inhibited the production of IL-17 and IFN-γ compared to untreated BMDC (Fig 4, p<0.05). We also observed a corresponding increase in IL-4 and IL-10 production under the same conditions. It is possible that treatment of anti-HSP27 and anti-HSP70 antibody shifts the response from a Th1/Th17 to a Th2 phenotype at least in vitro.

Figure 4.

Figure 4

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Effect of HSP27 and −70 on cytokines by draining lymph node cells after DNFB sensitization in C3H/HeN mice. C3H/HeN mice were pretreated with anti-HSP27 and anti-HSP70 or species matched control antibodies and sensitized with 25µl of 0.5% DNFB. Primed lymph node cells were isolated after 5 days from the draining lymph nodes of DNFB sensitized mice and placed in cultures with DNBS labeled BMDC. Treatment of BMDC with anti-HSP27/70 antibody significantly inhibited the production of IL-17 and IFN-γ compared to untreated BMDC (*p<0.05). There was a corresponding increase in IL-4 and IL-10 production under the same conditions. The results are the mean ± SD with 3 mice per group and each experiment was repeated twice with identical results. The data shown are from one representative experiment.

Inhibition of contact hypersensitivity to DNFB by anti-HSP antibodies is a local effect of the antibodies

In order to assess whether the effect of topically applied anti-HSP antibodies was a local or systemic one, separate panels of mice were treated with anti-HSP27 or anti-HSP70 on the abdomen or the back, and attempts were then made to sensitize both panels of mice on the abdomen. Mice that had been treated with anti-HSP27 or anti-HSP70 on the abdomen, showed significant inhibition of the DNFB contact hypersensitivity response (Fig. 5), confirming the results shown in Fig. 3. In contrast, mice treated with anti-HSP antibody on the back, but having received a sensitizing dose of DNFB on the abdomen, had no inhibition of contact hypersensitivity (p>0.05). These results show that the inhibitory effect of topical application of this dose of anti-HSP antibodies is a local occurrence, restricted to the site of antibody treatment.

Figure 5.

Figure 5

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Application of anti-HSP antibodies to the skin inhibits contact hypersensitivity to DNFB through a local effect. C3H/HeN mice were treated with anti-HSP27 or anti-HSP70 antibody on the abdomen or back as indicated. All mice, with the exception of the negative control, were then sensitized with DNFB on the abdomen. Application of hapten to the antibody treated site produced significant inhibition of the contact hypersensitivity response (**p<0.001). In contrast, administration of anti-HSP27 or HSP70 antibodies to the back but with hapten sensitization to the abdomen resulted in an ear swelling response that was not significantly different (p>0.05) from the PBS treated control. Results are expressed as change in auricular thickness ± SEM. There were four mice per group and each experiment was repeated twice with identical results. The data shown are from one representative experiment.

Induction of hapten-specific tolerance by HSP-27 and −70 antibodies

Experiments were then conducted to determine whether epicutaneous application of anti-HSP antibody followed by DNFB application had an effect on subsequent attempts to sensitize mice to that hapten. Animals that had been sensitized with DNFB on anti-HSP antibody treated skin were resensitized with 0.5% DNFB after a resting period of 14 days and were then ear challenged with DNFB 5 days after that. The ear swelling response of mice pretreated with anti-HSP antibody was suppressed compared to positive controls despite the fact that the second attempt to sensitize mice was through normal skin (Fig. 6). This indicated that mice treated with anti-HSP antibody followed by hapten application had become tolerant to DNFB.

Figure 6.

Figure 6

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Application of DNFB to sites of HSP treatment renders mice tolerant to that hapten. C3H/HeN mice were treated with HSP antibody on the shaved abdomen. DNFB was applied to the antibody site as described elsewhere. Fourteen days later, mice were sensitized with DNFB on the shaved back and were subsequently ear challenged after 5 days. Ear swelling responses indicate that the inhibition of CHS response produced by the HSP antibody persisted and was significant (*p<0.05, **p<0.001) in these mice. Results were expressed as change in auricular thickness ± SEM. There were four mice per group and each experiment was repeated twice with identical results. The data shown are from one representative experiment.

To determine whether suppression of the induction of contact hypersensitivity was hapten specific and to exclude the possibility that the mice treated with a combination of anti-HSP27 and anti-HSP70 antibodies and hapten were also non-responsive to other haptens given subsequently, the following experiment was performed. Panels of mice were treated with a combination of anti-HSP27 and anti-HSP70 antibodies followed immediately thereafter by application of DNFB to the antibody treated site. Two weeks later, animals were treated with either a sensitizing dose of DNFB or a different hapten (oxazolone). Five days after that they were ear challenged with the same hapten that had been applied five days earlier. Although mice treated with DNFB plus anti-HSP antibodies and then resensitized with DNFB had a significantly suppressed response to DNFB, mice treated with DNFB plus anti-HSP antibody and then sensitized with oxazolone behaved like normal mice in their ear swelling response to oxazolone (Fig. 7). The results indicate that these animals are able to render a full immune response to other haptens and that the combination of anti-HSP antibodies followed by hapten sensitization induces hapten specific tolerance.

Figure 7.

Figure 7

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Tolerance to DNFB following HSP antibody treatment is antigen specific. C3H/HeN mice were treated with HSP antibody on the abdomen after which 0.5% DNFB was applied to the same site. After 14 days, the mice were resensitized on the shaved back with either 0.5%DNFB or 3% oxazolone. Mice were ear challenged 5 days later with the same hapten that had been applied the back. Ear swelling responses indicate that the inhibition of CHS response produced by the HSP antibody was present in the DNFB back sensitized group (*p<0.05), but was not present in the oxazalone back sensitized mice (p>0.05). Results were expressed as change in auricular thickness ± SEM. There were four mice per group and each experiment was repeated twice with identical results. The data shown are from a representative experiment.

Tolerance to DNFB by HSP27 and HSP70 can be adoptively transferred

In other systems, tolerance to DNFB in mice is frequently accompanied by the development of regulatory T-cells which inhibit the response on subsequent encounter with hapten (15, 17). To examine whether the unresponsiveness to DNFB which occurred following HSP-antibody treatment was also associated with the development of these cells, adoptive transfer studies were performed. Mice, skin painted with a sensitizing dose of DNFB following treatment with anti-HSP-antibody or species matched control antibody, served as donors of spleen cells which were transferred to naive syngeneic recipients. These recipient animals were then sensitized and ear challenged with DNFB. Mice that received cells from animals that were unresponsive to DNFB by pretreatment with HSP-antibodies developed a significantly suppressed ear swelling response (p<0.001) compared to positive controls that had not received cells but which had been immunized and ear challenged to DNFB and to negative controls which had not received cells nor had been sensitized but had been ear challenged to DNFB (Fig. 8). Furthermore, suppression could not be adoptively transferred (p>0.05) with spleen cells from donors treated with species matched control antibody prior to sensitization to DNFB. These results indicate that unresponsiveness to DNFB could be adoptively transferred by spleen cells.

Figure 8.

Figure 8

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Inhibition of DNFB contact hypersensitivity by anti-HSP antibodies can be adoptively transferred by splenocytes. C3H/HeN mice were treated with either a combination of anti-HSP27 and anti-HSP70 or control antibody and the site of antibody application was then treated with 0.5% DNFB. Five days later 50×106 splenocytes from these mice were transferred into naïve syngeneic recipients. The recipients were then sensitized and ear challenged with DNFB. The adoptive transfer of splenocytes from HSP treated donor mice led to significant inhibition (**p<0.001) of contact hypersensitivity in the recipient naïve mice compared to the control recipient mice. Results were expressed as change in auricular thickness ± SEM. There were four mice per group and each experiment was repeated twice with identical results. The data shown are from one representative experiment.

HSP27 augments cytokine production and the induction of contact hypersensitivity by dendritic cells

There is evidence to indicate that HSPs augment the presentation of antigen to T-cells (23–26). Experiments were therefore conducted to determine whether the modulation of contact hypersensitivity response by HSPs occurred by influencing the function of dendritic cells. Initial studies in this regard revealed that topical administration of anti-HSP27 and anti-HSP70 antibodies had no significant effect on densities of epidermal Langerhans cells and there was no difference on the migration of dendritic cells to regional lymph nodes after hapten application (data not shown). Studies were next performed in which bone marrow derived dendritic cells were stimulated with HSP27 protein for 24h and then evaluated for the production of IL-1β, IL-12p35, IL-23p19, IL-12p40, all of which are involved in the presentation of hapten to T-cells.

To exclude the possibility that any changes in cytokine production were caused by contamination by LPS, polymyxin B was added to cultures. The addition of HSP27 to cultures augmented the production of IL-1β, IL-12p35 and IL-12p40, IL-6 and TNF-α but not IL-23p19 in C3H/HeN mice (Fig. 9).

Figure 9.

Figure 9

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Production of proinflammatory cytokines by HSP27 stimulated bone marrow derived dendritic cells (BMDCs) is dependant on TLR4. BMDCs from C3H/HeN and C3H/HeJ mice were treated with recombinant mouse HSP27. Polymyxin B (10µg/ml) was added to the culture to rule out any LPS contamination. There was a significant increase in IL-12p35, TNF-α, IL-1β (*p<0.05) and IL-12p40, IL-6 (**p<0.001) in C3H/HeN mice compared to C3H/HeJ mice. C3H/HeJ mice, on the other hand, showed an increase in IL-23p19 (*p<0.05) on stimulation with HSP27. There were two mice per group and each experiment was repeated twice with identical results. The data shown are from one representative experiment.

Several reports have shown that recombinant HSPs are frequently contaminated with TLR ligands (27, 28). To determine whether this was the case in our system, cultures were treated with both recombinant HSP27 protein and antibodies to HSP27 at the same time. The cultures were then examined for production of of IL-12p35, IL-12p40, and IL-23p19. Treatment of cultures increased production of those cytokines, whereas addition of antibodies to HSP27 abrogated that response, indicating that it was HSP27 that stimulated the BMDCs to produce the cytokines rather than a contaminant in the HSP27 preparation (Fig. 10).

Figure 10.

Figure 10

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Production of proinflammatory cytokines by HSP27 stimulated bone marrow derived dendritic cells (BMDCs) from C3H/HeN mice can be blocked by anti-HSP27 antibodies. Cultures were treated with HSP27 in a manner identical to that of Figure 9. Antibodies to HSP27 were added to separate cultures. There was a significant increase in IL-12p35, (*p<0.05) and IL-12p40, production and upon addition of HSP27 antibody (5mg/ml) there was significantly less production of these cytokines. There were two mice per group and each experiment was repeated twice with identical results. The data shown are from one representative experiment.

Requirement of functional TLR4 on dendritic cells for HSP27-induced cytokine production and augmentation of the induction of contact hypersensitivity

Studies were also conducted to evaluate the contribution of TLR4 to the augmented cytokine response in dendritic cells elicited by HSP27. To accomplish this, BMDC from C3H/HeJ mice, which have a functional deficiency in TLR4 signaling due to a mutation in the TLR4 gene, were compared to BMDC from C3H/HeN mice, which have normal TLR4 signaling (Fig. 9). In contrast to BMDC from C3H/HeN mice, there was no augmentation of IL-12p35 or IL-12p40 and only a modest increase in IL-1β, TNF-α and IL-6 in BMDC from C3H/HeJ mice. Even for IL-1β, TNF-α and IL-6, HSP27-induced production was significantly less than in BMDC from C3H/HeN mice. IL-23p19, however, was significantly increased in BMDC from C3H/HeJ following treatment with HSP27, whereas levels of this cytokine were unaffected in BMDC from C3H/HeN mice.

Treatment of hapten-conjugated BMDC with HSP27 augments their contact sensitizing capacity

We used DNBS conjugated HSP27 treated BMDC from C3H/HeN and C3H/HeJ mice to immunize naïve C3H/HeN and C3H/HeJ recipients. HSP27 stimulated DNBS labeled DCs from C3H/HeN mice were much more proficient at immunizing both C3H/HeN and C3H/HeJ mice to DNFB than control DCs labeled with DNBS but not treated with HSP27 (Fig. 11). On the other hand, HSP27 stimulated DNBS labeled DCs from C3H/HeJ mice were comparable to control DCs labeled with DNBS (Fig. 11). These results indicated that HSP27 alters the immunizing capacity of dendritic cells through a TLR4 dependent process in vivo.

Figure 11.

Figure 11

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Immunization with HSP27 stimulated DNBS labeled BMDCs from C3H/HeN mice augments the induction of CHS. Naïve and HSP27 stimulated BMDC from C3H/HeN and C3H/HeJ mice were labeled with DNBS and injected subcutaneously into naïve C3H/HeN mice or C3H/HeJ mice. CHS was then assessed according to the protocol as described in the Methods section. There was a significant increase in the CHS response (*p<0.05) on injection of DNBS labeled and HSP27 stimulated BMDC as compared to DNBS labeled naïve BMDC from C3H/HeN mice when they were transferred to C3H/HeN or C3H/HeJ mice. On the other hand, HSP27 stimulated DNBS labeled BMDC from C3H/HeJ mice were not effective in eliciting an enhanced CHS response when transferred to either C3H/HeN or C3H/HeJ mice on challenge. There were three mice per group and each experiment was repeated twice with identical results. The data are from a representative experiment.

DISCUSSION

Our results suggest that the initial steps that occur during the induction of contact hypersensitivity responses are more complex than previously conceptualized. Traditionally, it was thought that haptens were taken up by Langerhans cells and other cutaneous dendritic cells, pro-inflammatory cytokines were produced and following migration of dendritic cells to regional lymph nodes, presentation of antigen to subpopulations of T-lymphocytes occurred (29, 30). It now seems likely that heat shock proteins and elements of the innate immune system play a much greater role in the initial stages of this process than previously thought. Employing an in vivo model of allergic contact hypersensitivity, we found that HSP27 and HSP70 are key participants in the induction of immune responses to topically applied haptens. When antibodies to HSP27 and/or HSP70 were applied to the skin and then the hapten DNFB was applied to the same site, the induction of contact hypersensitivity was suppressed and antigen specific immunological unresponsiveness developed. Further analysis showed that incubation of dendritic cells with recombinant HSP27 augmented the production of IL-1β, IL-12p35 and IL-12p40, and those cells were much more proficient at immunizing animals to DNFB. Moreover, the effect of HSP27 was mediated through the TLR4 pathway, since increased production of IL-1β, IL-12p35 and IL-12p40, augmented the capacity of dendritic cells from C3H/HeN mice to initiate contact hypersensitivity responses, but this augmentation did not occur in dendritic cells of C3H/HeJ mice, which have a mutation in the TLR4 gene. These findings are consistent with the hypothesis that following topical application of hapten, the induction of contact hypersensitivity involves heat shock proteins, which in turn activate TLR4 in dendritic cells. TLR4 activation then serves as a stimulus for production of IL-1β, IL-12p35 and IL-12p40, by dendritic cells, which increases their effectiveness at initiating T-cell-mediated immune responses and controls the types of T-cells that elicit and regulate the contact hypersensitivity response.

We became interested in investigating the in vivo contribution of heat shock proteins 27 and 70 to contact hypersensitivity responses when we found that large amounts of these molecules were constitutively expressed in the skin of mice (6). In vitro studies have implicated the role of heat shock proteins in adaptive and innate immune responses (5, 31). These evolutionally conserved proteins chaperone antigenic moieties into the appropriate intracellular channels in dendritic cells for presentation of antigen to T-cells. They also promote dendritic cell maturation and they stimulate dendritic cell production of IL-1β, IL-12p35 and IL-12p40, all of which are known to promote Th1 and Tc1 cell-mediated immune responses. The immunomodulatory properties of HSPs have been exploited to stimulate immune responses by incorporating them into vectors used for vaccination against viral and tumor antigens (32–34). Moreover, HSP70 peptide complexes isolated from the brains of EAE mice have been used to promote NK-induced tolerance to the disease in healthy mice (35). HSP70 has previously been shown to function in association with MHC class I and II molecules as part of the endogenous pathway of antigen presentation. HSP70 is capable of binding to antigenic peptides and markedly increases the efficiency of MHC class I and II presentation (36). Heat shock proteins are expressed in all organisms and in different subcellular compartments (37). The ability of HSPs to bind antigenic peptides and deliver them to APCs forms the basis for their potential role in the generation of peptide-specific T lymphocyte responses. New biochemical and structural studies have recently emerged to support the role of HSPs in some, but not all, models of antigen processing and presentation. Antigen recognition by TLRs results in activation of the innate immune system and leads to the secretion of many inflammatory mediators such as TNF-α, IL-6, and several chemokines. Therefore, innate immunity is likely to play an important role both in the initiation and perpetuation of the inflammatory processes. It has been demonstrated that two members of the sHSP family, A crystallin and HSPB8, were able to activate DCs by inducing maturation and cytokine production in a rheumatoid arthritis model (38). Toxoplasma gondii–derived heat shock protein 70 (T.g.HSP70) has also been shown to stimulate murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade (39).

The contribution of heat shock proteins to in vivo models of autoimmune and inflammatory disease has received little attention. These proteins are overexpressed in the synovial tissue of rheumatoid arthritis patients and in involved tissues of animals with experimental autoimmune encephalitis and collagen-induced arthritis (35, 38, 40). Our experiments indicate that a deficiency of HSPs in vivo will alter the T-cell repertoire that develops in response to hapten. Greater numbers of immunoregulatory T-cells and fewer effector T-cells developed under conditions in which HSP27 was deficient and this was associated with a shift in the balance of IL-12 and IL-23 that were produced in response to hapten.

The role of TLR4 in the biologic activity of HSPs is not without precedent. Asea et al. have previously shown that TLR2 and TLR4 are required for cytokine production by purified human monocytes and the THP-1 monocyte cell line in response to HSP70. HSP70-induced IL-12 production by dendritic cells also has been shown to require TLRs (41). In addition, Flohé et al have shown that HSP60 treatment of bone marrow derived dendritic cells results in their maturation and release of TNF-α, IL-12 and IL-1β (42). However, dendritic cells from C3H/HeJ mice with a mutation at the TLR4 locus failed to respond to HSP60. In this study, we were able to demonstrate that TLRs are required for production of these cytokines in dendritic cells and that this has consequences in vivo.

Our findings highlight the complex interplay between the adaptive and innate immune system during the early stages of the development of contact hypersensitivity involving HSPs, TLR4, and production of cytokines known to augment presentation of antigen to T-cells. In support of this concept are the findings of Di Nardo et al., who have shown that cathelicidins, antimicrobial peptide components of the innate immune system, inhibit the development of DNFB contact hypersensitivity (43). Cathelicidins are antimicrobial peptides which have been shown to have both pro-and anti-inflammatory activities; with respect to contact hypersensitivity they interfere with DNFB-induced activation of TLR4. These findings are consistent with our results and provide additional evidence that the cellular composition of the inflammatory response with respect to regulatory T-cells, effector T-cells and the cytokines that they produce is determined at least in part by the different types and concentrations of molecules of the innate immune system that are present following the skin’s initial encounter with antigen.

Many aspects of the immunological consequences of a localized deficiency of HSP27 and HSP70 in the skin are reminiscent of the effect of low doses of ultraviolet radiation on cutaneous immune responses. Like UV exposure, reducing the activity of HSP27 and HSP70 inhibited the induction of contact hypersensitivity. In both cases this was due to a reduction in the antigen presenting capacity in a confined area of skin. Also in both systems, application of hapten to the UV or HSP antibody treated site resulted in antigen specific unresponsiveness (2). These findings raise the possibility that alterations in the expression of HSP27 and HSP70 occur after UV exposure and that such alterations contribute to UV-induced immune suppression.

Our findings indicate that treatment of dendritic cells with HSP27 resulted in the production of several cytokines that are involved in the antigen presentation process. While it is tempting to attribute the defect in antigen presentation that we observed in HSP deficient skin solely to an inability to synthesize these cytokines, we are mindful of the fact that heat shock proteins have additional effects on antigen presenting cells, including the channeling antigens into the appropriate pathways for antigen processing and display on MHC proteins, the maturation of dendritic cells and the production of other cytokines.

Significant progress has been made in elucidating the cellular and molecular mechanisms involved in the immune response to haptens applied topically to the skin. The finding that HSPs participate in the induction of contact hypersensitivity means that they may be attractive targets with which to manipulate the immune response to cutaneous antigens. Addition of HSPs may increase immune responses and therefore may be useful as adjuvants for vaccination, whereas inhibition of their activity may be beneficial in the prevention of autoimmune and immunological hypersensitivity diseases of the skin such as allergic contact hypersensitivity.

Acknowledgments

Grant Support: NIH Grants P30 AR050948 (CAE), P30 AR050948, Veterans Administration Merit Review Award 18-103-02 (CAE) and a grant from the Department of Defense.

Abbreviations

HSP27

heat shock protein 27

HSP70

heat shock protein 70

CHS

contact hypersensitivity

DNFB

1-Fluoro-2, 4-dinitrobenzene

DNBS

2,4-dinitrobenzene sulphonic acid

BMDC

bone marrow dendritic cells

TLR

toll-like receptor

Footnotes

DISCLOSURES

All authors concur with the submission and have no financial conflict of interest.

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