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Skin immune responses to peptide and protein antigen are TLR4 independent
Cellular Immunology 226 (2003) 116–123 www.elsevier.com/locate/ycimm
Skin immune responses to peptide and protein antigen are TLR4 independent Roopjeet Kahlon and Jan P. Dutz* Department of Medicine and BC Research Institute of Children and WomenÕs Health, University of British Columbia, Vancouver, BC, Canada V5Z 4E2 Received 30 September 2003; accepted 26 November 2003
Abstract Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide + cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4þ and CD8þ T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG1 , IgG2a , and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Toll like receptors; Skin; Rodent; Tetramer; Cytotoxic T lymphocytes
1. Introduction Activation of innate immunity is an important element of the mammalian immune response and contributes to the generation of a robust adaptive immune response. One of the most ancient and conserved effector mechanisms employed by the innate immune response is activation of the toll like receptor (TLR) pathway. The receptors of this pathway recognize conserved molecular motifs associated with microbial and bacterial entities. In addition, several endogenously produced ligands have been identified for the various TLRs. In humans 10 different TLRs, recognizing distinct microbial features, have been characterized [1,2]. While each TLR responds to a particular bacterial stimulus, the signaling events downstream all 10 TLRs converge on activation of the transcription protein NFjB. NFjB translocates into the nucleus and up-regulates the transcription of several * Corresponding author. Present address: The Skin Care Center, 835 West Tenth Avenue Vancouver, BC, Canada V5Z 4E8. Fax: +1-604-8753-9919. E-mail address: [email protected] (J.P. Dutz).
0008-8749/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2003.11.007
pro-inflammatory chemokines and cytokines that function to stimulate the adaptive arm of the immune system by recruiting and activating dendritic cells (DCs) [3]. While various immune related tissues of the body express TLRs, their presence may be particularly important within the skin. The skinÕs primary function is to act as a barrier between the host and its environment. Its location at this host-environment interface makes it susceptible to invasion by microbial entities and subjects it to physical trauma causing tissue injury and distress [4]. The presence of TLRs on skin resident immune cells may assist in mounting a rapid and efficient host defense against invading pathogens or tissue injury causing events. Accordingly, studies have shown that human basal layer and mid epidermal keratinocytes (KC) express TLR1 and TLR4, respectively [5]. Also, human epidermal resident Langerhans cells (LCs) have been reported to express TLR1 and TLR4, and human dermal DCs express TLR1, TLR2, and TLR4 [6]. In the murine system it has been shown that primary KC constitutively express TLR2 and TLR4 mRNA [7] and show surface expression of TLR1 and TLR4 [8]. Murine LCs have been shown to express TLR2, TLR4, and
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TLR9 [9]. Of the three TLRs expressed in the epidermal and dermal layers of the skin, TLR4 may be of particular importance to skin immunobiology. TLR4, in addition to being activated by bacterial lipopolysaccharide (LPS), can be activated by various endogenous ligands that are released by injured or distressed tissue. These endogenous ligands include heparan sulfate [10], oligosaccharides of hyaluronic acid (HA) [11], fibronectin [12], certain heat shock proteins [13–15], and the murine antimicrobial peptide beta defensin 2 [16]. Little is known about the role of TLR4 in the modulation of in vivo immune responses at cutaneous and mucosal surfaces. TLR signaling through MyD88 is required for the initiation of cutaneous allograft rejection demonstrating that this pathway can modulate immune responses to non-infectious ligands [17]. Fragments of HA can induce DC emigration from murine skin explant cultures in a TLR4 dependent manner [11]. This suggests that TLR4 expressed by murine skin-derived DCs can be activated in situ. Further, these results indicate that putative endogenous ligands, such as HA can affect DC behavior in the skin. Recently, it has been shown that peptides and proteins can be applied to the skin to elicit immune responses [18–21]. We have previously shown that mild tissue injury initiated by tape stripping the skin prior to peptide application can enhance the CD8þ T cell response achieved with peptide + adjuvant [22]. Tape stripping, in addition to removing the stratum corneum, has been shown to release IL-1 in murine as well as human epidermis [23,24]. IL-1 is a recognized endogenous danger signal that signals through the Toll/IL-1R pathway [25]. Since TS induces mild tissue injury it may release other endogenous ligands that signal through this same pathway. Specifically, mild tissue injury caused by TS may generate the release of endogenous ligands of TLR4 such as HA, fibronectin, and heat shock proteins. Given the expression of TLR4 on skin resident antigen presenting cells (APCs) and the possible activation of the pathway through TS prior to peptide application, we wanted to determine whether TLR4 signaling played a role in skinderived immune responses to peptide and protein antigens. Cellular immune responses to epicutaneous immunization with peptide + adjuvant subsequent to TS were compared in wild type and TLR4 mutant mice. We found that TS enhances the generation of both CD4þ and CD8þ T cell responses to epicutaneous peptide administration in the presence of CT. Wild type and TLR4 mutant mice mounted equivalent CD4þ and CD8þ T cell responses on Balb/c and C57BL6 genetic backgrounds, respectively. Skin-applied protein specific antibody responses were also found to be TLR4 independent. Thus, although TS enhances epicutaneous immune responses, it does so in a TLR4 independent manner and TLR4 signaling is not required for the induction of T cell or B cell responses through the skin.
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2. Materials and methods 2.1. Animals Balb/c and C57BL/10ScSn (B10) mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and held in a specific pathogen-free environment. C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Balb/c D011.1 mice, expressing a CD4þ T cell receptor (TCR) recognizing Ova323–339 in the context of I-Ad [26] and C57BL/6OT-1 mice expressing a CD8þ TCR recognizing Ova8 [27] in the context of Kb were obtained from Jackson laboratories and bred in our facility. C57BL/10ScNCr (TLR4lps-del) [28], and CC3H-Lpsd [29] were also obtained from Jackson and bred in our facility. CC3HLpsd mice are mice with a Balb/c genetic background into which the Lpsd allele has been introduced. To confirm the LPS hyporesponsive nature of these TLR4 deficient strains, the LPS susceptibility of these mice was determined in a lethality test and compared to the control strains as described [30]. All experiments were carried out on 6–8 week old mice and were approved by the Animal Care Committee of the University of British Columbia. 2.2. Peptide antigens The immunodominant H2-Kb restricted epitope of chicken ovalbumin is Ova254–267 (SIINFEKL—Ova8) [31], and the immunodominant class II H-2d restricted epitope is Ova323–339 (ISQAVHAAHAEINEAGR— Ova323). Peptides were synthesized using FMOC chemistry, purified by reverse phase high-performance liquid chromatography to >80% purity at the Nucleic Acid and Peptide Synthesis Facility of the University of British Columbia. 2.3. Adoptive transfer of transgenic T cells To follow immune responses to epicutaneously applied antigen, we used an adoptive transfer model in which na€ıve T cells bearing a T cell receptor (TCR) specific for the peptide antigen applied to the skin were transferred to na€ıve hosts prior to immunization [32,33]. Peptide antigen-specific CD8þ T cells were isolated from OT-1 TCR transgenic mice while CD4þ T cells were isolated from DO11.10 TCR transgenic mice. Transgenic T cells were isolated from the secondary lymphoid organs (peripheral LN and the spleen) using a positive selection protocol and CD4þ (for DO11.10 mice) or CD8þ (for OT-1 mice) microbeads (Miltenyi Biotech) according to manufacturerÕs instructions. Purified cells (5 million per mouse) were washed, re-suspended in PBS, labeled with 5 lM carboxyfluorescein succinimide ester (CFSE) and adoptively transferred into na€ıve hosts of the appropriate strain by tail vein injection.
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2.4. Epicutaneous immunization Peptide immunization was carried out one day following adoptive transfer of TCR transgenic T cells. Mice were sedated with 80–120 mg/kg ketamine and 8–10 mg/kg xylazine. Balb/c mice were immunized with 200 lg of Ova323 25 lg of whole cholera toxin (Sigma) in PBS with or without epidermal barrier disruption by tape stripping. C57BL/6 mice were immunized with 25 lg Ova8 25 lg of cholera toxin in PBS with or without barrier disruption. Tape stripping (TS) consisted of application and removal of cellophane tape (Scotch Brand 3710 adhesive tape) 8 times to the dorsal and ventral sides of the ear. Acetone was applied to ensure the removal of the lipid barrier. Immunogen was then applied in a 25 ll volume, distributed equally to the dorsal and ventral ear. Sedation was adequate to allow drying of the applied solution and prevent lose of peptide through grooming. Mice were immunized once and the skin draining LN were removed for analysis 4 days after the immunization for CD4þ T cells and 3 days post immunization for CD8þ T cells. These corresponded to time-points of optimal in vivo proliferation as detected in preliminary experiments. To generate a CD8þ T cell response in na€ıve mice and in the absence of the adoptive transfer of antigen-specific precursors, C57BL/6 mice were immunized as above and boosted once seven days later using the contra-lateral ear. The skin draining lymph nodes were removed for analysis 14 days after the first immunization. To generate an antibody response na€ıve Balb/c mice were anaesthetized and immunized with 200 lg of chicken ovalbumin protein (OVA V, Sigma, MO) and 25 lg of CT after TS. Mice were boosted on the contra-lateral ear 7 days later. Serum
was obtained 14 days post priming and stored at )20 °C for further analysis. 2.5. Antibody staining and cytofluorometric analysis The frequency of peptide specific T cells in the skin draining lymph nodes was determined by cytofluorimetry. Single cell preparations were prepared and labeled in PBS with 2% fetal calf serum. Antibodies used to identify peptide specific CD4þ T cells included KJ1-26 PE (Cederlane), a monoclonal Ab specific for the transgenic TCR expressed by DO11.10 CD4þ T cells [34], and anti-CD4 Cyc (BD Pharmingen). Peptide specific CD8þ T cells were identified using a PE conjugated Kb -Ova tetramer (graciously provided by Jacqueline D. Trudeau and Dr. Rusung Tan, synthesized according to Altman et al. [35]) and anti-CD8a FITC (Cederlane). Non-specific binding of the Kb -Ova tetramer was minimized by restricting the analysis to cells that did not stain for a B cell marker, CD45R using anti-CD45R/ B220 PerCP (BD Pharmingen). After appropriate staining, 200,000 size and forward scatter-gated events were acquired for analysis using four-channel fluorescence activated cell sorting (FACS) performed on a FACScalibur flow cytometer (Becton–Dickinson, San Jose, CA) and Cell Quest software (Becton–Dickinson). 2.6. Antibody ELISA OVA-specific IgG, IgG1 , IgG2a , and IgE titers were determined in serum of mice bled at necropsy. A previously described protocol [36] was used. The detection of antibodies was carried out using HRP rabbit antimouse antibodies (all from Zymed laboratories, San
c Fig. 1. Effect of tape stripping and TLR4 signaling on the frequency of cognate CD4þ T cell proliferation in response to epicutaneous peptide administration. Balb/c mice were immunized with 200 lg of Ova323 25 lg of cholera toxin with or without tape stripping one day following transfer of DO11.1 transgenic CD4þ T cells. Transfer only mice represent un-immunized controls. Lymphocytes were isolated from skin draining LN 4 days following transfer. (A) Density plots of lymphocytes from representative mice. Peptide specific CD4þ T cells were identified by staining with the TCR clonotypic antibody KJ1-26 and anti-CD4 monoclonal antibody and analyzed using FACS analysis. Numbers indicate percent of CD4þ T cells that express the peptide-specific clonotypic TCR. (B) Scatter gram indicating the percentage of clonotypic CD4þ T cells detected in the skin draining lymph nodes of individual mice from five independent experiments. (C) Wild type Balb/c and CC3H-Lpsd TLR4 mutant mice (TLR4def) were immunized following the adoptive transfer of D011.1 CD4þ T cells and the number of clonotypic T cells in the auricular draining lymph nodes was determined. Aggregate data from six independent experiments is presented. ANOVA (B) and Nonparametric studentÕs t test (C) were used to calculate p values. p < 0:05 denotes statistical significance between groups. Fig. 2. Effect of tape stripping and TLR4 signaling on the frequency of cognate CD8þ T cells in response to epicutaneous peptide administration. C57BL/6 mice were primed and boosted 7 days later on the contra-lateral ear with 25 lg of Ova8 25 lg of cholera toxin (CT) with or without tape stripping (TS). Lymphocytes from skin draining LN were collected 14 days after priming and peptide specific CD8þ T cells were enumerated by FACS analysis. (A) Representative density plots of CD8 and Ova-specific tetramer staining of lymphocytes from immunized mice. (B) Scatter gram indicating the percentage of Ova-specific CD8þ T cells (% antigen-specific T cells) detected in the skin draining lymph nodes of individual mice from three independent experiments. Nonparametric studentÕs t test was used to calculate p values. p < 0:05 denotes statistical significance between groups. (C) Na€ıve C57BL/10ScSn (wild type) and C57BL/10ScNCr (TLR4lps-del ) mice were immunized with 25 lg of Ova8 + 25 lg of CT after tape stripping one day following i.v. transfer of 5 106 CFSE labelled OT1.1 transgenic CD8þ T cells. Lymphocytes were isolated from skin draining LN 3 days later. Density plots of lymphocytes from representative mice are shown. Peptide specific CD8þ T cells were identified by staining with CFSE, Kb -Ova tetramer and anti-CD8 monoclonal antibody and analyzed using FACS analysis. (D) The bar graph illustrates the mean percentage of peptidespecific CD8þ T cells present in the skin draining lymph nodes in the two strains following immunization with error calculated as standard error of the mean. Data is representative of two independent experiments.
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Fig. 1.
Fig. 2.
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Fransisco CA) except for IgE detection where biotinylated anti-mouse IgE and streptavidin-HRP amplification was used (Pharmingen). The detection system was from Kirkegaard and Perry Laboratories (Gaithersburg MD). Commercial standards were used for IgG and IgG1 , (Sigma) and laboratory-generated reference standards were used for IgG2a and IgE. Titers were calculated based on linear regression analysis of the absorbances. Individual values represent the average of duplicate measurements. 2.7. Statistical analysis Statistically significant differences between groups were calculated using two-tailed StudentÕs t tests and ANOVA. A p < 0:05 denotes a statistically significant difference. All analysis were done using Prism 3 (GraphPad software, San Diego, CA).
3. Results and discussion 3.1. CD4þ T cell responses to epicutaneous immunization with cholera toxin and tape stripping are TLR4 independent To characterize the CD4þ T cell response to epicutaneously applied peptide we used an adoptive T cell transfer model [32,33]. The model makes use of a CD4þ TCR transgenic mouse, DO11.10, expressing a TCR specific for the MHC class II restricted epitope, Ova323, of the chicken protein ovalbumin as a source of na€ıve, antigen-specific T cells. These cells are transferred to na€ıve, non-transgenic mice that are then immunized. The transferred cells can be detected by a clonotypic antibody (KJ1-26) [34], allowing the number of antigenspecific cells present in the draining lymph nodes in response to an immunogen to be easily determined by cytoflourometric analysis. Tape stripping and cholera toxin have both been shown to enhance immune responses to cutaneously applied antigen [18,37]. We have demonstrated that the combined use of TS and CT leads to optimal CD8þ T cell responses to epicutaneously applied peptide [22]. We first chose to determine if these manipulations would enhance the immune response to a CD4þ T cell determinant peptide. One day following adoptive transfer, the Balb/c chimeric mice were therefore immunized on the ear with the class II restricted epitope Ova323 with or without TS the ear skin and with or without the adjuvant CT. The extent of proliferation of the transferred cells in response to the epicutaneous immunization was determined 4 days later by FACS analysis of lymphocytes collected from skin draining LN. The frequency of antigen-specific T cells was determined by simultaneous staining with the clonotypic TCR specific antibody, KJ1-26, and anti-CD4 monoclonal antibody (mAb) (Figs. 1A and B).
The greatest frequency of peptide specific CD4þ T cells was noted in mice immunized with cognate peptide with both TS and CT with a mean of 6.3 1.0% peptide specific CD4þ T cells. In comparison peptide immunization with CT alone resulted in 1.3 0.2% (p < 0:001) peptide specific CD4þ T cells. Similarly peptide immunization with TS alone resulted in 3.0 0.7% (p < 0:001) peptide specific CD4þ T cells. While immunization with Ova323 + TS resulted in a greater percentage of peptide specific CD4þ T cells than immunization with Ova323 + CT this difference was not statistically significant (p > 0:05). Mice adoptively transferred with the transgenic DO11.1 cells but otherwise un-manipulated served as the control group. In comparison to the control group, only peptide immunization with CT + TS resulted in a significant increase (p < 0:001) in peptide specific CD4þ T cells. Thus, optimal frequencies of antigen-specific CD4þ T cells are detected following immunization with TS and CT as adjuvants. These frequencies represent local T cell proliferation and not altered T cell traffic as they are concordant with the relative serial dilution of carboxyfluoresceine succinimide ester (CFSE) labeled antigen specific T cells (data not shown). The results indicate a marked enhancement of the immune response to peptide and cholera toxin administration following TS. Putative TLR4 ligands include molecules released during tissue injury such as induced by TS. To determine the role of TLR4 signaling in the generation of cellular immune responses to epicutaneous peptide, the optimal CD4þ T cell response was compared between wild type Balb/c and TLR4 mutant CC3H-Lpsd mice (these are Balb/c congenic mice that carry the C3H/HeJ Lpsd allele [29]). C3H/HeJ mice have a mis-sense proline to histidine point mutation in the cytoplasmic domain of the tlr4 gene inhibiting signaling downstream of TLR4 and rendering them hyporesponsive to endotoxin stimulation [28]. Control and TLR4 deficient mice were immunized with Ova323 and CT after TS. There was no significant difference in peptide specific CD4þ T cell proliferation between wild type and TLR4 mutant mice (Fig. 1C). Thus TLR4 signaling is not required for the generation of CD4þ T cell responses to epicutaneous peptide applied following TS and with CT. 3.2. CD8þ T cell responses to epicutaneous immunization with cholera toxin and tape stripping are TLR4 independent We have previously shown that optimal CD8þ T cell responses to epicutaneously applied peptide can be obtained with CT administration and following TS [22]. Since the immunodominant class I MHC restricted epitope of ovalbumin presented by Kb is well defined [31] and C57BL/6 mice represent an IFNc rich environment as compared to Balb/c mice, they were used to charac-
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terize the CD8 T cell responses [38]. Na€ıve C57BL/6 (B6) mice were immunized with 25 lg of Ova8 with or without TS and/or CT and in the absence of transgenic TCR cells. Mice were boosted on the contra-lateral ear 7 days later and skin draining LN collected 14 days after the initial priming for enumeration of peptide specific CTL. Fluorescently labeled multimeric complexes of class I MHC complexed with peptide can be used to detect antigen specific CD8þ T cells ex vivo [39]. Ova8 specific CD8þ T cells were thus visualized using cell surface staining with Kb -Ova tetramer and anti-CD8 mAb (Fig. 2A). Immunization with both CT and TS elicited an 11-fold greater response than TS alone and a threefold greater response than CT alone: Mice immunized with CT + TS displayed 0.65 0.06% Ova8 specific CD8þ T cells. In comparison, immunization with CT or TS alone resulted in 0.23 0.03% (p ¼ 0:0004) and 0.05 0.01% (p < 0:0001) peptide specific CD8þ T cells, respectively. Consequently, similar to the effect seen with the CD4þ T cell response, optimal cognate CD8þ T cell priming is achieved with both TS and CT. To determine if TLR4 signaling contributes to the optimal CD8þ T cell response to epicutaneous peptide following TS and adjuvant application, we compared the CD8þ T cell response between wild type and TLR4 mutant mice. In these experiments, we employed an adoptive transfer system similar to the one described for the CD4þ T cell response. Again, adoptive transfer allowed a rapid and highly reproducible analysis of the
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local T cell response to epicutaneously administered antigen. Here OT1.1 TCR transgenic mice recognizing Ova8 were adoptively transferred into wild type C57BL/ 10ScSn (B10) and TLR4 knockout C57BL/10ScNCr mice. The mice were immunized one day following adoptive transfer with Ova8 and CT subsequent to TS. Responses in the skin draining LN were characterized 3 days post immunization. Enumeration of peptide specific CD8þ T cells was accomplished by simultaneous staining with Kb -Ova tetramer and anti-CD8 monoclonal antibody. Similar frequencies of peptide specific CD8þ T cells were identified in wild type (22.2 1.3%) and TLR4 knockout mice (18.7 2.6%), respectively (Fig. 2B). The extent of proliferation of adoptively transferred cells as determined by CFSE staining did not differ (data not shown). 3.3. Humoral immune responses to epicutaneous immunization with CT and TS are TLR4 independent We have previously demonstrated that TS enhances the antibody responses to epicutaneously applied protein antigen [22]. To study the role of TLR4 signaling in modulating humoral responses to epicutaneous immunization, the antibody response in wild type Balb/c mice was compared to that achieved in TLR4 mutant CC3HLpsd mice. Mice were immunized and boosted seven days later on the contra-lateral ear with 200 lg of full Ova protein + 25 lg of CT after TS the ear. Three
Fig. 3. The role of TLR4 signaling in humoral immune responses to epicutaneous protein immunization. Wild type (Balb/c) and TLR4 mutant mice (CC3H-Lpsd, TLR4def) were immunized with 200 lg ovalbumin protein + 25 lg CT after TS. Mice were primed and boosted 14 days later on the contra-lateral ear. Three weeks after priming serum was collected and assayed for Ova-specific antibodies using ELISA. Scatter grams of data from 3 individual mice per group are shown. Representative data from two independent experiments is represented.
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weeks after priming serum was collected and assayed for Ova-specific antibodies. OVA-specific total IgG, IgE, IgG1 , and IgG2a titers were assayed and found to be equivalently induced in both wild type and TLR4 mutant mice (Fig. 3). 3.4. Discussion The results of this study indicate that both CD4þ and CD8þ skin initiated cellular immune responses to peptide antigen are enhanced following tape stripping and in the presence of cholera toxin as adjuvant. This observation extends the previous reports of the value of this simple manoeuvre to enhance immune responses to skin-applied antigens [22,40,41] and details the adjuvant effect of mild skin injury. As TLR4 polymorphisms have been associated with susceptibility to gram )ve bacterial infection, the skin expression of TLR4 has been implicated in the cutaneous defence against gram )ve bacteria [42]. Murine LC have been shown to express TLR4 [9]. A number of endogenous TLR4 ligands have recently been described and Toll receptor signaling pathways have been implicated in the generation of alloreactive immune responses in the skin [17]. We therefore asked whether TLR4 signaling modulated responses to epicutaneous peptides or proteins in the presence of adjuvant and tape stripping. We found that CD4þ and CD8þ T cell responses were similarly evoked in wild type and TLR4 mutant mice. Similarly, humoral immune responses to epicutaneously applied protein antigen were also found to be TLR4 independent. We conclude that the adjuvant-like properties demonstrated by these two modalities in our epicutaneous immunization model do not stem from activation of innate immunity through TLR4. Consistent with this, Takeuchi et al. have recently demonstrated that the adjuvant properties of CT are TLR4 independent and result primarily from the engagement of the GM1-ganglioside receptor [43]. Interestingly, TLR4 signaling has recently been shown to enable Th2 immune responses to protein antigen in the lung. In this tissue, TLR4 signaling is initiated by low concentrations of lipopolysaccharide present in protein preparations [44]. Our results indicate that TLR4 signaling, in contradistinction to its role in facilitating pulmonary immune responses to protein antigen, may be dispensable in the initiation of skin immune responses. Indeed, keratinocytes, through their release of IL-1, have allied pathways of signaling tissue damage and infection. It has recently been suggested that cutaneous associated DC may downregulate TLR4 expression in order to prevent inordinate immune stimulation by commensal and saprophytic bacteria [45]. Further, the recognition of endogenous danger signals by TLR4 in the skin, an organ at constant risk of minor trauma and abrasion by virtue of its location at the hostenvironment interface, might lead to inappropriate loss
of tolerance to self. The conditions other than the presence of bacterial lipopolysaccharide that trigger productive TLR4 signaling in the skin remain to be determined. Further, given the recently demonstrated importance of MyD88 dependent signaling in cutaneous allograft rejection [17], other TLR receptors and ligands may have an important role in the control of skin immune responses.
Acknowledgments We thank Jacqueline D. Trudeau and Dr. Rusung Tan for the gift of the Kb -OVA reagent. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR). RK is supported by an award from the CIHR/Michael Smith Foundation for Health Research (MSFHR) Transplant Research Training Program. JPD is a Junior Scholar of the Arthritis Society and a UBC Galderma Scholar.
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Skin immune responses to peptide and protein antigen are TLR4 independent Roopjeet Kahlon and Jan P. Dutz* Department of Medicine and BC Research Institute of Children and WomenÕs Health, University of British Columbia, Vancouver, BC, Canada V5Z 4E2 Received 30 September 2003; accepted 26 November 2003
Abstract Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide + cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4þ and CD8þ T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG1 , IgG2a , and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Toll like receptors; Skin; Rodent; Tetramer; Cytotoxic T lymphocytes
1. Introduction Activation of innate immunity is an important element of the mammalian immune response and contributes to the generation of a robust adaptive immune response. One of the most ancient and conserved effector mechanisms employed by the innate immune response is activation of the toll like receptor (TLR) pathway. The receptors of this pathway recognize conserved molecular motifs associated with microbial and bacterial entities. In addition, several endogenously produced ligands have been identified for the various TLRs. In humans 10 different TLRs, recognizing distinct microbial features, have been characterized [1,2]. While each TLR responds to a particular bacterial stimulus, the signaling events downstream all 10 TLRs converge on activation of the transcription protein NFjB. NFjB translocates into the nucleus and up-regulates the transcription of several * Corresponding author. Present address: The Skin Care Center, 835 West Tenth Avenue Vancouver, BC, Canada V5Z 4E8. Fax: +1-604-8753-9919. E-mail address: [email protected] (J.P. Dutz).
0008-8749/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2003.11.007
pro-inflammatory chemokines and cytokines that function to stimulate the adaptive arm of the immune system by recruiting and activating dendritic cells (DCs) [3]. While various immune related tissues of the body express TLRs, their presence may be particularly important within the skin. The skinÕs primary function is to act as a barrier between the host and its environment. Its location at this host-environment interface makes it susceptible to invasion by microbial entities and subjects it to physical trauma causing tissue injury and distress [4]. The presence of TLRs on skin resident immune cells may assist in mounting a rapid and efficient host defense against invading pathogens or tissue injury causing events. Accordingly, studies have shown that human basal layer and mid epidermal keratinocytes (KC) express TLR1 and TLR4, respectively [5]. Also, human epidermal resident Langerhans cells (LCs) have been reported to express TLR1 and TLR4, and human dermal DCs express TLR1, TLR2, and TLR4 [6]. In the murine system it has been shown that primary KC constitutively express TLR2 and TLR4 mRNA [7] and show surface expression of TLR1 and TLR4 [8]. Murine LCs have been shown to express TLR2, TLR4, and
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TLR9 [9]. Of the three TLRs expressed in the epidermal and dermal layers of the skin, TLR4 may be of particular importance to skin immunobiology. TLR4, in addition to being activated by bacterial lipopolysaccharide (LPS), can be activated by various endogenous ligands that are released by injured or distressed tissue. These endogenous ligands include heparan sulfate [10], oligosaccharides of hyaluronic acid (HA) [11], fibronectin [12], certain heat shock proteins [13–15], and the murine antimicrobial peptide beta defensin 2 [16]. Little is known about the role of TLR4 in the modulation of in vivo immune responses at cutaneous and mucosal surfaces. TLR signaling through MyD88 is required for the initiation of cutaneous allograft rejection demonstrating that this pathway can modulate immune responses to non-infectious ligands [17]. Fragments of HA can induce DC emigration from murine skin explant cultures in a TLR4 dependent manner [11]. This suggests that TLR4 expressed by murine skin-derived DCs can be activated in situ. Further, these results indicate that putative endogenous ligands, such as HA can affect DC behavior in the skin. Recently, it has been shown that peptides and proteins can be applied to the skin to elicit immune responses [18–21]. We have previously shown that mild tissue injury initiated by tape stripping the skin prior to peptide application can enhance the CD8þ T cell response achieved with peptide + adjuvant [22]. Tape stripping, in addition to removing the stratum corneum, has been shown to release IL-1 in murine as well as human epidermis [23,24]. IL-1 is a recognized endogenous danger signal that signals through the Toll/IL-1R pathway [25]. Since TS induces mild tissue injury it may release other endogenous ligands that signal through this same pathway. Specifically, mild tissue injury caused by TS may generate the release of endogenous ligands of TLR4 such as HA, fibronectin, and heat shock proteins. Given the expression of TLR4 on skin resident antigen presenting cells (APCs) and the possible activation of the pathway through TS prior to peptide application, we wanted to determine whether TLR4 signaling played a role in skinderived immune responses to peptide and protein antigens. Cellular immune responses to epicutaneous immunization with peptide + adjuvant subsequent to TS were compared in wild type and TLR4 mutant mice. We found that TS enhances the generation of both CD4þ and CD8þ T cell responses to epicutaneous peptide administration in the presence of CT. Wild type and TLR4 mutant mice mounted equivalent CD4þ and CD8þ T cell responses on Balb/c and C57BL6 genetic backgrounds, respectively. Skin-applied protein specific antibody responses were also found to be TLR4 independent. Thus, although TS enhances epicutaneous immune responses, it does so in a TLR4 independent manner and TLR4 signaling is not required for the induction of T cell or B cell responses through the skin.
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2. Materials and methods 2.1. Animals Balb/c and C57BL/10ScSn (B10) mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and held in a specific pathogen-free environment. C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Balb/c D011.1 mice, expressing a CD4þ T cell receptor (TCR) recognizing Ova323–339 in the context of I-Ad [26] and C57BL/6OT-1 mice expressing a CD8þ TCR recognizing Ova8 [27] in the context of Kb were obtained from Jackson laboratories and bred in our facility. C57BL/10ScNCr (TLR4lps-del) [28], and CC3H-Lpsd [29] were also obtained from Jackson and bred in our facility. CC3HLpsd mice are mice with a Balb/c genetic background into which the Lpsd allele has been introduced. To confirm the LPS hyporesponsive nature of these TLR4 deficient strains, the LPS susceptibility of these mice was determined in a lethality test and compared to the control strains as described [30]. All experiments were carried out on 6–8 week old mice and were approved by the Animal Care Committee of the University of British Columbia. 2.2. Peptide antigens The immunodominant H2-Kb restricted epitope of chicken ovalbumin is Ova254–267 (SIINFEKL—Ova8) [31], and the immunodominant class II H-2d restricted epitope is Ova323–339 (ISQAVHAAHAEINEAGR— Ova323). Peptides were synthesized using FMOC chemistry, purified by reverse phase high-performance liquid chromatography to >80% purity at the Nucleic Acid and Peptide Synthesis Facility of the University of British Columbia. 2.3. Adoptive transfer of transgenic T cells To follow immune responses to epicutaneously applied antigen, we used an adoptive transfer model in which na€ıve T cells bearing a T cell receptor (TCR) specific for the peptide antigen applied to the skin were transferred to na€ıve hosts prior to immunization [32,33]. Peptide antigen-specific CD8þ T cells were isolated from OT-1 TCR transgenic mice while CD4þ T cells were isolated from DO11.10 TCR transgenic mice. Transgenic T cells were isolated from the secondary lymphoid organs (peripheral LN and the spleen) using a positive selection protocol and CD4þ (for DO11.10 mice) or CD8þ (for OT-1 mice) microbeads (Miltenyi Biotech) according to manufacturerÕs instructions. Purified cells (5 million per mouse) were washed, re-suspended in PBS, labeled with 5 lM carboxyfluorescein succinimide ester (CFSE) and adoptively transferred into na€ıve hosts of the appropriate strain by tail vein injection.
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2.4. Epicutaneous immunization Peptide immunization was carried out one day following adoptive transfer of TCR transgenic T cells. Mice were sedated with 80–120 mg/kg ketamine and 8–10 mg/kg xylazine. Balb/c mice were immunized with 200 lg of Ova323 25 lg of whole cholera toxin (Sigma) in PBS with or without epidermal barrier disruption by tape stripping. C57BL/6 mice were immunized with 25 lg Ova8 25 lg of cholera toxin in PBS with or without barrier disruption. Tape stripping (TS) consisted of application and removal of cellophane tape (Scotch Brand 3710 adhesive tape) 8 times to the dorsal and ventral sides of the ear. Acetone was applied to ensure the removal of the lipid barrier. Immunogen was then applied in a 25 ll volume, distributed equally to the dorsal and ventral ear. Sedation was adequate to allow drying of the applied solution and prevent lose of peptide through grooming. Mice were immunized once and the skin draining LN were removed for analysis 4 days after the immunization for CD4þ T cells and 3 days post immunization for CD8þ T cells. These corresponded to time-points of optimal in vivo proliferation as detected in preliminary experiments. To generate a CD8þ T cell response in na€ıve mice and in the absence of the adoptive transfer of antigen-specific precursors, C57BL/6 mice were immunized as above and boosted once seven days later using the contra-lateral ear. The skin draining lymph nodes were removed for analysis 14 days after the first immunization. To generate an antibody response na€ıve Balb/c mice were anaesthetized and immunized with 200 lg of chicken ovalbumin protein (OVA V, Sigma, MO) and 25 lg of CT after TS. Mice were boosted on the contra-lateral ear 7 days later. Serum
was obtained 14 days post priming and stored at )20 °C for further analysis. 2.5. Antibody staining and cytofluorometric analysis The frequency of peptide specific T cells in the skin draining lymph nodes was determined by cytofluorimetry. Single cell preparations were prepared and labeled in PBS with 2% fetal calf serum. Antibodies used to identify peptide specific CD4þ T cells included KJ1-26 PE (Cederlane), a monoclonal Ab specific for the transgenic TCR expressed by DO11.10 CD4þ T cells [34], and anti-CD4 Cyc (BD Pharmingen). Peptide specific CD8þ T cells were identified using a PE conjugated Kb -Ova tetramer (graciously provided by Jacqueline D. Trudeau and Dr. Rusung Tan, synthesized according to Altman et al. [35]) and anti-CD8a FITC (Cederlane). Non-specific binding of the Kb -Ova tetramer was minimized by restricting the analysis to cells that did not stain for a B cell marker, CD45R using anti-CD45R/ B220 PerCP (BD Pharmingen). After appropriate staining, 200,000 size and forward scatter-gated events were acquired for analysis using four-channel fluorescence activated cell sorting (FACS) performed on a FACScalibur flow cytometer (Becton–Dickinson, San Jose, CA) and Cell Quest software (Becton–Dickinson). 2.6. Antibody ELISA OVA-specific IgG, IgG1 , IgG2a , and IgE titers were determined in serum of mice bled at necropsy. A previously described protocol [36] was used. The detection of antibodies was carried out using HRP rabbit antimouse antibodies (all from Zymed laboratories, San
c Fig. 1. Effect of tape stripping and TLR4 signaling on the frequency of cognate CD4þ T cell proliferation in response to epicutaneous peptide administration. Balb/c mice were immunized with 200 lg of Ova323 25 lg of cholera toxin with or without tape stripping one day following transfer of DO11.1 transgenic CD4þ T cells. Transfer only mice represent un-immunized controls. Lymphocytes were isolated from skin draining LN 4 days following transfer. (A) Density plots of lymphocytes from representative mice. Peptide specific CD4þ T cells were identified by staining with the TCR clonotypic antibody KJ1-26 and anti-CD4 monoclonal antibody and analyzed using FACS analysis. Numbers indicate percent of CD4þ T cells that express the peptide-specific clonotypic TCR. (B) Scatter gram indicating the percentage of clonotypic CD4þ T cells detected in the skin draining lymph nodes of individual mice from five independent experiments. (C) Wild type Balb/c and CC3H-Lpsd TLR4 mutant mice (TLR4def) were immunized following the adoptive transfer of D011.1 CD4þ T cells and the number of clonotypic T cells in the auricular draining lymph nodes was determined. Aggregate data from six independent experiments is presented. ANOVA (B) and Nonparametric studentÕs t test (C) were used to calculate p values. p < 0:05 denotes statistical significance between groups. Fig. 2. Effect of tape stripping and TLR4 signaling on the frequency of cognate CD8þ T cells in response to epicutaneous peptide administration. C57BL/6 mice were primed and boosted 7 days later on the contra-lateral ear with 25 lg of Ova8 25 lg of cholera toxin (CT) with or without tape stripping (TS). Lymphocytes from skin draining LN were collected 14 days after priming and peptide specific CD8þ T cells were enumerated by FACS analysis. (A) Representative density plots of CD8 and Ova-specific tetramer staining of lymphocytes from immunized mice. (B) Scatter gram indicating the percentage of Ova-specific CD8þ T cells (% antigen-specific T cells) detected in the skin draining lymph nodes of individual mice from three independent experiments. Nonparametric studentÕs t test was used to calculate p values. p < 0:05 denotes statistical significance between groups. (C) Na€ıve C57BL/10ScSn (wild type) and C57BL/10ScNCr (TLR4lps-del ) mice were immunized with 25 lg of Ova8 + 25 lg of CT after tape stripping one day following i.v. transfer of 5 106 CFSE labelled OT1.1 transgenic CD8þ T cells. Lymphocytes were isolated from skin draining LN 3 days later. Density plots of lymphocytes from representative mice are shown. Peptide specific CD8þ T cells were identified by staining with CFSE, Kb -Ova tetramer and anti-CD8 monoclonal antibody and analyzed using FACS analysis. (D) The bar graph illustrates the mean percentage of peptidespecific CD8þ T cells present in the skin draining lymph nodes in the two strains following immunization with error calculated as standard error of the mean. Data is representative of two independent experiments.
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Fig. 1.
Fig. 2.
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Fransisco CA) except for IgE detection where biotinylated anti-mouse IgE and streptavidin-HRP amplification was used (Pharmingen). The detection system was from Kirkegaard and Perry Laboratories (Gaithersburg MD). Commercial standards were used for IgG and IgG1 , (Sigma) and laboratory-generated reference standards were used for IgG2a and IgE. Titers were calculated based on linear regression analysis of the absorbances. Individual values represent the average of duplicate measurements. 2.7. Statistical analysis Statistically significant differences between groups were calculated using two-tailed StudentÕs t tests and ANOVA. A p < 0:05 denotes a statistically significant difference. All analysis were done using Prism 3 (GraphPad software, San Diego, CA).
3. Results and discussion 3.1. CD4þ T cell responses to epicutaneous immunization with cholera toxin and tape stripping are TLR4 independent To characterize the CD4þ T cell response to epicutaneously applied peptide we used an adoptive T cell transfer model [32,33]. The model makes use of a CD4þ TCR transgenic mouse, DO11.10, expressing a TCR specific for the MHC class II restricted epitope, Ova323, of the chicken protein ovalbumin as a source of na€ıve, antigen-specific T cells. These cells are transferred to na€ıve, non-transgenic mice that are then immunized. The transferred cells can be detected by a clonotypic antibody (KJ1-26) [34], allowing the number of antigenspecific cells present in the draining lymph nodes in response to an immunogen to be easily determined by cytoflourometric analysis. Tape stripping and cholera toxin have both been shown to enhance immune responses to cutaneously applied antigen [18,37]. We have demonstrated that the combined use of TS and CT leads to optimal CD8þ T cell responses to epicutaneously applied peptide [22]. We first chose to determine if these manipulations would enhance the immune response to a CD4þ T cell determinant peptide. One day following adoptive transfer, the Balb/c chimeric mice were therefore immunized on the ear with the class II restricted epitope Ova323 with or without TS the ear skin and with or without the adjuvant CT. The extent of proliferation of the transferred cells in response to the epicutaneous immunization was determined 4 days later by FACS analysis of lymphocytes collected from skin draining LN. The frequency of antigen-specific T cells was determined by simultaneous staining with the clonotypic TCR specific antibody, KJ1-26, and anti-CD4 monoclonal antibody (mAb) (Figs. 1A and B).
The greatest frequency of peptide specific CD4þ T cells was noted in mice immunized with cognate peptide with both TS and CT with a mean of 6.3 1.0% peptide specific CD4þ T cells. In comparison peptide immunization with CT alone resulted in 1.3 0.2% (p < 0:001) peptide specific CD4þ T cells. Similarly peptide immunization with TS alone resulted in 3.0 0.7% (p < 0:001) peptide specific CD4þ T cells. While immunization with Ova323 + TS resulted in a greater percentage of peptide specific CD4þ T cells than immunization with Ova323 + CT this difference was not statistically significant (p > 0:05). Mice adoptively transferred with the transgenic DO11.1 cells but otherwise un-manipulated served as the control group. In comparison to the control group, only peptide immunization with CT + TS resulted in a significant increase (p < 0:001) in peptide specific CD4þ T cells. Thus, optimal frequencies of antigen-specific CD4þ T cells are detected following immunization with TS and CT as adjuvants. These frequencies represent local T cell proliferation and not altered T cell traffic as they are concordant with the relative serial dilution of carboxyfluoresceine succinimide ester (CFSE) labeled antigen specific T cells (data not shown). The results indicate a marked enhancement of the immune response to peptide and cholera toxin administration following TS. Putative TLR4 ligands include molecules released during tissue injury such as induced by TS. To determine the role of TLR4 signaling in the generation of cellular immune responses to epicutaneous peptide, the optimal CD4þ T cell response was compared between wild type Balb/c and TLR4 mutant CC3H-Lpsd mice (these are Balb/c congenic mice that carry the C3H/HeJ Lpsd allele [29]). C3H/HeJ mice have a mis-sense proline to histidine point mutation in the cytoplasmic domain of the tlr4 gene inhibiting signaling downstream of TLR4 and rendering them hyporesponsive to endotoxin stimulation [28]. Control and TLR4 deficient mice were immunized with Ova323 and CT after TS. There was no significant difference in peptide specific CD4þ T cell proliferation between wild type and TLR4 mutant mice (Fig. 1C). Thus TLR4 signaling is not required for the generation of CD4þ T cell responses to epicutaneous peptide applied following TS and with CT. 3.2. CD8þ T cell responses to epicutaneous immunization with cholera toxin and tape stripping are TLR4 independent We have previously shown that optimal CD8þ T cell responses to epicutaneously applied peptide can be obtained with CT administration and following TS [22]. Since the immunodominant class I MHC restricted epitope of ovalbumin presented by Kb is well defined [31] and C57BL/6 mice represent an IFNc rich environment as compared to Balb/c mice, they were used to charac-
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terize the CD8 T cell responses [38]. Na€ıve C57BL/6 (B6) mice were immunized with 25 lg of Ova8 with or without TS and/or CT and in the absence of transgenic TCR cells. Mice were boosted on the contra-lateral ear 7 days later and skin draining LN collected 14 days after the initial priming for enumeration of peptide specific CTL. Fluorescently labeled multimeric complexes of class I MHC complexed with peptide can be used to detect antigen specific CD8þ T cells ex vivo [39]. Ova8 specific CD8þ T cells were thus visualized using cell surface staining with Kb -Ova tetramer and anti-CD8 mAb (Fig. 2A). Immunization with both CT and TS elicited an 11-fold greater response than TS alone and a threefold greater response than CT alone: Mice immunized with CT + TS displayed 0.65 0.06% Ova8 specific CD8þ T cells. In comparison, immunization with CT or TS alone resulted in 0.23 0.03% (p ¼ 0:0004) and 0.05 0.01% (p < 0:0001) peptide specific CD8þ T cells, respectively. Consequently, similar to the effect seen with the CD4þ T cell response, optimal cognate CD8þ T cell priming is achieved with both TS and CT. To determine if TLR4 signaling contributes to the optimal CD8þ T cell response to epicutaneous peptide following TS and adjuvant application, we compared the CD8þ T cell response between wild type and TLR4 mutant mice. In these experiments, we employed an adoptive transfer system similar to the one described for the CD4þ T cell response. Again, adoptive transfer allowed a rapid and highly reproducible analysis of the
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local T cell response to epicutaneously administered antigen. Here OT1.1 TCR transgenic mice recognizing Ova8 were adoptively transferred into wild type C57BL/ 10ScSn (B10) and TLR4 knockout C57BL/10ScNCr mice. The mice were immunized one day following adoptive transfer with Ova8 and CT subsequent to TS. Responses in the skin draining LN were characterized 3 days post immunization. Enumeration of peptide specific CD8þ T cells was accomplished by simultaneous staining with Kb -Ova tetramer and anti-CD8 monoclonal antibody. Similar frequencies of peptide specific CD8þ T cells were identified in wild type (22.2 1.3%) and TLR4 knockout mice (18.7 2.6%), respectively (Fig. 2B). The extent of proliferation of adoptively transferred cells as determined by CFSE staining did not differ (data not shown). 3.3. Humoral immune responses to epicutaneous immunization with CT and TS are TLR4 independent We have previously demonstrated that TS enhances the antibody responses to epicutaneously applied protein antigen [22]. To study the role of TLR4 signaling in modulating humoral responses to epicutaneous immunization, the antibody response in wild type Balb/c mice was compared to that achieved in TLR4 mutant CC3HLpsd mice. Mice were immunized and boosted seven days later on the contra-lateral ear with 200 lg of full Ova protein + 25 lg of CT after TS the ear. Three
Fig. 3. The role of TLR4 signaling in humoral immune responses to epicutaneous protein immunization. Wild type (Balb/c) and TLR4 mutant mice (CC3H-Lpsd, TLR4def) were immunized with 200 lg ovalbumin protein + 25 lg CT after TS. Mice were primed and boosted 14 days later on the contra-lateral ear. Three weeks after priming serum was collected and assayed for Ova-specific antibodies using ELISA. Scatter grams of data from 3 individual mice per group are shown. Representative data from two independent experiments is represented.
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weeks after priming serum was collected and assayed for Ova-specific antibodies. OVA-specific total IgG, IgE, IgG1 , and IgG2a titers were assayed and found to be equivalently induced in both wild type and TLR4 mutant mice (Fig. 3). 3.4. Discussion The results of this study indicate that both CD4þ and CD8þ skin initiated cellular immune responses to peptide antigen are enhanced following tape stripping and in the presence of cholera toxin as adjuvant. This observation extends the previous reports of the value of this simple manoeuvre to enhance immune responses to skin-applied antigens [22,40,41] and details the adjuvant effect of mild skin injury. As TLR4 polymorphisms have been associated with susceptibility to gram )ve bacterial infection, the skin expression of TLR4 has been implicated in the cutaneous defence against gram )ve bacteria [42]. Murine LC have been shown to express TLR4 [9]. A number of endogenous TLR4 ligands have recently been described and Toll receptor signaling pathways have been implicated in the generation of alloreactive immune responses in the skin [17]. We therefore asked whether TLR4 signaling modulated responses to epicutaneous peptides or proteins in the presence of adjuvant and tape stripping. We found that CD4þ and CD8þ T cell responses were similarly evoked in wild type and TLR4 mutant mice. Similarly, humoral immune responses to epicutaneously applied protein antigen were also found to be TLR4 independent. We conclude that the adjuvant-like properties demonstrated by these two modalities in our epicutaneous immunization model do not stem from activation of innate immunity through TLR4. Consistent with this, Takeuchi et al. have recently demonstrated that the adjuvant properties of CT are TLR4 independent and result primarily from the engagement of the GM1-ganglioside receptor [43]. Interestingly, TLR4 signaling has recently been shown to enable Th2 immune responses to protein antigen in the lung. In this tissue, TLR4 signaling is initiated by low concentrations of lipopolysaccharide present in protein preparations [44]. Our results indicate that TLR4 signaling, in contradistinction to its role in facilitating pulmonary immune responses to protein antigen, may be dispensable in the initiation of skin immune responses. Indeed, keratinocytes, through their release of IL-1, have allied pathways of signaling tissue damage and infection. It has recently been suggested that cutaneous associated DC may downregulate TLR4 expression in order to prevent inordinate immune stimulation by commensal and saprophytic bacteria [45]. Further, the recognition of endogenous danger signals by TLR4 in the skin, an organ at constant risk of minor trauma and abrasion by virtue of its location at the hostenvironment interface, might lead to inappropriate loss
of tolerance to self. The conditions other than the presence of bacterial lipopolysaccharide that trigger productive TLR4 signaling in the skin remain to be determined. Further, given the recently demonstrated importance of MyD88 dependent signaling in cutaneous allograft rejection [17], other TLR receptors and ligands may have an important role in the control of skin immune responses.
Acknowledgments We thank Jacqueline D. Trudeau and Dr. Rusung Tan for the gift of the Kb -OVA reagent. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR). RK is supported by an award from the CIHR/Michael Smith Foundation for Health Research (MSFHR) Transplant Research Training Program. JPD is a Junior Scholar of the Arthritis Society and a UBC Galderma Scholar.
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