Published online before print November 5, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* St. John’s Institute of Dermatology, King’s College London, London, United Kingdom;
Syngenta Central Toxicology Laboratory, Macclesfield, Cheshire, United Kingdom;
School of Dentistry, Cardiff University, Cardiff, United Kingdom; and
Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow, United Kingdom
1 Correspondence: St. John’s Institute of Dermatology, Guy’s Hospital, St. Thomas Street, London, SE1 9RT, UK. E-mail: richard.groves{at}kcl.ac.uk
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
were equally able to mobilize LC from epidermis, indicating that migration in response to these cytokines is not dependent on IL-18 and suggesting that IL-18 acts upstream of these cytokines in the initiation of antigen-induced LC migration. Moreover, IL-1β but not IL-18 was able to rescue the defective CHS response observed in caspase-1–/– mice, which have no functional IL-1β or IL-18. These data indicate that IL-18 is a key proximal mediator of LC migration and CHS, acting upstream of IL-1β and TNF-, and may play a central role in regulation of cutaneous immune responses.
Key Words: oxazolone • lymph nodes • caspase-1
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To present antigen encountered in the skin to the immune system, LC must migrate from epidermis to draining lymph nodes [3
]. A considerable body of data indicates that this migratory response is under the control of at least two cytokines derived from epidermal cells, IL-1β and TNF-. Intradermal injection of TNF- or IL-1β into murine skin results in migration of LC from epidermis and their subsequent accumulation in draining lymphoid tissue [4
, 5
]. Similar experiments in human skin confirm that TNF- is also a potent initiator of LC migration from epidermis in man [6
]. Moreover, neutralizing antibodies against either cytokine are capable of blocking migration of LC following topical application of antigen [5
]. That this inhibition is of functional relevance is exemplified by the observation that intradermal injection of anti-IL-1β antibody prior to sensitization can suppress the murine CHS response effectively [7
].
We have shown previously that the cysteine protease caspase-1 plays an important regulatory role governing murine LC migration and CHS [8
]. This enzyme is responsible for processing IL-1β and the structurally related cytokine IL-18 from their inactive precursor forms into mature bioactive cytokines capable of receptor binding and initiation of their respective signaling pathways. We have begun to investigate the role of IL-18 in LC migration and have shown that like IL-1β and TNF-, intradermal injection of IL-18 is capable of initiating LC migration from epidermis in mice, with subsequent accumulation of mature dendritic cells (DC) in draining lymph nodes [9
]. Moreover, migratory LC can release functional IL-18 [10
], and antibodies against IL-18 are able to inhibit antigen-induced LC migration [9
]. Taken together, these data strongly suggest that like IL-1β and TNF-, IL-18 is involved in the regulation of LC migration from epidermis to lymph node. Compatible with this is the observation that human DC express functional IL-18R [11
].
Current data suggest that IL-1β and TNF- are required for LC migration, and their interdependence is complex. Thus, antibodies to TNF- are able to inhibit IL-1β-induced LC migration, and anti-IL-1β antibodies are able to inhibit TNF--induced migration [5
]. To date, a more proximal cytokine signal has not been identified, and in the current study, we sought to determine whether IL-18 might provide such a signal. We were also interested in defining the role played by IL-18 in cutaneous inflammatory responses typified by CHS and have compared this with nonantigen-specific inflammation induced by skin irritants.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(specific activity 2x108 U/mg by L929 cytotoxicity assay) were purchased from R&D Systems (Oxon, UK), PeproTech EC Ltd. (London, UK), and Genzyme Diagnostics (West Malling, Kent, UK), respectively. Cytokines were diluted in sterile PBS containing 0.1% BSA as carrier and were administered locally by intradermal injection into ear pinnae (30 µl). Biotin-conjugated rat anti-mouse I-Ad/I-Ed mAb (clone 2G9) and streptavidin-FITC conjugates were purchased from PharMingen (San Diego, CA, USA). All other reagents, including contact sensitizers 2,4-dinitrofluorobenzene (DNFB) and oxazolone (OX) and skin irritant sodium lauryl sulfate (SLS), were purchased from Sigma Chemicals Ltd. (Poole, Dorset, UK) unless otherwise indicated.
Animals
IL-18 knockout (IL-18–/–) and wild-type (WT) mice have been described previously in detail [12
]. Caspase-1–/– and WT control mice were the kind gift of Dr. Winnie Wong (BASF Corp., Worcester, MA, USA) and have also been described in detail [13
, 14
]. All animals were housed in a conventional animal facility with a 12-h light/dark cycle. Mice were used between 6 and 12 weeks of age and in individual experiments, were age-matched to within 2 weeks. All experiments were carried out under the provisions of the Animals (Scientific Procedures) Act 1986, UK.
CHS
Groups of at least three mice were sensitized by application of 150 µl 0.5% DNFB in 4:1 acetone/olive oil (AOO) to abdominal skin. Five days later, 25 µl 0.25% DNFB was applied to dorsal and ventral surfaces of the right ear and AOO alone to the left ear. At intervals thereafter (24, 48, and 72 h), challenge-induced ear swelling (relative to the AOO-treated ear) was measured using a modified, spring-loaded micrometer (Mitutoya Inc., Japan). In parallel experiments, OX was used in the same volumes and diluent at concentrations of 1% (sensitization) and 0.5% (elicitation). In cytokine rescue experiments, the dorsal surface of the right ear was pretreated by intradermal injection of 30 µl IL-18 (50 ng), IL-1β (50 ng), or diluent alone 30 min prior to sensitization with allergen (30 µl 1% OX or 0.5% DNFB). Five days later, the left ear of the mice was challenged with 30 µl 0.5% OX or 0.25% DNFB, respectively, and ear swelling (relative to prechallenge) was measured as described above. All experiments were repeated at least three times, and representative data are shown.
Irritant dermatitis
Groups of at least three mice received 25 µl of the skin irritant SLS (10%), dissolved in dimethylformamide (DMF) on the dorsal and ventral surfaces of the right ear and DMF alone to the left ear. At intervals thereafter (2, 6, and 24 h), ear swelling (relative to the DMF-treated ear) was measured using a modified, spring-loaded micrometer (Mitutoya Inc.). All experiments were repeated at least three times, and representative data are shown.
Immunohistochemical staining of LC
LC numbers and morphology were evaluated en face in epidermal sheets prepared as described previously [8
]. Briefly, ears were harvested and split into dorsal and ventral halves using fine forceps. Dorsal ear halves were then incubated in 0.02 M EDTA at 37°C for 90 min to allow separation of epidermis and dermis. Epidermal sheets were peeled away carefully from dermis, washed twice in PBS, fixed in acetone at –20°C for 20 min, and washed again. Epidermal sheets were then incubated with biotinylated rat anti-mouse I-Ad/I-Ed antibody diluted to 5 µg/ml in PBS with 0.1% BSA for 45 min at room temperature, washed, and subsequently incubated for a further 45 min with FITC-labeled streptavidin diluted 1:100 in PBS with 0.1% BSA. Following further washing, epidermal sheets were mounted whole in glycerol, slides were coded, and LC were counted in 10 high-power fields in the central portion of the ear using an eye-piece with a calibrated grid (0.25 mmx0.25 mm at x40 magnification) on a fluorescence microscope (Axiophot, Zeiss, New York, NY, USA). The evaluator was blinded to the nature of slides examined. Results are expressed as mean ± SEM numbers of LC/mm2 epidermis.
Statistical analyses
The statistical significance of differences of means of experimental groups was calculated using a two-tailed Student’s t-test. Mean differences were considered to be significantly different when P < 0.05. All data are presented as mean ± SEM, and error bars are indicated on figures where SEM >5% of the mean.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (25K): [in a new window] |
Figure 1. The CHS response is suppressed in IL-18–/– mice, which were sensitized with OX or DNFB and challenged 5 days later on one ear as described. Marked ear swelling developed in WT mice ( ) but was suppressed significantly in IL-18–/– mice ( ); *, P < 0.05. Challenged but unsensitized control animals ( , WT Uns;  , IL-18–/– Uns) were used as contols; n = 3 mice/group. Results are representative of three independent experiments.
|
 View larger version (17K): [in a new window] |
Figure 2. ICD is normal in IL-18–/– mice. Ten percent SLS (25 µl) in DMF was applied to ear skin of WT and IL-18–/– mice as described. Ear swelling developed in both groups, maximal at 24 h, and no significant difference could be identified between WT and IL-18–/– mice; n = 3 mice/group. Results are representative of three independent experiments.
|
 View larger version (16K): [in a new window] |
Figure 3. LC migration in response to allergen stimulation is impaired in IL-18–/– mice. LC numbers in epidermal sheets prepared following topical application of skin allergens DNFB or OX and the skin irritant SLS were enumerated by immunohistochemical staining for MHC class II antigens. Application of DNFB or OX resulted in a significant fall in epidermal LC numbers in WT mice, but this decline was absent in IL-18–/– mice. In contrast, application of SLS induced a marked fall in epidermal LC density in WT and IL-18–/– mice. Results are expressed as the mean number of LC/mm2 (±SEM); n = 4 ears/group; *, P < 0.05; **, P < 0.01, compared with vehicle control. Results are representative of three independent experiments.
|
 View larger version (39K): [in a new window] |
Figure 4. Intradermal administration of rMuIL-18 prior to sensitization with oxazolone fully restores CHS in IL-18–/– mice. Ear skin of WT and IL-18–/– mice was pretreated locally by intradermal administration of 30 µl IL-18 (50 ng), IL-1β (50 ng), or diluent alone, 30 min prior to sensitization with 0.5% OX, and 5 days later, ears were challenged as described. IL-18–/– mice pretreated by intradermal injection of diluent alone or untreated showed suppressed ear swelling as expected, but this attenuated response was restored to levels seen in WT mice following pretreatment with IL-18 or IL-1β. Note that neither IL-18 nor IL-1β pretreatment enhanced ear swelling in WT mice; n = 5 mice/group. ns, Not significant; **, P < 0.01, compared with vehicle control. Results are representative of three independent experiments.
|
IL-18 acts proximally to IL-1β and TNF- in initiation of LC migration and murine CHS
We were next interested in determining the relationship between IL-18 and the other cytokines (notably, IL-1β and TNF-), implicated previously in induction of LC migration. Thus, 50 ng IL-1β, IL-18, or TNF- was injected into IL-18–/– or WT ear skin as described previously, and remaining epidermal LC at 4 h (IL-1β and IL-18) or 30 min (TNF-) were enumerated by immunostaining of epidermal sheet preparations. All three cytokines induced an equivalent reduction of epidermal LC numbers in IL-18–/– and WT mice (Fig. 5
), indicating that the migratory response to these cytokines is not dependent on secondary secretion of IL-18.
 View larger version (12K): [in a new window] |
Figure 5. Intradermal injection of TNF-, IL-1β, or IL-18 induces LC migration in WT and IL-18–/– mice, which received 50 ng MuTNF-, IL-1β, or IL-18 by intradermal injection into ear pinnae. Control mice were untreated or received diluent alone. Epidermal sheets were prepared 4 h (IL-1β and IL-18) or 30 min (TNF-) after treatment, and LC numbers were determined following immunofluorescent labeling of MHC class II-positive cells. Administration of all three cytokines resulted in a similar decline in LC numbers in WT and IL-18–/– mice. BSA alone was without effect. Results are expressed as the mean number of LC/mm2 (±SEM). **, P < 0.01, compared with vehicle control; n = 4 ears/group. Results are representative of three independent experiments.
|
 View larger version (40K): [in a new window] |
Figure 6. IL-1β but not IL-18 is able to restore the normal CHS in caspase-1–/– mice. MuIL-1β or IL-18 (50 ng) in 0.1% BSA was administered by intradermal injection locally to ear pinna skin of WT and caspase-1–/– mice 30 min prior to sensitization with 1% OX on the same ear. Five days later, the contralateral ear pinna was challenged with 0.5% OX, and swelling was measured 24 h after challenge. WT mice exhibited marked ear swelling, whereas caspase-1–/– mice without cytokine pretreatment showed significant suppression of this response. Preinjection of caspase-1–/– ear skin with IL-1β fully restored normal ear swelling, but IL-18 and BSA were without effect. Neither cytokine affected ear swelling significantly in WT mice. *, P < 0.05; n = 5 mice/group. Data are representative of three independent experiments.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, two cytokines that have previously been shown to govern LC migration from epidermis to lymph node. IL-18 bears strong structural relationships to IL-1β [15 ]. Both molecules require proteolytic cleavage by caspase-1 to generate the mature functional cytokine from an inactive precursor form [16 ]. We have demonstrated previously that caspase-1 plays a key regulatory role in LC migration [8 ], presumably by modulation of processing and release of IL-1β and IL-18. Within the epidermal microenvironment, keratinocytes [17 ] and LC [18 , 19 ] express IL-18 and caspase-1, and thus, either cell may be the source of this signal. IL-18 may also be released in a noncaspase-1-dependent manner following the ligation of Fas ligand (FasL) [20 ]. Keratinocytes have been shown to express Fas and FasL in a number of inflammatory states [21 , 22 ], and thus, this mechanism may also be of importance in the generation of mature IL-18 in the context of human inflammatory skin disease.
Our data indicate that IL-18 has a critical role in the regulation of inflammation within the skin. Thus, it is unsurprising that unregulated, transgenic overexpression of IL-18 in skin leads to uncontrolled inflammation [23 ]. Interfering with IL-18 activity is therefore an attractive therapeutic option in inflammatory skin disease, and pharmacologic use of IL-18-binding protein has been shown to inhibit contact sensitization in mice [24 ]. The data presented in this paper would suggest that because of the proximal signaling role played by IL-18 in LC mobilization, this approach would have significant advantages over other targets for cytokine blockade.
It has been well-established that IL-1β and TNF- are required for the induction of LC migration in response to allergen [4
, 5
, 25
, 26
]. The current hypothesis is that following allergen contact with epidermis, LC release IL-1β, which stimulates production of TNF- by neighboring keratinocytes that express the signal-transducing type-1 IL-1R. LC, which express IL-1R1 [27
] and TNFR2 [28
], are stimulated by the presence of both cytokines to enter the migration pathway, with loss of E-cadherin expression and induction of matrix metalloproteinase-9 and other markers associated with maturation and antigen presentation [3
]. Both of these cytokines can induce the expression of the other under appropriate conditions [29
], and it is therefore difficult to determine their relative importance to LC migration. However, the data presented herein, taken together with antibody inhibition studies published previously [9
], indicate clearly that IL-18 acts upstream of TNF- and IL-1β.
Our current data do not define the source of IL-18 release, although keratinocytes or LC may be responsible. Keratinocytes [30
, 31
] and DC [11
] have been shown to express and signal through the IL-18R, and DC are responsive to this cytokine in other ways [32
]; thus, either may be the potential responder cell within the epidermis. Alternatively, IL-18 may stimulate the release of preformed TNF- from mast cells, which could promote LC migration subsequently [33
]. That IL-18 should be a proximal mediator in LC migration is in accord with previous data indicating that IL-18 can induce synthesis and release of a number of other cytokines, including TNF-, IL-6, IL-8, and IL-1β. In some cells, this is a direct effect [34
]; in others, it requires the presence of TNF- [35
]. Although formal proof from cellular studies is awaited, it is therefore highly likely that IL-18 could induce production of both downstream cytokines involved in regulation of LC migration within the epidermis.
Our finding that IL-18 has no apparent role in ICD is of interest for a number of reasons. First, it indicates that the role for IL-18 in CHS is not simply part of a general requirement for IL-18 in skin inflammation. Second, it implies that IL-18, which is stored preformed in considerable quantities in murine and human keratinocytes [17
], is not immediately bioactive. Were IL-18 directly proinflammatory in skin, we would have predicted that at least part of the ICD ear-swelling response, which occurs as a result of release of preformed, proinflammatory cytokines such as IL-1 from damaged keratinocytes [36
], would have been compromised in IL-18–/– mice. This was not the case. An alternative explanation is that bioactive IL-18 does not have a direct, proinflammatory effect in skin, although we consider this unlikely, as IL-18 is capable of inducing expression of endothelial adhesion molecules [37
], as well as promoting synthesis and release of other cytokines including TNF-, which is known to be proinflammatory when released in the cutaneous microenvironment [34
]. Finally, and perhaps most important, the data suggest that the presence of IL-18 is not obligatory for LC migration in all circumstances. Although the mechanisms and biological role of LC migration in irritant dermatitis are unclear, there is increasing evidence that preformed IL-1 release is important in initiating this response [38
]. It therefore seems likely that given such a signal, LC are able to avoid the requirement for IL-18 in migration in some situations.
A considerable body of evidence is now accumulating that indicates that IL-18 plays a key role in epithelial defense against microorganisms. Several skin-specific viruses (e.g., human papilloma virus [39 , 40 ] and pox viruses [41 , 42 ]) have evolved means of suppressing IL-18 in the skin by production of inhibitory binding proteins analogous to the endogenously produced IL-18-binding protein. Moreover, mice deficient in IL-18 are prone to experimental infection by a number of organisms including Leishmania and Staphylococcus aureus [12 ], both of which are major skin pathogens. A central role for IL-18 in the initiation of LC migration following antigen penetration of epidermis fits in well with this role and underscores the key role played by IL-18 in epithelial defense.
Received June 21, 2004; revised August 8, 2007; accepted August 30, 2007.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Arch. Dermatol. Res. 289,277-284[CrossRef][Medline] and interleukin-1β for migration Immunology 92,388-395[CrossRef][Medline] induces Langerhans cell migration in humans Br. J. Dermatol. 141,192-200[CrossRef][Medline]- and IL-1β-dependent mechanism Immunology 102,323-330[CrossRef][Medline] Immunology 81,395-401[Medline]
release by murine dendritic cells J. Immunol. 167,957-965-inducing factor) induces IL-8 and IL-1β via TNF production from non-CD14+ human blood mononuclear cells J. Clin. Invest. 101,711-721[Medline]
B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways J. Biol. Chem. 276,37069-37075 and IL-1β during irritant- and allergen-induced cutaneous immune responses Toxicol. Appl. Pharmacol. 182,126-135[CrossRef][Medline] production in human peripheral blood mononuclear and NK cells J. Immunol. 167,497-504
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
