Roderick C. McKenzie

Differential modulation of interleukin‐1α (IL‐1α) and interleukin‐1β(IL‐1β in human epidermal keratinocytes by UVB

Abstract Conflicting reports exist concerning ultraviolet‐B (UVB) effects on keralinocyte (KC) interleukin‐l (IL‐1) expression. To clarify the modulatery effects of UVB on IL‐1, the following study was undertaken. Normal human epidermal KCs cultured in a standard low Ca2+ and serum‐free medium were irradiated in quiescent phase with UVB. In this study, we used semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) to determine the mRNA level of interleukin‐1α (IL‐1α) and interleukin‐1β (IL‐β). After exposure to 100 or 300 J/m2 UVB, a transient increase in mRNA levels was observed within I hour for IL‐1α and 3 to 6 h for IL‐β Following this transient induction, mRNA levels for both IL‐1α and IL‐1β returned to steady‐state levels after 100 J/m2. After 300 J/m2 irradiation, IL‐1α and IL‐1β levels were downregulated compared to unirradiated cultures at 24‐h post‐irradiation. The half‐life for IL‐1α and IL‐1β was estimated using actinomycin D treatment. Both IL‐1α and IL‐1β mRNAs half‐lives (t1/2) decreased faster in irradiated cells (t1/2 = 30 minutes for IL‐1α and 2 h for IL‐1β) compared to unirradiated cells (t1/2= 1 h and 4 h, respectively). These results suggest that IL‐1α and IL‐1β mRNA expression are differentially regulated by UVB. In contrast to down‐regulation of mRNA levels, a significant increase in IL‐1α protein levels, measured by ELISA. was observed in culture supernatant from 6 h to 24 h after 300 J/m2 UVB irradiation. Cycloheximide treatment did not abrogate this increase in IL‐1α protein level. Since this dose of UVB irradiation decreased the stability of IL‐1α and IL‐1β mRNA, this suggests that the release of IL‐1α after UVB irradiation was due to leakage from UVB‐damaged cells and not from de novo protein synthesis.

The role of cis-urocanic acid in UVB-induced suppression of contact hypersensitivity

Ultraviolet light B (UVB) is well recognized to suppress the contact hypersensitivity (CHS) response and it has been postulated that cis-urocanic acid (UCA) is a mediator of the immunosuppression. This study was designed to examine the effect of UCA on CHS and to clarify its role in UVB-induced immunosuppression in C57BL/6 mice. Intradermal injection of 0.5-50 micrograms cis-UCA into the ear 2 h before DNFB sensitization resulted in a 60-70% reduction of CHS assessed by ear swelling, whereas trans-UCA did not have a significant effect on CHS except at a high dose (50 micrograms) which showed a 20-40% suppression. Intraperitoneal injection of anti-cis-UCA antibody before administration of cis-UCA abrogated the suppression. To examine the effect of UCA on UVB-induced immunosuppression, some mice were pre-treated with anti-cis-UCA antibody and then exposed to UVB (960 J/m2). After 3 days the mice were sensitized either on the irradiated abdominal skin or on the unirradiated dorsal surface of the right ear followed by the challenge on the left ear. The CHS response was significantly suppressed in UVB-irradiated mice both locally (abdominal sensitization, suppression was 45%, P < 0.001) and systemically (ear sensitization, suppression was 53%, P < 0.0025). The CHS response was partially restored in both abdominal sensitized mice and ear sensitized mice by pre-treatment with anti-cis-UCA antibody. These results confirmed the immunosuppressive effects of cis-UCA on CHS and suggest that cis-UCA plays a role in UVB-induced local and systemic immunosuppression.

Interleukin-8 and melanoma growth-stimulating activity (GRO) are induced by ultraviolet B radiation in human keratinocyte cell lines

Abstract Ultraviolet radiation can induce the transcription and release of cytokines From keratinocytes (KCs). These cytokines have the potential to modulate local and systemic immunologic responses. In this paper we report that northern blotting showed that human KC and KC lines expressed a 1.2‐1.4 kb transcript for the chemokine and melanoma growth‐stimulatory protein, GRO‐α and that ultraviolet B radiation (UVB) could upregulate the expression of GRO‐α mRNA and protein in the KC line A431. The GRO‐α gene response to UVB was maximal at 48h post‐irradiation with 70 J/m2. Reverse transcription‐polymerase chain reaction (RT‐PCR) revealed a 4.5‐fold increase in GRO‐α mRNA over basal levels (p<0.001). GRO‐α protein was measured in the culture media by enzyme‐linked immunosorbent assay (ELISA). Media from unirradiated cultures contained 1166±83 pg/ml GRO‐α protein. After UVB, a time‐dependent increase in GRO‐α protein was seen in the culture media from 6‐48h. At 48h post‐irradiation the GRO‐α protein content was 27583±678 pg/ml, or 23 times the basal level. This protein release could be inhibited by 70% when the cells were pre‐incubated with 10 μg/ml interleukin‐1 receptor antagonist (IL‐1RA). We also show that another potent leukocyte chemoattractant, Interleukin‐8 (IL‐8), was induced in A431 cells by UVB. The induction of IL‐8 mRNA began as early as 3h post‐irradiation, when it reached twice basal levels (p<0.05) and reached 4.5‐fold basal levels at 48h post‐irradiation (p<0.005). Analysis of the conditioned media from the irradiated cells by ELISA revealed that IL‐8 protein accumulation was increased from 162± 16 pg/ml (unirradiated cultures) to 993± 120 pg/ml 24h post‐irradiation. The accumulation increased to 1383± 178 pg/ml, or 9‐fold the basal level by 48h post‐irradiation. Thus keratinocytes are capable of generating chemoattractant factors which enhance melanoma growth after UVB irradiation, which may contribute to the inflammatory infiltrate found in the dermis of sunburned skin.

Inhibition of ultraviolet B radiation-induced interleukin 10 expression in murine keratinocytes by selenium compounds

Summary Background Selenium is an essential trace nutrient necessary for the normal function of the immune system. Selenium compounds protect mice against ultraviolet (UV) B‐induced tumours, probably by preventing oxidative damage to the host skin cells and to the host immune system. One possible mechanism of protection is that selenium can prevent oxidative stress‐induced release of cytokines such as interleukin (IL)‐10, which could suppress cell‐mediated immunity.

Lymphocyte function‐associated antigen‐1 is required for maximum elicitation of allergic contact dermatitis

Cell adhesion molecules (CAMs) are known to play an important role in cell‐mediated immunity and inflammation. To investigate the importance of one of the CAMs, lymphocyte function‐associated antigen‐1 (LFA‐1) in allergic contact dermatitis (ACD), a specific anti‐LFA‐1 monoclonal antibody was injected into the ears of mice after sensitization but prior to challenge with dinitrofluorobenzene (DNFB). BALB/c mice were sensitized on the dorsal skin with DNFB, and challenged on the ears 6 days later. The effect of the antibody on the elicitation phase of contact sensitization was determined by its intradermal injection into the pinnae of the mice at doses of 1–40μg. At 24 and 48 h after challenge, ear swelling was dose‐dependently suppressed by injection of LFA‐1 antibody at doses of 2–40 μg, which are less than 10% of the systemic dose required for effective suppression of delayed‐ type hypersensitivity in previous studies. Maximal inhibition of ear swelling (44%) was observed after injection of 20 μg of anti‐LFA‐1 antibody. Inhibition of ear swelling was accompanied by a reduction in dermal oedema and leucocyte infiltration into the dermis. Our results suggest that LFA‐1 plays a significant role in the elicitation of ACD reactions, and also that our test system is a sensitive and useful means of evaluating the blocking effect of antibody to adhesion molecules thought to be involved in the elicitation phase of ACD.

cis‐Urocanic Acid does not Induce the Expression of Immunosuppressive Cytokines in Murine Keratinocytes

Abstract trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10–100 μg/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-β and tumor necrosis factor (TNF)-α messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, β-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-α mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 μg/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-α and TGF-β, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.

Effects of contact sensitizers neomycin sulfate, benzocaine and 2,4-dinitrobenzene 1-sulfonate, sodium salt on viability, membrane integrity and IL-1α mRNA expression of cultured normal human keratinocytes

The toxic effect of three potential contact sensitization chemicals [the aminoglycosidic antibiotic neomycin sulfate, the local anaesthetic benzocaine and the primary sensitizer 2,4-dinitrobenzene 1-sulfonate, sodium salt (DNBS)], on cultured human keratinocytes was examined. The three chemicals were compared with respect to their cytotoxic potential (determined by crystal violet staining assay), their membrane disruptive potential ([3H]arachidonic acid release assay), and their effects on interleukin 1 alpha (IL-1 alpha) mRNA expression [reverse transcription-polymerase chain reaction (RT-PCR)]. At the concentrations used, neomycin sulfate (0.004-0.32%) and benzocaine (0.0165-0.165%) did not show relevant cytotoxicity or membrane perturbation. On the other hand, DNBS (0.001-1%) caused a significant dose-dependent cytotoxic response at concentrations higher than 0.1%, while the [3H]arachidonic acid release assay indicated absence of membrane perturbation activity in all the range of DNBS concentrations examined. The effects of the three sensitizers on IL-1 alpha mRNA expression were varied; neomycin sulfate caused a dose-dependent induction of IL-1 alpha mRNA, benzocaine did not significantly affect its signal, and DNBS suppressed IL-1 alpha gene expression.

Cutaneous Toxicity of Surfactants in Normal Human Keratinocytes Assessed by Cytotoxicity, Arachidonic Acid Release, and Regulation of Interleukin-lα mRNA

Development and validation of in vitro methods for cutaneous irritation testing is aimed at establishing simple and reliable assays that eliminate the need for animals in toxicity testing. It is therefore important to identify in vitro end points that are predictive of in vivo toxicity. Identification of proinflamma-tory mediators and cytokines in keratinocytes may represent an early event in cutaneous inflammation. Three end points were evaluated: cytotoxicity [(crystal violet staining (CVS)], the release of [3H]arachidonic acid (AAR), and the regulation of the proinflammatory cytokine interleukin-1α (IL-lα) message in keratinocytes as a potential in vitro assay for surfactants. Cultured normal human epidermal keratinocytes were treated with various concentrations of three surfactants (SDS, Triton X-100, and Tween 20) of different in vivo potencies. The CVS50 1 h (surfactant concentrations yielding 50% viability) and AAR50 (surfactant concentrations causing 50% release of labelled arachidonic acid), both...

Inhibition of Cytokine Gene Expression in Mouse Skin by Subcutaneous Injection of Cyclosporine

Cyclosporine A (CsA) has been shown to be an effective therapeutic agent for a wide variety of cutaneous diseases yet its exact mechanism of action is still unclear, although one well-defined effect of CsA is the inhibition of T-cell-derived cytokine expression. We recently demonstrated in vitro that CsA inhibits cell proliferation and suppresses cytokine gene expression in keratinocytes. In this study, we report the in vivo effects of CsA on skin cytokine gene expression as determined by reverse-transcriptase polymerase chain reaction. C57BL6 mice (female, 8-10 weeks old) were subcutaneously injected with CsA in olive oil (0, 5 and 10 mg/kg) every other day for 3 weeks. Treatment with 5 mg/kg CsA inhibited both interleukin (IL)-1 alpha and tumor necrosis factor alpha gene expression by about 70 and 90%, respectively, relative to vehicle control levels. However, IL-6 gene expression did not significantly change. Injection of 10 mg/kg CsA inhibited expression of all three genes by 80-90% relative to control levels. These data show that CsA can inhibit constitutive cytokine gene expression in mouse skin.