Phospholipase Cγ1 is required for normal irritant contact dermatitis responses and sebaceous gland homeostasis.

Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte-specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.

Modulation of cutaneous inflammation by angiotensin-converting enzyme.

Cutaneous neurogenic inflammation is a complex biological response of the host immune system to noxious stimuli. Present evidence suggests that zinc metalloproteases may play an important role in the regulation of neurogenic inflammation by controlling the local availability of neuropeptides, such as substance P (SP), that are capable of initiating or amplifying cutaneous inflammation after release from sensory nerves. To address the hypothesis that the dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) is capable of modulating skin inflammation, we have analyzed murine allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) using wild-type C57BL/6J (ACE(+/+)) or genetically engineered mice with a heterozygous deletion of somatic ACE (ACE(+/-)). In 2,4-dinitro-1-fluorobenzene-sensitized ACE(+/-) mice, ACD was significantly augmented in comparison to ACE(+/+) controls as determined by the degree of ear swelling after exposure to hapten. Likewise, systemic treatment of ACE(+/+) mice with the ACE inhibitor captopril before sensitization or elicitation of ACD significantly augmented the ACD response. In contrast, local damage and neuropeptide depletion of sensory nerves following capsaicin, injection of a bradykinin B(2), or a SP receptor antagonist before sensitization significantly inhibited the augmented effector phase of ACD in mice with functionally absent ACE. However, in contrast to ACD, the response to the irritant croton oil was not significantly altered in ACE(+/-) compared with ACE(+/+) mice. Thus, ACE by degrading bradykinin and SP significantly controls cutaneous inflammatory responses to allergens but not to irritants, which may explain the frequently observed exacerbation of inflammatory skin disease in patients under medication with ACE inhibitors.

Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis.

Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.

Effect of triterpenoids on the inflammation induced by protein kinase C activators, neuronally acting irritants and other agents.

In order to establish the mode of the anti-inflammatory activity of triterpenoids, 11 naturally occurring compounds were assayed on mouse ear oedema induced by the protein kinase C activators, mezerein, 12-O-tetradecanoylphorbol-13-acetate (TPA), two 12-deoxyphorbol-13-monoesters (13-tetradecanoate (DPT) and 13-phenylacetate (DPP)) and bryostatin 1, and by resiniferatoxin, xylene and arachidonic acid. The effects on bradykinin-induced paw oedema and on the rat skin inflammation caused by hydrogen peroxide were also examined. The oedema induced by mezerein and DPT was reduced to different extents by the triterpenoids administered epicutaneously (0.5 mg per ear). Against DPT-induced oedema, lupane and oleanane derivatives were the most effective compounds. Oleananes and lupanes possessing a carboxyl group were active against bryostatin 1-induced oedema. Most of the triterpenoids were ineffective against the neurogenic inflammation caused by resiniferatoxin and xylene. Many triterpenoids, especially oleanane and lupane alcoholic derivatives, were active against the plantar oedema induced by bradykinin and on the intradermal inflammation induced by hydrogen peroxide. In conclusion, the anti-inflammatory activity of triterpenoids may depend on inhibition of protein kinase C, without any involvement of neurogenic inflammatory mechanisms.

Protein kinase C isoform levels in normal and sodium dodecyl sulphate-irritated mouse skin.

Protein kinase C (PKC) comprises a family of related phospholipid-dependent serine/threonine protein kinases. PKC is important in signal transduction, regulating cell proliferation and differentiation. Recently, it has also been suggested that PKC may play a part in the pathogenesis of contact dermatitis. However, the expression of PKC isoforms in the skin of mice with irritant contact dermatitis (ICD) has not been examined. In this study, ICD was induced in mouse skin by applying 5%, 10% and 20% sodium dodecyl sulphate (SDS) in Finn chambers on the backs of mice and fixing with surgical dressings for 24 h. Depending upon the SDS concentration, mild to strong skin irritant reactions were observed 24 h after removal of the irritant patches. The intensity of the reactions increased with the increasing concentration of SDS. PKC isoforms alpha, beta, gamma and delta were all detected in normal mouse skin by Western immunoblotting. The specificity of the PKC isoforms detected was identified further by competitive Western immunoblotting. Compared with normal mouse skin treated with double-distilled water, the levels of PKC isoforms alpha, beta, gamma and delta in the SDS-irritated mouse skin was decreased by 24.8-75.8%. These results suggest that, in SDS-ICD, mouse skin PKC isoforms alpha, beta, gamma and delta are down-regulated. The significance of this decrease is under further investigation.

Down-regulation of protein kinase C isoforms in irritant contact dermatitis.

Protein kinase C (PKC) isoforms are important in cell signal transduction associated with regulation of cell proliferation and differentiation. In this study, alterations of PKC isoform levels in irritant patch test reactions were detected by Western immunoblots. 4 chemically and structurally different irritants, 4% and 8% hydrochloric acid (HCl); 1% and 2% sodium hydroxide (NaOH); 20% and 40% nonanoic acid (NON) in 1-propanol; and 5% and 10% sodium dodecyl sulfate (SDS) were applied on the backs of mice in Finn Chambers and fixed with surgical dressings. The patches were kept on the skin for 24 h and removed. 24 h after removal, mild erythema with thickening of skin without vesicles was observed in all irritated skin, except that 4% HCl and 1% NaOH treated skin showed unremarkable skin reaction. No visible skin reaction was detected in vehicle-treated skin. PKC isoform alpha, beta, gamma, and delta levels in irritated skin revealed a 10% to 65% decrease compared to vehicle treated skin. These results indicate that in HCl, NaOH, NON and SDS-induced irritation, activation of the PKC related cell signal transduction cascade may be involved, and that PKC mediated events may be a common phenomenon in irritant contact dermatitis.

Immunocytochemical demonstration of reduced Cu,Zn-superoxide dismutase levels following topical application of dithranol and sodium lauryl sulphate: an indication of the role of oxidative stress in acute irritant contact dermatitis.

Oxidative stress is known to be implicated in the inflammation induced by the anti-psoriatic irritant, dithranol. In this study, we wished to investigate whether this is reflected in the levels of the antioxidant enzyme, Cu,Zn-superoxide dismutase, as detected by quantitative immunocytochemistry, and whether similar changes also occur following exposure to an irritant not normally associated with reactive oxygen species generation, namely sodium lauryl sulphate. Analysis of biopsies from patch test sites revealed that significant reductions in the epidermal levels of Cu,Zn-superoxide dismutase were induced by both dithranol and sodium lauryl sulphate, although the time course of diminished activity was different for each irritant. Our findings suggest that oxidative stress plays a general role in the pathophysiology of acute irritant contact dermatitis.

Inducible nitric oxide synthase demonstrated in allergic and irritant contact dermatitis.

Eight allergic patch test reactions, eight irritant skin reactions induced by 3% sodium lauryl sulphate and six normal controls were biopsied. Biopsies were immunohistochemically stained with a mouse monoclonal antibody to inducible nitric oxide synthase (iNOS), and staining was quantified by computerised image analysis. Human chondrocytes induced to express iNOS were used as a positive control. A significant increase in iNOS was found in both irritant and allergic contact dermatitis. There were no differences in the distribution of expression of iNOS. The antibody used was confirmed by Western blotting not to cross-react with the endothelial isoform of nitric oxide synthase (NOS) but did cross-react with a 150 kDa protein, which may be neuronal NOS or an isoform of neuronal NOS.