Cowhage can induce itch in the atopic dog

Abstract

Itch is a cardinal symptom of atopic dermatitis in humans and dogs. Until now, experimental induction of itch in dogs has proven difficult. The objectives of this study were to determine whether protease-rich spicules, protein extracts and the protease mucunain of the tropical legume cowhage provoked itch and inflammation when rubbed onto canine skin. Native spicules variably induced itch manifestations in about half of the dogs, while challenges with protease-deactivated spicules remained negative. The epicutaneous application of cowhage extract and mucunain after microneedle roller usage also induced pruritus and inflammation. Importantly, there was an interindividual inconsistency in pruritus and inflammation induction and also marked differences in pruritus intensity after challenge. In conclusion, cowhage spicules, protein-rich extracts and mucunain can all induce pruritus and inflammation in dogs as in other species, but the inconsistency of provocation is currently a limitation of this challenge type for future studies of pruritus in dogs.

Background

In dogs, atopic dermatitis (AD) is defined as an inflammatory and pruritic allergic skin disease with characteristic clinical features, associated most commonly with IgE antibodies to environmental allergens (1); it appears homologous to human extrinsic AD (2). Whereas localized atopic skin lesions can be reproduced experimentally in dogs by allergen painting (3,4), attempts to experimentally induce itch in dogs with non-allergenic substances have been unsuccessful. Indeed, intradermal injections of histamine, serotonin, tryptase, substance P, interleukin-2 (5) or mast cell degranulating anti-IgE antibodies (6) all failed to induce itch in dogs.

In the skin, the interplay between proteases and protease inhibitors mediates important roles for both desquamation and inflammation. Most biological activities of proteases are due to their binding to one of the four protease-activated receptors (PARs) (7). The PAR-2 receptor, expressed by cutaneous keratinocytes, fibroblasts, endothelial cells and afferent nerve endings (8), is likely relevant for the pathogenesis of human AD, as suggested by its high detection in lesional AD skin and by itch induction with PAR-2 agonists (7,9). Spicules covering the pods of the tropical legume cowhage (Mucuna pruriens) have long been shown to evoke intense itch and variable inflammation after contact with primate and rodent skin (10). Cowhage-induced itch is due to mucunain, a spicule-coating cysteine protease that activates PAR-2 and PAR-4 (11). Fifty years ago, a brief article reported that itch was inconsistently induced in dogs with a 5% cowhage-derived ointment (12), but there were no further reports of cowhage-induced pruritus in dogs.

Questions addressed

The objective of these studies was to develop a canine itch model using cowhage and proteases.

Experimental design

Cowhage spicule challenges

Eleven Maltese–Beagle atopic dogs were randomized for challenges with either spicules from native cowhage (M. pruriens) or cowhage with proteases inactivated by autoclaving for 100 min at 120°C (11). Spicules were applied one week apart, alternating between the left or right groin of each dog. Hair was removed with clippers 24 h beforehand. On the day of challenge, the stratum corneum was removed with ten tape strips, and a small pinch of spicules (approximately 100) was placed on the skin, rubbed for 5 s with a gloved digit and then covered with a drop of mineral oil to prevent spicule shedding. Itch was graded visually by a second investigator, blinded to the challenge type, using a composite pruritus visual analogue scale (CPVAS) (Figure S1). In this study, CPVAS values were added during four consecutive 5-min epochs (20 min total). A positive itch induction was defined as a CPVAS ≥ 0.5. Erythema was recorded visually as present or absent, 20 min after challenge.

Cowhage extracts and mucunain challenges

Five dogs were used for the following experiments separated by intervals longer than 7 days. After hair clipping and ten tape strips, a 1-mm microneedle roller (New Spa, Tallahasse, FL, USA) was criss-crossed twice, in two orthogonal directions, onto the skin of the left axilla. The following solutions were then applied epicutaneously and rubbed using a gloved digit: 100 μl of saline, 100 μL of a cowhage protein extract made as described previously (11) and 150 μg of recombinant mucunain in 100 μl of saline (Table 1). Pruritus, erythema and oedema were visually recorded as present or absent in the 20 min following challenge.

Table 1.

Epicutaneous challenges of dog axilla skin with saline, cowhage extracts and recombinant mucunain

	Pruritus	Erythema	Edema
100 μl saline EC after 10 TS and microneedle roller	0/5	0/5	0/5
100 μl cowhage extract (6 mg/ml protein) EC after 10 TS and microneedle roller	3/5	0/5	0/5
100 μl cowhage extract (6 mg/ml protein) EC after 10 TS and microneedle roller	3/5	3/5	4/5
100 μl cowhage extract (8 mg/ml protein) EC after 10 TS and microneedle roller	3/5	3/5	2/5
150 μg recombinant mucunain EC after 10 TS and microneedle roller	2/5	3/5	0/5

EC, epicutaneously; TS, tape strips.

Results

Cowhage spicule challenges

Itch was induced in five (45%) and none (0%) of the 11 dogs challenged with the native and inactivated spicules, respectively (Fig. 1). This difference was statistically significant (Fisher’s test, P < 0.05). Composite PVAS values were significantly higher after challenge with native compared with inactivated spicules (Wilcoxon test, P < 0.05). Erythema was seen in 7 of 11 dogs (64%) stimulated with the native cowhage, but in none with inactivated spicules (Fisher’s test, P < 0.05). Erythema scores and CPVAS values were not significantly correlated (Spearman’s r = 0.26; P > 0.05).

Figure 1.

Composite pruritus visual analogue scale values in 11 dogs after application of native (black) or denatured (white) cowhage spicules to the abdominal skin.

Cowhage extracts and mucunain challenges

Whereas the application of saline after tape strips and microneedle roller did not lead to visible inflammation or pruritus, that of the three applications of cowhage extract and recombinant mucunain did, but inconsistently so (Table 1). Indeed, the three challenges with the cowhage extract did not always induce pruritus manifestations, erythema or oedema in the same dogs.

Conclusions

In this study, we induced pruritus manifestation in approximately half of the 11 dogs with native cowhage spicules, but in none of those challenged with spicules covered with proteases that had been heat-denatured. Pruritus and inflammation were also induced with a protein extract made from cowhage spicules and with recombinant mucunain. Together, these observations suggest that in dogs, like in humans (11), cowhage-induced itch is due to the protease activity of mucunain but not to the mechanical pricking by spicules.

In contrast to humans (13), monkeys (14) and mice (15), in whom itch and scratching are consistently induced by the topical application of cowhage, the challenge of canine-haired skin with the native spicules or cowhage extracts variably caused pruritus. This inconsistency was found not only in the lack of itch induction in some dogs, but also in the high variability in the pruritus scores. This unpredictability of itch induction had been reported in a previous study, in which some dogs were found not to show signs after application of a 5% cowhage ointment (12). The reasons for this interspecies difference in the consistency of itch induction currently remain unknown.

Our observations suggest that proteases can induce itch and inflammation in dogs and that the PAR-2 pathway might have a role in atopic itch in dogs as it has in humans. However, because of their inconsistency in pruritus elicitation, cowhage challenges do not seem to be a valid model for itch studies in this species.