DOI: 10.1111/j.1365-3164.2010.00938.x
Stratum corneum removal facilitates experimental sensitization to mite allergens in atopic dogs
Thierry Olivry*,†, Jessica Wofford*, Judy S. Paps* and Stanley M. Dunston* *Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA † Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA Correspondence: Thierry Olivry, Department of Clinical Sciences, College of Veterinary Medicine, Veterinary Teaching Hospital, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA. E-mail:
[email protected] Sources of Funding This study was self-funded. Conflict of Interest No conflicts of interest have been declared.
Abstract In humans with atopic dermatitis and in mouse models of IgE-mediated allergic diseases, evidence is mounting that the stratum corneum (SC) provides an important barrier against environmental allergens. At this time, it is not known whether the SC has a similar role in dogs, especially in those with atopic dermatitis. The objectives of this pilot study were to determine whether SC removal led to earlier and stronger sensitization of atopic dogs to Dermatophagoides farinae (Df) house dust mites. Five Maltese-beagle atopic (MBA) dogs were sensitized epicutaneously after the SC was removed with ten tape strips (TS group), while sensitization was done without tape strips in five other MBA dogs (nontape stripping; NTS group). During this 16 week study, sensitization was assessed with allergen-specific IgE serology, intradermal testing with Df allergens and determination of stimulation indices of blood mononuclear cells cultured with Df and stained for CD4 and the activation markers CD25 or CD30. Compared with dogs from the NTS group, those of the TS group exhibited earlier rises in Df-specific IgE serum levels, usually had higher allergen-specific IgE titres, showed higher intradermal test reactivity and had earlier increases and higher percentages of CD25- or CD30-positive activated allergen-specific peripheral CD4-positive T lymphocytes. These observations implicate a role of the SC as a barrier limiting sensitization to exogenous allergens in this experimental atopic dog model. Accepted 1 September 2010
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Introduction An intact skin barrier is critical to prevent desiccation from excessive water loss and penetration of exogenous substances detrimental to the body. The main responsibility for assuming this protective role lies with the outermost epidermal layer, the stratum corneum (SC), a zone composed of cornified keratinocytes (corneocytes) surrounded by complex lipid lamellae.1,2 In recent years, the SC and its barrier function roles have taken centre stage in the pathogenesis of atopic dermatitis (AD) in humans.2–4 This focus on epidermal barrier dysfunction stemmed from the study of human genetic disorders that affect the skin barrier and are associated with AD. For example, genetic variations leading to the hyperactivity of SC desquamating enzymes, or to the reduction of function of SC protease inhibitors, all appear to enhance barrier dysfunction and cause AD or the AD-like Netherton syndrome.4,5 The main link between barrier dysfunction and AD occurred with the unexpected discovery that mutations in the filaggrin gene (FLG) predisposed not only to ichthyosis vulgaris but also to AD.6,7 A recent systematic review and meta-analysis established that FLG gene null mutations increased the risk of developing allergic sensitizations, AD, as well as allergic rhinitis or asthma associated with AD.8 Filaggrin gene null mutations are suspected to cause human AD by reducing filaggrin-derived components of the ‘natural moisturizing factor’, decreasing SC acidity and leading to an enhanced activity of desquamating proteases and ⁄ or altering the flattening of corneocytes secondary to the impaired aggregation of the keratin cytoskeleton, this abnormal cellular shape thereby impacting the impermeability of the SC.2,4 The importance of filaggrin in maintaining a functional skin barrier is supported by the study of flakytail mice, which exhibit a phenotype of diffuse mild scaling and elevated serum IgE due to a homozygous Flg mutation.9 These filaggrin-deficient mice have an abnormal skin barrier function, low-grade dermal inflammation, enhanced bidirectional penetration of water-soluble tracers in the SC, structural defects in the extrusion of lipid-secreting lamellar bodies and a reduction in inflammatory thresholds to both irritants and contact allergens. Importantly, these mice exhibit an AD-like dermatosis when challenged with lower hapten doses than wild-type mice.10 As pro-allergic Th2 cytokines, such as interleukin (IL)-4 and 13, not only reduce the expression of filaggrin but also that of the cornified envelope proteins involucrin and loricrin in humans with AD,11–13 it is hypothesized that
ª 2010 The Authors. Journal compilation ª 2010 ESVD and ACVD, Veterinary Dermatology, 22, 188–196.
Stratum corneum removal in atopic dogs
genetic barrier defects lead to atopic cutaneous inflammation that further results in decreased SC protein production and an aggravated barrier dysfunction. This theory forms the basis of the new paradigm of ‘outside– inside–outside’ pathogenesis of human AD.2 There is increasing evidence that SC and upper epidermal defects might exist in the skin of atopic dogs. At this time, it remains unknown whether these anomalies are seen in every dog with AD, if they are transient or permanent, or if they are present before and ⁄ or after the development of active skin lesions. Arguments in favour of an abnormal barrier include the observations that SC lipid lamellae are disorganized, shorter and thinner in the skin of atopic dogs compared with those of normal canine skin,14–16 that abnormal lamellar bodies are present in skin lesions of some dogs with experimentally induced AD,16 and that decreased amounts and an abnormal composition of epidermal ceramides exist in the SC of atopic dogs.17,18 At the time of writing, genetic mutations of canine FLG have not been reported in dogs. Nevertheless, in a recent study of filaggrin immunostaining in the epidermis of dogs with natural and spontaneous AD, four of 18 dogs (22%) had normal N-terminal but no detectable C-terminal filaggrin. These observations suggest the existence of loss-of-function mutations of FLG leading to the translation of a truncated filaggrin protein lacking a functional C-terminus in some dogs with AD.19 Even if a barrier dysfunction were present in the skin of dogs with AD, it is not known whether such a defect would enhance the risk for development of sensitization to environmental allergens, or if it would augment signs of allergy after epicutaneous allergen contact. To test the hypothesis that an experimental barrier defect enhances the development of atopic sensitization, we monitored immunological parameters following the epicutaneous application of Dermatophagoides farinae (Df) house dust mites on Maltese-beagle atopic (MBA) dogs at sites where the SC was present or had been removed by tape stripping.
tivity to food allergens and to develop AD-like signs upon challenge with such allergens.20,21 Moreover, our previous studies confirmed the easy sensitization of these dogs to Df allergens and the development of AD-like skin lesions following epicutaneous mite allergen challenges.22 These dogs do not usually develop skin lesions, apart from occasional bacterial folliculitis, if not provoked with an allergen to which they are hypersensitive. There were three males and two females in one group and two males and three females in the other. The mean age was 7.6 years (range: 3–11 years). This study was designed as an experiment of two groups of five MBA dogs of similar average ages (Figure 1).
Sensitization protocol In the first group (tape strip; TS), before each epicutaneous allergen application, hair was clipped 2 days before the stratum corneum was removed from the right axilla and groin with ten consecutive tape strips done using a one inch-(2.5 cm) wide clear adhesive tape. Each successive strip used a new tape area to ensure appropriate adhesiveness and SC removal. This number of tape strips was selected from a pilot experiment which had confirmed that topically applied fluorescein was completely removed from interfollicular epidermis with no further colour change obtained with five additional strips (Figure 2). Moreover, this number of strips was shown previously to result in almost complete removal of the SC with a corresponding doubling of transepidermal water loss values in normal dogs.17 As a result, this TS method was felt to appropriately recreate an experimental skin barrier defect. In the second group (nontape strip; NTS), the hair was clipped as above, but the stratum corneum was not removed from either axilla or groin. In both groups, the allergen preparation contained 500 lg of lyophilized Df powder, made from milled cultures of whole mites (Greer Laboratories, Lenoir, NC, USA), suspended in 20 lL of mineral oil. This suspension was rubbed onto the previously tape-stripped or clipped right axilla once weekly for eight applications over 2 months (Figure 1); the same volume of mineral oil was applied to the right groin at the same time. An epicutaneous Df allergen booster was done approximately 1 month after the last previous application, with or without tape stripping as done previously.
Materials and methods North Carolina State University Institutional Animal Care and Use Committee (IACUC) approved all phases of the experimental design of the study beforehand.
Animals and study design Ten MBA dogs were enrolled in this study. This inbred line of dogs was selected because it is known to exhibit spontaneous hypersensi-
# TS:
0
5
Figure 1. Study time line. BMC, blood mononuclear cells; HDM, house dust mites; IDT, intradermal testing; IgE, Df-specific IgE serology; TS, tape stripping.
10
15
Figure 2. Fluorescein stain before and after tape stripping. A drop of fluorescein (10% fluorescein sodium sterile solution for injection; Akorn Pharmaceutical, Lake Forest, IL, USA) was applied to the skin and allowed to dry. Tape stripping (TS) was performed and photographs were taken before (0) and after five, ten and 15 strips. After ten and 15 strips, fluorescein only remained in the follicular infundibula, indicating appropriate removal of the stratum corneum.
ª 2010 The Authors. Journal compilation ª 2010 ESVD and ACVD, Veterinary Dermatology, 22, 188–196.
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Allergen-specific IgE serology Before sensitization and monthly thereafter for 3 months, blood was collected and serum separated for determination of Df-specific IgE titres. Such titres were determined by a fluorometric ELISA. Briefly, microplates were coated at 4 !C overnight with 4.5 lg per well of Df house dust mite antigen extract (Greer Laboratories) diluted in 0.05 M bicarbonated coating buffer. After blocking with phosphatebuffered saline (PBS) containing 10% fetal bovine serum (FBS) at room temperature for 1 h, sera from MBA dogs, diluted with the blocking buffer, were tested in triplicate at doubling dilutions starting at 1:10, and incubated at room temperature for 2 h. Intermediate washes were done with PBS containing 0.1% Tween 20 (PBST). Sera from specific-pathogen-free dogs were used as negative controls, while a pool of canine sera with known high IgE reactivity to Df (Greer Laboratories) was used to establish a standard curve. The plates were then incubated at room temperature for 1 h with 1:1000 dilution of biotinylated mouse anti-dog IgE clone 5.91 in PBST.23 Finally, streptavidin–b-galactosidase (Sigma Aldrich, St Louis, MO, USA) and 4-methylumbelliferyl-D-galactoside (Sigma Aldrich) were used as an enzymatic reaction stopped with 0.1 M glycine–NaOH (pH 10.2) after 1 h incubation at 37 !C. The fluorescence intensity was read on a Fluoroskan Acent FL (Thermo Fisher Scientific, Inc., Waltham, MA, USA) using excitation and emission filters of 355 and 460 nm, respectively. Results are expressed as arbitrary units (a.u.) based on the standard curve established with dilutions of the positive serum control pool that had been attributed arbitrary values (100 units at 1:40 dilution). The threshold of positivity (200 a.u.) was established as twice the 99th percentile of Df-specific serum IgE values of ten normal dogs without pruritus and skin diseases. Altogether, this ELISA had intra- and interassay coefficients of variation of 12% and 11%, respectively.
Allergen-specific T lymphocyte activation Prior to sensitization and 1 week after the end of induction (week 9) and challenge (week 13), blood was collected from all dogs. Unless otherwise noted, all reagents were purchased from Fisher Scientific (Waltham, MA, USA). Blood mononuclear cells (BMCs) were isolated using Lymphoprep in Leucosep tubes (VWR International – Greiner Bio-One, Suwanee, GA, USA). Briefly, 15 mL of Lymphoprep was centrifuged through the filter in a Leucosep tube and 5–10 mL of blood was layered over the filter. After centrifugation, the cells remaining above the filter were removed and washed with PBS. The remaining red blood cells were lysed with ACK lysing buffer. Cells were then resuspended in 4 mL PBS, layered over 4 mL FBS in a 15 mL tube, and centrifuged. Cells were resuspended in complete RPMI medium (RPMI with 10% FBS, 5 mM glutamate and antibiotics; Mediatech, Manassas, VA, USA) for culture or in flow staining buffer (PBS with 2% FBS and 1 mM EDTA) if they were to be stained immediately. Live cells were counted using trypan blue exclusion. Blood mononuclear cells were cultured at 2 · 106 cells ⁄ mL in complete RPMI with 5 ng ⁄ mL recombinant human IL-2 (Ebioscience, San Diego, CA, USA) with or without crude Df antigen (Greer Laboratories) at 50 lg ⁄ mL. Concanavalin A (Con A, 1.5 lg ⁄ mL) was used as a positive control for activation, and crude cat allergen extract (Greer Laboratories) served as a negative control. After 7 days, activated BMCs were harvested, and wells were washed once with flow staining buffer. Cells were resuspended in a blocking buffer containing 5% each of mouse, rabbit and goat serum in flow staining buffer and incubated for 15–20 min on ice before mouse anti-CD4-AlexaFluor647 (1:50 dilution; Serotec, Raleigh, NC, USA), mouse antihuman CD25RPE (1:25 dilution; Dako, Carpinteria, CA, USA) and mouse antihuman CD30-FITC (1:25 dilution; Dako) were added. The antihuman CD25 antibody has been validated for used in dogs,24,25 and it also proved useful to recognize activated T lymphocytes after allergen stimulation in this line of dogs.21 As CD25 is expressed not only by activated but also regulatory T lymphocytes in dogs, as in other species,24,25 we also stained for CD30, a cell membrane protein of the tumour necrosis factor receptor family expressed on activated but not rested T and B lymphocytes.26 The antibody used recognizes epitopes of sequence shared between humans and dogs. Prelimin-
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ary studies showed the neoexpression of this antigen in canine activated T lymphocytes that also expressed CD25 (K. Masuda and T. Olivry, unpublished data). Cells were stained for 30 min on ice and washed twice with buffer before being fixed in 2% paraformaldehyde in PBS. Cells were kept at 4 !C for 1 day before flow cytometric analysis on an LSR II (BD Biosciences, San Jose, CA, USA). Lymphocytes were gated based on forward- and side-scatter characteristics, and CD4-positive live lymphocytes were analysed for surface expression of CD25 and CD30. Positive gating was verified using isotype control stains for CD25 and CD30 (mouse IgG1-RPE and mouse IgG1-FITC, respectively) on CD4-positive cells. Control stains for CD4 (mouse IgG2a-AlexaFluor647) were also performed to ensure specificity. Results are expressed as stimulation indices, defined as the percentage of positive cells (among CD4-positive cells) in the treated conditions minus the percentage of positive cells in the untreated control conditions. Negative stimulation indices were possible, meaning that cell activation with the allergens was less than that of the untreated cells.
Intradermal testing Before sensitization and at the end of the study (week 16), intradermal testing (IDT) was performed, in a blinded fashion, using 0.05 mL injections of commercially available Df extract diluted at 1:1000 and 1:10 000 (w ⁄ v), histamine phosphate (0.275 mg ⁄ mL) and saline (all from Greer Laboratories). Immediate skin reactions were recorded after 20 min, and a global wheal score (GWS), which assessed the extent and intensity of the reactions, was determined as reported previously.27 For extent determination, the average diameter (D) in orthogonal directions was measured in millimetres. For intensity, erythema (E) and firmness (F) were assessed subjectively on a threepoint scale as follows: 1 (no erythema, flaccid wheal), 2 (weak erythema, firm wheal) or 3 (strong erythema, firm wheal). The GWS was calculated as follows: GWS = D · E · F.
Clinical reactions Skin lesions that developed after the epicutaneous Df allergen application were graded at the site of allergen (right axilla) and control application (right groin) 24, 48 and 72 h after challenge by an investigator unaware of the intervention groups. Erythematous macules, oedema, papules ⁄ pustules and excoriations were scored as 0 (absent), 1 (faint, mild) or 2 (strong, severe). The grades for each lesion were summed to yield a lesional score (LS). The maximal LS was 8 points. After the booster, total scores that consisted of the addition of LS obtained at 24, 48 and 72 h were calculated.
Analyses of the data Owing to the small number of dogs in this study, only descriptive comparisons between groups will be made and statistical analyses will not be reported.
Results Allergen-specific IgE serology Dermatophagoides farinae-specific serum IgE titres above the threshold of positivity (200 a.u.) were not detected in any dog before epicutaneous sensitization began (Figure 3a). The number of dogs with IgE serum titres above the positive threshold increased progressively and was always higher in the TS compared with the NTS group (Figure 3a). One week after the allergen booster (i.e. at week 12), three dogs from the TS group had values near or above 1000 a.u., while only one dog from the NTS group had such elevated values (Figure 3b). After 12 weeks, IgE serum titres were almost always higher in dogs from the TS group, but there was a single dog from the NTS group that had a very high IgE titre (98 000 a.u.; Table 1 and Figure 3b).
ª 2010 The Authors. Journal compilation ª 2010 ESVD and ACVD, Veterinary Dermatology, 22, 188–196.
Stratum corneum removal in atopic dogs
Figure 3. Dermatophagoides farinae allergen-specific IgE serology. (a) Number of dogs with Df-specific IgE serum titres above 200 arbitrary units (a.u.). (b) Individual Df-specific IgE values at month 3, 1 week after the booster. Bars indicate the median values.
Table 1. Dermatophagoides farinae allergen-specific IgE serum levels (a.u.) Group
Month 0
Month 1
Month 2
Month 3
TS NTS
18 (0–55) 39 (0–113)
0 (0–636) 28 (0–342)
112 (27–12 800) 130 (61–920)
960 (120–6316) 126 (16–98 000)
(a)
Data represent medians (range). a.u., arbitrary units; NTS, no tape stripping; TS, tape stripping.
Allergen-specific T lymphocyte activation The culture of BMCs with the positive control Con A resulted in stimulation indices, among CD4-positive T cells, that averaged 31% and 12% for CD25 and CD30, respectively. Average indices obtained after culture with the irrelevant cat allergen were
