Interleukin-31: its role in canine pruritus and naturally occurring canine atopic dermatitis
Background – Interleukin-31 (IL-31) is a member of the gp130/interleukin-6 cytokine family that is produced by cell types such as T helper 2 lymphocytes and cutaneous lymphocyte antigen positive skin homing T cells. When overexpressed in transgenic mice, IL-31 induces severe pruritus, alopecia and skin lesions. In humans, IL-31 serum levels correlate with the severity of atopic dermatitis in adults and children.
Hypothesis/Objective – To determine the role of IL-31 in canine pruritus and naturally occurring canine atopic dermatitis (AD).
Animals – Purpose-bred beagle dogs were used for laboratory studies. Serum samples were obtained from laboratory animals, nondiseased client-owned dogs and client-owned dogs diagnosed with naturally occurring AD.
Methods – Purpose-bred beagle dogs were administered canine interleukin-31 (cIL-31) via several routes (intravenous, subcutaneous or intradermal), and pruritic behaviour was observed/quantified via video monitoring. Quantitative immunoassay techniques were employed to measure serum levels of cIL-31 in dogs.
Results – Injection of cIL-31 into laboratory beagle dogs caused transient episodes of pruritic behaviour regardless of the route of administration. When evaluated over a 2 h period, dogs receiving cIL-31 exhibited a significant increase in pruritic behaviour compared with dogs that received placebo. In addition, cIL-31 levels were detectable in 57% of dogs with naturally occurring AD (≥13 pg/mL) but were below limits of quantification (<13 pg/mL) in normal, nondiseased laboratory or client-owned animals.
Conclusions – Canine IL-31 induced pruritic behaviours in dogs. Canine IL-31 was detected in the majority of dogs with naturally occurring AD, suggesting that this cytokine may play an important role in pruritic allergic skin conditions, such as atopic dermatitis, in this species.
Canine atopic dermatitis (AD) is a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic clinical features.1 One clinical feature that dogs with AD commonly display is pruritus, which can have a significant impact on the quality of life for the pet as well as for the owner. However, the underlying pathways and mechanisms involved in triggering pruritic behaviours are not clear, hampering the development of effective antipruritic therapies.
Interleukin-31 (IL-31) is a recently identified cytokine implicated in pruritic skin conditions such as human AD. When initially characterized in transgenic mice, overexpression of IL-31 led to the development of several hallmark signs of AD, which included increased inflammatory cell infiltration into the skin, severe pruritus, alopecia and skin lesions.2 Interleukin-31 has been shown to be produced by activated T helper type 2 lymphocytes and by cutaneous lymphocyte antigen positive (CLA+) skin homing T cells from human AD patients, suggesting that these cells may represent a major source of this cytokine. Interleukin-31 has been found to be elevated preferentially in pruritic versus nonpruritic human skin conditions, and serum levels of IL-31 correlate with disease severity in human adults as well as children with AD.2–8
Interleukin-31 binds to a heterodimeric receptor consisting of IL-31 receptor A and oncostatin M receptor β. Upon ligand binding to this receptor complex, signal transduction cascades such as the Janus kinase-signal transducer and activator of transcription (JAK-STAT), mitogen-activated protein kinase (MAPK) and phosphati-dylinositol 3-kinase (PI3K) pathways are activated.9 Receptors for IL-31 are found on a variety of cells, such as keratinocytes, macrophages and eosinophils, and participate in regulating immune responses in these cell types.9–11 Of great interest is the finding that these receptors are present on a subset of small-sized nociceptive neurons of mouse and human dorsal root ganglia, suggesting that this cytokine may directly activate pruritogenic signals in peripheral nerves.4,12
The cloning of canine interleukin-31 (cIL-31) has been previously reported.13 These investigators were able to detect cIL-31 mRNA in freshly isolated canine peripheral blood mononuclear cells after concanavalin A treatment, suggesting that IL-31 may be produced by canine T cells; however, they were not able to detect cIL-31 mRNA in skin biopsy specimens from dogs diagnosed with AD, which calls into question the role of IL-31 in canine AD. To extend investigations of canine IL-31 to assessments of biological activity and protein levels in disease, the present study was conducted to evaluate the role of IL-31 in canine pruritus using purpose-bred beagle dogs and to evaluate whether IL-31 is present in the serum of animals with naturally occurring AD.
Materials and methods
Cloning and expression of cIL-31
Using total RNA isolated from canine testicular tissue and oligo-(dT)20 primers, complementary DNA was synthesized with the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA.) according to the manufacturer’s protocol. Polymerase chain reactions were performed to amplify the cIL-31 gene from complementary DNA using primers TEF-1237 (5′- AGAT-CTGCCACCATGCTCTCCCACACAGGACCATCCAG-3′) and TEF-1240 (5′-GGTACCCTACTGAGGTCCAGAGTTTAGTGAC-3′). The PCR product was cloned into pCR®-Blunt II-TOPO® according to the manufacturer’s protocols (Life Technologies, Grand Island, NY, USA) and further subcloned into the expression construct pSOO524. The cIL-31 expression construct was either transiently transfected into FreeStyle™ 293 suspension culture cells following the manufacturers’ protocol (Life Technologies) or stably transfected into CHO cells using a site-specific integration system.14
Protein purification and analysis of recombinant cIL-31
Canine interleukin-31 was produced by cultured FreeStyle™ 293 cells or CHO cells. Conditioned media from these cells was collected, dialysed with buffer (20 mmol/L Tris, pH 8.0, and 40 mmol/L NaCl) and purified by anion exchange chromatography (Q Sepharose). Protein identity was confirmed by N-terminal sequencing and by liquid chromatography-mass spectrometry (LC-MS) analysis of a tryptic digest of the protein.
The DH82 canine monocytic cell line (American Type Culture Collection, Manassas, VA, USA) was used to evaluate cIL-31 cytokine function. DH82 cells were plated into CoStar 96-well flat-bottomed cell culture plates (Corning, Tewksbury, MA, USA) at a density of 1 × 105 cells per well in MEM growth media (Life Technologies) containing 15% heat-inactivated fetal bovine serum, 2 mmol/L GlutaMax, 1 mmol/L sodium pyruvate, 50 μg/L gentamicin and 10 ng/mL canine interferon-γ (R&D Systems, Minneapolis, MN, USA) for 24 h at 37°C in humidified air supplemented with 5% CO2. The following day, cells were exposed to MEM growth media without serum or interferon-γ for 2 h. Following serum deprivation, cells were treated with cIL-31 for 5 min. Cytokine treatment was terminated by removing medium and then adding AlphaScreen SureFire™ lysis buffer (Perkin Elmer, Waltham, MA, USA) and freezing samples at -20°C.
Signal transduction pathway activation
Cell lysates were used to evaluate phosphorylation of signal transducer and activator of transcription 3 (STAT3) and extracellular signalregulated kinase 1/2 (ERK1/2). Activation of STAT3 was detected using the Perkin Elmer AlphaScreen SureFire™ STAT3 p-Y705 kit, and activation of ERK1/2 was detected using the Perkin Elmer AlphaScreen SureFire™ MAPK p-T202/Y204 kit, following the manufacturer’s protocol. Specifically, 4 μL of cIL-31-treated cell lysates was sequentially incubated with streptavidin-coated donor beads bound with biotinylated capture antibody, then with protein A-coated acceptor beads bound with antibody that recognized the phophorylation site on the target protein. Assay plates were placed on a Perkin Elmer Envision plate reader to cause excitation of the donor beads at 680 nm. Upon excitation of a donor bead, a singlet oxygen transfer occurs from the donor to an acceptor bead. Any acceptor bead in close proximity to a donor bead (due to the binding of capture and detection antibodies to the desired target protein) emits light at 520–620 nm as a result of a cascade of energy transfer triggered by the singlet oxygen. Light emission was detected by the Envision plate reader. Data were expressed as mean relative signal units, and the EC50 for induction of phosphorylated STAT3 (pSTAT3) and phosphorylated MAPK (pMAPK) was determined by a nonlinear fit model in GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).
Assessment of pruritus in animals
All animal procedures were approved by the Institutional Animal Care and Use Committee (Pfizer Animal Health, Kalamazoo, MI, USA) and were performed in compliance with the Animal Welfare Act, Regulations, 9 CFR Parts 1, 2 and 3, and with the Guide for the Care and Use of Laboratory Animals, issued by the US Institute for Laboratory Animal Research Commission of Life Sciences (National Academy Press, Washington, DC, 1996).
Purpose-bred beagle dogs (Marshall BioResources, North Rose, NY, USA) were used in these experiments. Dogs were acclimated for at least 1 h to single-housed runs equipped with ceiling cameras. To evaluate the effects of cIL-31 administration via various routes on pruritic behaviour, cIL-31 (10 μg) or vehicle control [phosphate-buffered saline (PBS) containing equivalent amounts of mammalian host cell proteins to those present in the cIL-31 preparation] was administered intradermally (i.d.), subcutaneously (s.c.) or intravenously (i.v.). Pruritic behaviours (e.g. scratching, licking, chewing, scooting, head shaking and body rubbing) were monitored using video surveillance. Pruritic behaviours were measured as the time (in seconds) over a 4 h baseline period or 4 h after cIL-31 administration by one or more observers who were blinded to the treatment.
To evaluate the pruritic effects of cIL-31 in a statistically powered study, vehicle control-treated animals were compared with cIL-31-treated animals. Pruritic behaviour was evaluated for 2 h starting approximately 30 min after vehicle control or cIL-31 injection (1.75 μg/kg, i.v.) by one or more observers who were blinded to the treatment. Observed pruritic behaviour was measured using a categorical scoring system. ‘Yes/no’ determinations of displayed pruritic behaviour were made during consecutive, discrete 1 min intervals. The number of minutes categorized as ‘yes’ for displayed pruritic behaviours for an animal was then summed. The maximal achievable score for a 2 h (120 min) observation period was 120.
Canine serum samples
Blood was collected in 5 mL plastic BD Vacutainer™ SST™ tubes (Beckton Dickinson & Co., Franklin Lakes, NJ, USA) with owners’ signed informed consent when required, allowed to clot then separated according to the manufacturer’s protocol. Serum was collected from the following populations of dogs and frozen prior to measurements of serum cIL-31.