|Year : 2018 | Volume
| Issue : 2 | Page : 59-64
Immunohistochemical study of interleukin-31 in patients with atopic dermatitis
Tarek E Amin1, Nashwa N Elfar1, Fersan A Sallam2
1 Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Pathology Department, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||09-Jul-2017|
|Date of Acceptance||25-Feb-2018|
|Date of Web Publication||17-Aug-2018|
Nashwa N Elfar
Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Tanta, 3111
Source of Support: None, Conflict of Interest: None
Background Atopic dermatitis (AD) is a common chronic inflammatory skin disease of unknown etiology. Reduction of pruritus as one of the hallmarks of AD is the most effective therapeutic strategy for improving quality of life and for preventing aggravation of skin lesion. Interleukin-31 (IL-31) has been suggested to play a role in pathogenesis of AD through itch stimulation and correlation with development of pruritus.
Aim The aim of this work was to study the immunohistochemical expression of IL-31 in skin lesions of patients with AD.
Patients and methods The current study comprised 20 patients with AD. The severity of AD was assessed according to Six Area, Six Sign Atopic Dermatitis score. From the patients, 3-mm punch biopsy specimens were taken from lesional skin and nearby apparently normal skin to serve as control. Histopathological examination and immunohistochemical staining for IL-31 were done.
Results IL-31 expression was significantly increased in lesional skin of patients with AD in comparison with nearby apparently normal skin of the same patients. IL-31 expression in the lesional skin of patients with AD showed a significant positive correlation with severity of the disease evaluated by Six Area, Six Sign Atopic Dermatitis score.
Conclusion IL-31 expression was increased in the lesional skin of patients with AD compared with control specimens, and it was positively correlated with severity of AD. IL-31 may be a therapeutic target for AD and associated pruritus.
Keywords: atopic dermatitis, immunohistochemical, interleukin 31
|How to cite this article:|
Amin TE, Elfar NN, Sallam FA. Immunohistochemical study of interleukin-31 in patients with atopic dermatitis. Egypt J Dermatol Venerol 2018;38:59-64
|How to cite this URL:|
Amin TE, Elfar NN, Sallam FA. Immunohistochemical study of interleukin-31 in patients with atopic dermatitis. Egypt J Dermatol Venerol [serial online] 2018 [cited 2018 Sep 19];38:59-64. Available from: http://www.ejdv.eg.net/text.asp?2018/38/2/59/231230
| Introduction|| |
Atopic dermatitis (AD) is a chronic, highly pruritic inflammatory skin disease, and is one of the most common skin disorders in children . Its presentation varies from an acute eczematous relapsing eruption in early life to a characteristic lichenified dermatitis in older patients. AD often occurs in people with personal or family history of other atopic disease, that is, bronchial asthma, rhinitis, and hay fever . Interleukin-31 (IL-31) is a helical cytokine, belonging to the gp130/IL-6 cytokine family. IL-31 is expressed preferentially by activated T helper 2 common differentiating antigen 4 (CD4+) T cells. The activity of human IL-31 is mediated through a heterodimeric receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor .
IL-31 heterodimeric receptors are present in many tissues including human epidermal keratinocytes, bronchial epithelial cells, testis, brain, bone marrow, thymus, skin, and intestine . Target cells expressing IL-31RA for IL-31 in these tissues include activated monocytes, macrophages, myeloid progenitor cells, keratinocytes, eosinophils, and epithelial cells . IL-31 has been shown to induce skin inflammation, pruritus, and severe dermatitis in overexpressing transgenic mice . Schulz et al.  found that a mutation in the IL-31 gene is associated with intrinsic AD.
So, the aim of this study was to evaluate immunohistochemical expression of IL-31 in skin of patients with AD to elucidate its possible role in pathogenesis of the disease.
| Patients and methods|| |
A total of 20 patients with AD were included in this randomized controlled study. All patients were recruited from the Outpatient Clinic of Dermatology and Venereology Department, Tanta University Hospital. The study was approved by the Research Ethics Committee. All participants signed an informed consent before participation in the study. The diagnosis of patients with AD was based on Hanifin and Rajika  diagnostic criteria.
Patients who had received any systemic or topical treatment for AD at least 12 weeks before the study, who had a history of other dermatological or systemic diseases or associated autoimmune diseases, who were on systemic steroids or immunosuppressive drugs, pregnant women, and lactating women were excluded.
All patients were subjected to the following:
- Complete history (name, age, sex, marital status, occupation, onset, course, duration of the disease, drug intake, other atopic diseases, and family history of atopic diseases) and digital photographs were taken.
- Thorough general and dermatological examination was done for every patient to determine the clinical distribution, morphology, and activity of the lesions in all patients. The severity of AD was assessed through Six Area, Six Sign Atopic Dermatitis (SASSAD) score . SASSAD assesses six clinical signs of disease severity (erythema, exudation, dryness, cracking, excoriation, and lichenification) at six body sites (head/neck, trunk, arms, hands, legs, and feet). Each clinical sign on a given body site is graded on a scale of 0–3, and the scale confers a maximum score of 108. AD is classified as mild if SASSAD score is below 20, moderate if SASSAD score is 20–40, and severe if SASSAD score is more than 40.
- Histopathological examination: from all the patients, 3-mm punch biopsy specimens were taken. Biopsies were obtained from lesional skin and from nearby apparently normal skin of the same patients, which served as control, under local infiltration anesthesia and complete aseptic technique. The biopsies were immediately fixed in 10% formalin and embedded in paraffin, and hematoxylin and eosin (H&E) staining was done to confirm the histopathological diagnosis of AD. Then, immunohistochemical staining for expression of IL-31 was done using rabbit polyclonal antibody to IL-31 (Lot≠E6292; ZymoGenetics Inc., Seattle, Washington, USA) .
- Interpretation of immunohistochemical staining: IL-31 expression (immunoreactivity) was evaluated by using Quick score , which considers both the intensity of staining and frequency of positive cells in a semiquantitative pattern.
The intensity of immunostaining (I) was scored as follows: negative (0), weak (1), moderate (2), and strong (3).
The percentage of positive cells (P) was scored as follows: 0–4% (1), 5–19% (2), 20–39% (3), 40–59% (4), 60–79% (5), and 80–100% (6). Quick score was then calculated by multiplying the intensity by the percentage of positive cells (I)×(P), with a final score ranging from 0 to 18, where more than 10 was assigned as strong, 6 to 10 as moderate, 1 to 5 as mild, and 0 as negative.
Statistical presentation and analysis of the present study was conducted. Continuous variables are presented as mean±SD and discrete variables are shown as percentages. Both χ2 and Fischer χ2 testing were used for intergroup comparisons, and P less than 0.05 was considered significant. Software (SPSS, version 16.0 statistical package for Microsoft Windows; SPSS Inc., Chicago, Illinois, USA) was used throughout.
| Results|| |
All clinical results were summarized in [Table 1].
Regarding the severity of AD as assessed by SASSAD score, 14 (70%) patients had mild AD, with range of SASSAD score of 8–19 and a mean of 13.64±4.01, and six (30%) patients had moderate AD, with range of SASSAD score of 25–37 and a mean of 31.50±4.59. The total score of the patients ranged from 8 to 37, with a mean of 19.0±9.33 ([Table 2]).
|Table 2 Six Area, Six Sign Atopic Dermatitis score in the studied patients with atopic dermatitis|
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Hematoxylin and eosin staining
Stained sections with H&E of nearby apparently normal skin of patients with AD (control) showed normal appearance of the skin with no infiltration by inflammatory cells. Lesional skin biopsies stained with H&E showed variable degree of acanthosis, spongiosis, parakeratosis, elongation of rete ridges, and proliferating blood vessels with perivascular inflammatory infiltrate, mainly lymphocytes.
Immunohistochemical expression of interleukin-31
IL-31-stained sections of nearby apparently normal skin showed epidermal immunoreactivity of IL-31 and no dermal immunoreactivity of IL-31 ([Figure 1]). IL-31-stained sections of lesional skin of AD showed epidermal immunoreactivity of IL-31 in all samples, whereas dermal infiltrating cells revealed mild expression of IL-31 in eight (40%) patients, moderate expression of IL-31 in six (30%) patients, and strong expression of IL-31 in six (30%) patients. Lesional skin of AD showed high-significant expression of IL-31 compared with nearby apparently normal skin (P=0.001*) ([Table 3] and [Figure 2],[Figure 3],[Figure 4]).
|Figure 1 Nearby apparently normal skin of patients with atopic dermatitis showing epidermal immunoreactivity of interleukin-31 and no dermal immunoreactivity of interleukin-31 (Papanicolaou, PAP, ×200).|
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|Table 3 Immunoreactivity of interleukin-31 expression in the studied patients|
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|Figure 2 Mild expression of interleukin-31 in the infiltrating cells in lesional skin of patients with atopic dermatitis (dark arrows) (Papanicolaou, PAP, ×400).|
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|Figure 3 Lesional skin of patients with atopic dermatitis showing moderate expression of interleukin-31 in the infiltrating cells (Papanicolaou, PAP, ×400).|
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|Figure 4 Lesional skin of patients with atopic dermatitis showing strong expression of interleukin-31 in the infiltrating cells (Papanicolaou, PAP, ×400).|
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Regarding the relation between IL-31 expression and SASSAD score in the studied patients with AD, it revealed that among patients with mild AD, six patients showed mild expression, six patients showed moderate expression, and two patients showed strong expression of IL-31. However, among the patients with moderate AD, two patients showed mild expression and four patients showed strong expression of IL-31 ([Table 4]). There was a statistically significant positive correlation between the intensity of IL-31 expression in patients with AD regarding SASSAD score (Graph 1).
|Table 4 Relation between immunoreactivity of interleukin-31 with Six Area, Six Sign Atopic Dermatitis score in studied patients|
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| Discussion|| |
AD is a common chronic inflammatory skin disease of unknown etiology that usually starts in early infancy, though an adult-onset variant is recognized. Reduction of itching and pruritus as one of the hallmarks of AD is the most effective therapeutic strategy for improving the quality of life and for preventing aggravation of skin lesions . Much effort has been made to identify the major pruritogen in AD; the mechanisms of itch have not been fully elucidated . Skin-infiltrating T cells have been suggested to play a major role through production and release of different mediators . Among these mediators, IL-31 has been suggested to play a role .
The aim of this study was to evaluate immunohistochemical expression of IL-31 in skin of patients with AD in a trial to elucidate its possible role in the pathogenesis of the disease.
In the present study, there was significant increase of IL-31 expression in patients with AD at sites of the lesions when compared with the nearby apparently normal skin of the same patients. This result agreed with Kim et al. , who found that skin biopsies from patients with AD showed an increased IL-31 level. Moreover, Nobbe et al.  reported increased IL-31 expression in the inflammatory infiltrates in the skin biopsies of participants with AD. Cornelissen et al.  found enhanced expression of the gene that encodes the cytokine IL-31 in skin lesions of patients with AD.
In human keratinocytes, IL-31 induces several chemokine genes that have been associated with atopic skin inflammation. Hence, elevated levels of IL-31 in AD lesions may enhance skin inflammation through the induction of chemokines, which subsequently lead to the recruitment of T cells. In turn, activated skin-infiltrating T cells may become new sources of IL-31, thereby amplifying atopic skin inflammation and pruritus .
In this study, IL-31-stained sections of lesional skin of AD showed mild expression of IL-31 in eight (40%) patients, moderate expression of IL-31 in six (30%) patients, and strong expression of IL-31 in six (30%) patients. Dillon et al.  reported a moderate homogenous immunoreactivity of keratinocytes for IL-31RA in patients with AD and emphasize a constitutive expression of the receptor components in keratinocytes, whereas they found a mean of less than 5% immunoreactivity for IL-31RA in the dermis. The immunoreactivity in the dermal infiltrate mainly reflects the presence of macrophages, which have been shown to express the receptor components. On the contrary, Nobbe et al.  observed that 87% of samples from patients with AD showed immunoreactivity for IL-31 in cells infiltrating the dermis.
In the present study, there was a statistically significant positive correlation between the intensity of IL-31 expression in patients with AD regarding SASSAD score. This result agreed with Lei et al. , who found that IL-31 expression was positively correlated with severity of AD. Moreover, Sokołowska-Wojdyło et al.  suggested that some specific haplotypes of the IL-31 may be linked to the severity of AD. On the contrary, Neis et al.  found no correlations between IL-31 mRNA level and either disease severity or serum immunoglobulin E level in patients with AD. Nobbe et al.  reported that cutaneous IL-31and IL-31RA protein expression did not correlate with pruritus in patients with AD.
The mechanisms underlying dermatitis and pruritus are complex and not fully understood . Transgenic mice that overexpress IL-31 develop severe pruritus and skin lesions similar to AD . In an AD-like murine model (NC/Nga mice), high IL-31 mRNA expression is associated with scratching behavior , whereas an anti-IL-31 antibody reduces scratching tendency . It is unknown whether IL-31 acts directly through its receptor on sensory nerve endings or indirectly, for example, via keratinocytes. IL-31RA and oncostatin M receptor were found in afferent cutaneous nerve fibers/dorsal root ganglia . IL-31RA is a functional neural cytokine receptor involved in acute and chronic itch. In this respect, IL-31RA represents the long hypothesized ‘missing link’ in a direct neuroimmune cross talk between T cells and sensory nerves in itch. This finding emphasizes that not only mast cells through histamine or tryptase release  but also T cells through cytokines can directly communicate with sensory nerves to induce itch. Thus, blocking the effects of IL-31/IL-31RA may have a beneficial effect not only for the inhibition of inflammation but also to ameliorate directly the deleterious effects of T-cell-mediated itch . In AD, IL-31 mainly plays a role in inducing pruritus but not directly causing lesions per se. The development of lesions may arise over time from the excessive scratching behavior of the patients .
Epidermal keratinocytes from patients with AD were found to produce significantly higher level of CCL5, a potent eosinophil chemoattractant, following stimulation with tumor necrosis factor-α. All these chemokines have been implicated in the recruitment of T helper 2 lymphocytes and eosinophils into the skin of patients with AD, eventually contributing to the inflammatory processes associated with the pathogenesis of AD . In the inflammatory infiltrate of patients with AD, CD45RO+ CLA+ lymphocytes were found to express high levels of IL-31 messenger RNA . Infiltrating cells such as macrophages, which were found to be more numerous in skin from patients with AD, also expressed IL-31RA . Moreover, staphylococcal superantigens, which represent a general trigger factor for AD, rapidly induced IL-31 mRNA expression in the skin and in peripheral blood mononuclear cells of atopic individuals . Moreover, IL-31 induces proinflammatory cytokines following stimulation of staphylococcal exotoxins in human macrophages .
| Conclusion|| |
IL-31 expression was increased in lesional skin of patients with AD compared with control specimens, and it was positively correlated with severity of AD. IL-31 may be a therapeutic target for AD and associated pruritus. The limitation in our study was the small number of patients. Further larger studies are suggested to explore the role of IL-31 expression with prognosis and response to therapy.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Krakowski AC, Eichefield LF, Dohil MA. Management of atopic dermatitis in the pediatric population. Pediatr 2008; 122:812–824.
Simpson EL, Hanifin JM. Atopic dermatitis. J Am Acad Dermatol 2005; 53:115–128.
Dillon SR, Sprecher C, Hammond A, Bilsborough J, Rosenfeld-Franklin M, Presnell SR et al.
Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice. Nat Immunol 2004; 5:752–760.
Chattopadhyay S, Tracy E, Liang P, Robledo O, Rose-John S, Baumann H. Interleukin-31 and oncostatin-M mediate distinct signaling reactions and response patterns in lung epithelial cells. J Biol Chem 2007; 282:3014–3026.
Sonkoly E, Muller A, Pivarcsi A, Soto H, Kemeny L, Alenius H et al.
IL-31: a new link between T cells and pruritus in atopic skin inflammation. J Allergy Clin Immunol 2006; 117:411–417.
Schulz F, Marenholz I, Folster-Holst R, Chen C, Sternjak A, Baumgrass R et al.
A common haplotype of the IL-31 gene influencing gene expression is associated with nonatopic eczema. J Allergy Clin Immunol 2007; 120:1097–1102.
Hanifin JM, Rajaka G. Diagnostic features of atopic dermatitis. Acta Derm Venereol Suppl 1980; 92:44–47.
Berth-Jones J. Six areas, six signs atopic dermatitis (SASSAD) severity score: a simple system for monitoring disease activity in atopic dermatitis. Br J Dermatol 1996; 135:25–30.
Nobbe S, Dziunycz P, Mühleisen B, Bilsborough J, Dillon SR, French LE et al.
IL-31 expression by inflammatory cells is preferentially elevated in atopic dermatitis. Acta Derm Venereol 2012; 92:24–28.
Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The Mdm2 oncogene product forms a complex with the P53 protein and inhibits P53-mediated transactivation. Cell 1992; 69:1237–1245.
Finlay AY. Quality of life in atopic dermatitis. J Am Acad Dermatol 2001; 45:S64–S66.
Greaves MW, Khalifa N. Itch: more than skin deep. Int Arch Allergy Immunol 2004; 135:166–172.
Kim S, Kim HJ, Yang HS, Kim E, Huh IS, Yang JM. IL-31 serum protein and tissue mRNA levels in patients with atopic dermatitis. Ann Dermatol 2011; 23:468–473.
Cornelissen C, Lüscher-Firzlaff J, Baron JM, Lüscher B. Signaling by IL-31 and functional consequences. Eur J Cell Biol 2012; 91:552–566.
Lei Z, Liu G, Huang Q, Lv M, Zu R, Zhang GM et al.
SCF and IL-31 rather than IL-17 and BAFF are potential indicators in patients with allergic asthma. Allergy 2008; 63:327–332.
Sokołowska-Wojdyło M, Gleń JO, Zabłotna M. The frequencies of haplotypes defined by three polymorphisms of the IL-31 gene: −1066, −2057, and IVS2+12 in Polish patients with atopic dermatitis. Int J Dermatol 2015; 54:62–67.
Neis MM, Peters B, Dreuw A, Wenzel J, Bieber T, Mauch C et al.
Enhanced expression levels of IL-31 correlate with IL-4 and IL-13 in atopic and allergic contact dermatitis. J Allergy Clin Immunol 2006; 118:930–937.
Ogg G. Role of T cells in the pathogenesis of atopic dermatitis. Clin Exp Allergy 2009; 39:310–316.
Takaoka A, Arai I, Sugimoto M, Yamaguchi M, Tanaka M, Nakaike S. Expression of IL 31 gene transcripts in NC/Nga mice with atopic dermatitis. Eur J Pharmacol 2005; 516:180–181.
Grimstad Ø, Sawanobori Y, Vestergaard C, Bilsborough J, Olsen UB, Grønhøj-Larsen C et al.
Anti-interleukin-31-antibodies ameliorate scratching behavior in NC/Nga mice: a model of atopic dermatitis. Exp Dermatol 2009; 18:35–43.
Bando T, Morikawa Y, Komori T, Senba E. Complete overlap of interleukin-31 receptor A and oncostatin M receptor beta in the adult dorsal root ganglia with distinct developmental expression patterns. Neuroscience 2006; 142:1263–1271.
Steinhoff M, Vergnolle N, Young SH, Tognetto M, Amadesi S, Ennes HS et al.
Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat Med 2000; 6:151–158.
Steinhoff M, Cevikbas F, Yeh I, Chong K, Buddenkotte J, Ikoma A. Evaluation and management of a patient with chronic pruritus. J Allergy Clin Immunol 2012; 130:1015–1016.
Homey B, Steinhoff M, Ruzicka T, Leung DY. Cytokines and chemokines orchestrate atopic skin inflammation. J Allergy Clin Immunol 2006; 118:178–189.
Bilsborough J, Leung DY, Maurer M, Howell M, Boguniewcz M, Yao L et al.
IL-31 is associated with cutaneous lymphocyte antigen-positive skin homing T cells in patients with atopic dermatitis. J Allergy Clin Immunol 2006; 117:418–425.
Kasraie S, Niebuhr M, Werfel T. Interleukin (IL)-31 induces pro-inflammatory cytokines in human monocytes and macrophages following stimulation with staphylococcal exotoxins. Allergy 2010; 65:712–721.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]