MATERIALS AND METHODS

Patient selection

Twenty samples of hypertrophic burn scars were obtained from patients who had visited the Hallym University Hangang Sacred Heart Hospital in South Korea. Subjects were included in the study if they were at least seven years of age at the time of examination. Subjects were excluded if they (1) had a pre-existing disease known to frequently cause pruritus, such as chronic systemic or dermatologic disease, (2) were pregnant, (3) were unable to specifically describe their pruritus, (4) were on concurrent systemic medications that may affect their pruritic symptoms such as antihistamines, morphine, or systemic steroids, (5) had any psychotic diseases, (6) had cancer, (7) had an active infection, or (8) had been treated with immunomodulators, UV irradiation, or hydrogen peroxide within three months before surgery. All subjects provided informed consent. The protocols of this study were approved by Hallym University Hangang Sacred Heart Hospital Institutional Review Board (IRB approval No. 2011-186).

Histopathological analysis

Skin samples were obtained through punch biopsy (6 mm) during surgery from twenty patients between 2011 and 2012. Control tissue samples were obtained from non-burned skin areas adjacent to the burn injury site from the same patients. Tissue samples from hypertrophic scars were taken from the burn lesions of the same patients. The tissue samples were placed in 10% neutral buffered formalin for 18 hours. Paraplast (Sigma-Aldrich, St. Louis, MO, USA) was used for paraffin embedding. Serial sections 5-μm in thickness were processed [24]. The IL-31 antibody (Novus Biologicals, Littleton, CO, USA), IL-31RA antibody (Abnova, Taipei, Taiwan), and OSMR antibodies (Novus Biologicals) were used for staining purposes and sections were assessed by a pathologist. Immunohistochemistry results were semi-quantitatively evaluated for IL-31, IL-31RA, and OSMR based on the estimated epidermal positive basal cell percentage. Cells were counted in the stroma using a built-in 10 × 10 grid within the Nikon microscope (Plan-Apo, Nikon, Tokyo, Japan) with each section measuring 25 μm (0.025 mm) at × 400 magnification and the upper edge of the grid placed at the epidermal junction [25]. Three areas with the greatest density of inflammatory cells with the most distinct inflammation were counted per visual field and the cells were regarded as either immunoreactive or non-immunoreactive [19]. The IL-31RA epidermis, IL-31 epidermis basal layer, and OSMR epidermis basal layer staining intensities were rated as 1 when the positivity could be seen at 400, 2 at 200, 3 at 100, and 4 at 40-fold magnification with use of a Nikon microscope (Plan-Apo) with a digital camera (Nikon, DS-Ri2, Nikon digital SLR camera FX-format CMOS sensor optimized for microscopy) [24]. One pathologist and one dermatologist assessed the epidermal thickness, dermal thickness, and IL-31, IL-31RA, and OSMR infiltration through hematoxylin eosin staining through use of a Nikon microscope (Plan-Apo) with a digital camera (Nikon, DS-Ri2, Nikon digital SLR camera FX-format CMOS sensor optimized for microscopy), and the average values were obtained.

Statistical analysis

SPSS ver. 21.0 for Windows (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Normal skin and burn scar tissue comparisons were performed using the paired t test and the Wilcoxon signed-rank test. Pearson and Spearman rho was used to examine the correlation between IL-31, IL31-RA, and OSMR percentages and intensities. Values were regarded as significant if the p-value was < .05.

RESULTS

This study included tissue samples from twenty subjects. The characteristics of the burn subjects are presented in Table 1.

Comparison of burn scar and normal skin tissue characteristics are presented in Table 2. There was a significant difference in dermal thickness in burn scar tissue compared with normal skin (4,826.85 ± 1,955.34 μm and 1,809.50 ± 745.54 μm, respectively; p < .001) and the epidermal thickness was significantly thicker in the burn scar tissue compared with normal skin (5,112.25 ± 2,522.42 μm vs. 1,729.88 ± 806.55 μm, respectively; p<.001). The epidermal basal layer percentage of IL-31 was significantly greater in burn scar tissue compared with normal skin (22.4% and 12.7%, respectively; p < .001) (Fig. 1). However, there was no significant difference in IL-31 epidermal basal layer intensity in burn scar tissue compared with normal skin (p > .05). The IL-31RA epidermal percentage was significantly greater in burn scar tissue compared with normal skin (29.8% and 23.9%, respectively; p < .001) (Fig. 1). The comparison of IL-31, IL-31RA, and OSMR percentage levels between control and scar tissue is shown in Fig. 2. There were no significant differences in IL-31 epidermis basal layer intensity (p > .05). The OSMR epidermal basal layer cytoplasm percentage was greater in burn scar tissue compared with normal skin (46.50% and 25.00%, respectively; p < .001) (Fig. 1). There was also a significant difference in OSMR epidermis basal layer intensity in burn scar tissue compared with normal skin (p < .005). Pearson and Spearman rho analyses showed that the correlations between the percentages and intensities of IL-31, IL-31RA, and OSMR in burn scar tissue were significant (p < .05), indicating that the greater the infiltration percentage, the higher the intensity, based on a scale of 1 to 4 (Table 3).

There was also a significant difference in the number of mast cells, which was greater in burn scar tissue compared with normal skin (p < .05) (Table 2).

DISCUSSION

The objective of this study was to examine the expression of IL-31, IL-31RA, and OSMR in hypertrophic scar tissue and compare the results with those from normal tissue. IL-31 is a T-cell cytokine which acts through a heterodimeric receptor such IL-31RA and OSMR, which are expressed by epithelial cells [26].

Our study has found that IL-31, IL-31RA, and OSMR are expressed by epidermal basal cells from both normal and burn scar tissue samples. However, IL-31 and IL-31RA expression was significantly higher in the burn scar epidermal basal cells (p < .001) compared with normal skin tissue. The OSMR epidermal basal layer cytoplasm percentage was also significantly greater than in normal skin. Our findings are relatable to previous studies that have found greater levels of IL-31 and its receptors in subjects with various pruritic skin conditions compared with non-pruritic and normal skin [17-20,23]. Nobbe et al. [19] reported that the majority of the immunoreactivity stainings for IL-31RA and OSMR were cytoplasmic staining patterns.

Cutaneous T-cell lymphoma patients also commonly suffer from pruritus and have recently been found to have elevated IL-31 expression in the epidermis and dermis and increased IL-31RA and OSMR expression in the epidermis only [27].

It appears that IL-31 and its receptors have their respective roles in pruritus induction. IL-31 is postulated to stimulate the keratinocytes and infiltrating cells to release other mediators involved in the induction of pruritus. Moreover, IL-31 appears to cause pro-inflammatory cytokines to be released from macrophages, eosinophils, and monocytes [26,28].

Expression of IL-31RA and OSMR has been found in keratinocytes and the dorsal root ganglia, which are the locations where the cutaneous sensory neuron soma reside and their sensory fibers are directed towards the skin [29]. These sensory neurons may be involved in the itch sensation and are thought to be stimulated by IL-31. IL-31 may serve as a possible connection between the immune and sensory nervous system [30]. Therefore, although the presence of elevated IL-31 and its relation to pruritus is significant, the roles of IL-31RA and OSMR should not be neglected and should be taken into consideration in the discussion of pruritus.

This study found that burn scar thickness and epidermal thickness was significantly increased compared with normal tissue. When a burn injury occurs on the skin, the skin barrier function is damaged or destroyed [31]. Similarly, for patients with AD, the skin-barrier function is most commonly impaired, indicating that the epithelial defense system serves a significant role in the pathogenesis of AD [32]. When the skin barrier is altered or damaged in AD patients, this is partially associated with the distribution of the stratum corneum liquid composition, which allows harmful substances to penetrate cutaneously and subsequently signals for proliferation and differentiation of the epidermis [31]. Singh et al. [33] found that intradermal injection of recombinant mouse IL-31 (rIL-31) produced epidermal thickness and that elevated levels of IL-31 by T cells subsequently induced thickening of the epidermis and inflammatory infiltrates [33].

There was no significant difference in intensity levels for all variables, except for the OSMR basal layer intensity. This discrepancy may be attributed to the small sample size, but could show significance in future studies with larger sample sizes.

Although this study did not specifically look for pruritus in those with hypertrophic scars, the results show characteristics similar to pruritic skin conditions, such as AD, in which there are significantly greater IL-31, IL-31RA, and OSMR expression levels in the skin.

There was no significant difference in intensities between the control and burn scar tissues, aside from the OSMR basal layer intensity. However, Pearson and Spearman rho results showed significant correlations between infiltration percentages and intensities in burn scar tissues, where the greater the percentage of infiltration, the greater the grade in intensity, based on the scale of 1 to 4. As stated earlier, the IL-31RA epidermis, IL-31 epidermis basal layer, and OSMR epidermis basal layer staining intensity was rated as 1 when the positivity could be seen at 400, 2 at 200, 3 at 100, and 4 at 40-fold magnification with use of a Nikon microscope. In this case, an intensity grade of 4 is equal to 40-fold magnification; this indicates that the intensity is strong with low magnification because if the percentage of infiltration is high enough, a smaller amount of magnification is required to observe the infiltration through the microscope.

The mean value of the mast cells was significantly greater in the burn scar compared with the normal tissue samples. This finding may be significant for several reasons. Antimicrobial peptide proteins (AMPs), such as human β-defensins (hBDs), provide innate immunity and defense against microbial invasion, which is a common risk factor with burn injury, and are formed in the deep portions of burned skin [34,35]. Mast cells are located within the dermis and have been found to be another source of IL-31 [34]. AMPs have been found to cause mast cells to release IL-31 mRNA, and may lead to protein production and release [34]. Niyonsaba et al. [36] has also stated that mast cells are involved in the pathological process of other disorders of the skin in which AMP concentrations are enhanced [36]. In the case of wounding and lichen planus, direct contact between skin-derived AMPs and mast cells occurs, which subsequently activates due to basal membrane impairment between the dermis and epidermis. Sites of infection and inflammation show elevated amounts of hBDs and leucine-leucine 37-amino acid peptide (LL-37) in the human epithelium. LL-37 is induced in the keratinocytes of humans during contact dermatitis [37] and hBDs may contribute to skin inflammatory responses by causing mast cells to secrete IL-31 along with other factors that are pruritogenic. These findings may support the link between increased IL-31 levels and postburn hypertrophic scar pruritus in the present study.

This study had some limitations. The sample size was small and subject complaints of pruritus were not fully assessed. In addition, no other techniques were used to confirm our findings. However, this study has shown that there are pathological similarities between post-burn pruritus and other skin diseases known to cause pruritus, such as AD, prurigo nodularis, and cutaneous T-cell lymphoma, all of which exhibit increased expression levels of IL-31.

Therefore, the findings of this study may enlighten the understanding of pruritic events occurring in post-burn hypertrophic scars. Further studies with larger sample sizes that investigate the relationship between IL-31 and receptor expression levels with subjective and objective measurements of pruritus are warranted.

Acknowledgments

Fig. 1.

(A) Interleukin 31 (IL-31) control. (B) IL-31 scar. (C) IL-31 receptor alpha (IL-31RA) control. (D) IL-31RA scar. (E) Oncostatin M receptor (OSMR) control. (F) OSMR scar. IL-31, IL-31RA, and OSMR infiltration.

Fig. 2.

Comparison of interleukin 31 (IL-31), IL-31 receptor alpha (IL-31RA), and oncostatin M receptor (OSMR) percentage values between the normal and scar tissue (n=20). *p<.05.

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Figure & Data

References

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