Although many dermatological diseases are not life threatening, they pose a unique challenge to the human psyche and carry a distinctive psychosocial burden. Patients with dermatological diseases are often unable to hide their condition from public scrutiny. Specifically, acne vulgaris is a ubiquitous disease with a known potential to cause significant psychological repercussions 1.
Clinical evidences suggest that components of the nervous system, such as psychological and neurogenic factors, can influence the course of acne. The disease has been reported to be initiated and/or exacerbated as a result of emotional or psychosocial stress, suggesting the involvement of neural-derived factors in its pathogenesis 2.
Neuropeptides (NPs) are a heterogeneous group of biologically active peptides that are present in neurons of both the central and the peripheral nervous system. However, functional receptors for NPs have been found to be expressed in human skin, particularly in the sebaceous glands. These receptors modulate the production of inflammatory cytokines and regulate proliferation, differentiation, lipogenesis, and androgen metabolism in human sebocytes 3.
Substance P (SP) is a mediator of neurogenic inflammation that is expressed by nerve fibers including periglandular fibers 2. It mediates its effect by binding to its receptor neurokinin-1 (NK-1R) 4. It was shown that SP can alter the morphology and ultrastructure of sebaceous glands in skin organ cultures as it promotes the development of cytoplasmic organelles, stimulates sebaceous germinative cells, increases the size of individual sebaceous cells, and increases the number of sebum vacuoles in sebocytes 5. In addition, it promotes the proliferation and differentiation of sebaceous glands 6.
The aim of the present work was to study the pilosebaceous units (PSUs) and the expression of SP and its receptor NK-1R in comedonal and inflammatory acne lesions in comparison with control skin and to verify the relation of the expression of this NP and its receptor with stress in order to clarify their role in the pathogenesis of acne.
Patients and methods
Thirty patients with acne vulgaris attending the dermatology outpatient clinic of Kasr El Aini Hospital, Cairo University, were recruited for this case–control study during the period from February 2011 to May 2011. The study was approved by the Dermatology Research Ethical Committee of the National Research Centre. Twenty age-matched and sex-matched healthy individuals served as controls. An informed consent was signed by all patients and controls.
Acne patients 18 years old or more of both sexes with inflammatory and comedonal lesions involving the back were included.
Acne patients receiving topical treatment for acne in the last 2 weeks or receiving systemic treatment for acne in the last 4 weeks before taking the skin biopsy, patients receiving medications that may exacerbate acne such as steroids, patients receiving sedating or antidepressant medications as they may mask the effect of stress on acne, pregnant and lactating women, and patients younger than 18 years old were excluded.
Each patient in the study was subjected to the following:
- Full assessment of history.
- Dermatological examination: the severity of acne was determined using the Global Acne Grading System 7.
- Perceived Stress Scale (PSS) 8: all the patients were subjected to an Arabic script of the PSS, which is used to measure the perception of stress and its correlation with the severity of acne.
- Skin biopsy: two 3 mm punch biopsies were obtained from nonexposed skin of the back: one from a comedonal (noninflammatory) lesion and the other from a papular (inflammatory) lesion.
Control individuals were subjected to a 3 mm punch skin biopsy from the back.
Histopathological and immunohistochemical studies
Skin biopsies were fixed in 10% formalin and embedded in paraffin. Sections of 5 μm thickness were prepared both for routine hematoxylin and eosin (H&E) staining and for immunohistochemical detection of SP and NK-1R. The antibodies used in this study included monoclonal mouse IgG antihuman SP antibody (Clone# 266815, Catalog# MAB4375; R&D System, Minneapolis, Minnesota, USA) and polyclonal rabbit IgG antihuman NK-1R antibody (Catalog# PA3-301; Thermo Scientific, Rokford, Illinois, USA). The following detection kit was used: Rabbit HRP/DAB detection IHC kit (Catalog# ab64261; Abcam, Cambridge, UK). Immunohistochemical detection was carried out using the same method described by Schulz et al.9.
Morphometric analysis of pilosebaceous units and sebocytes in routinely stained hematoxylin and eosin sections
The morphometric analysis was carried out at the Pathology Department, National Research Center, using the Leica Qwin 500 Image Analyzer (Leica Imaging Systems Ltd, Cambridge, UK), which consists of a Leica DM-LB microscope with a JVC color video camera attached to a computer system Leica Q 500IW. The entire area of PSUs was measured in comedonal lesions, inflammatory lesions, and in controls. Then, 10 sebocytes were chosen randomly from each PSU in comedonal lesions, inflammatory lesions, and in controls; the sizes of sebocytes were measured and the mean value was calculated.
Evaluation of immunohistochemical detection of substance P and neurokinin-1 receptor
The slides were evaluated using a semiquantitative scale (0–3) assessing two parameters 9. Each section was divided into 10 high power fields. Each parameter was assessed in each of the 10 high power fields and a mean value was calculated.
Parameter 1: The percentage of positive (immunoreactive) sebocytes:
- Score 0: less than 10% immunoreactive sebocytes (very weak immunoreactivity).
- Score 1: 10–30% immunoreactive sebocytes (weak immunoreactivity).
- Score2: 30–50% immunoreactive sebocytes (moderate immunoreactivity).
- Score 3: more than 50% immunoreactive sebocytes (strong immunoreactivity).
Slides showing moderate and strong immunoreactivity were considered positive (scores 2 or 3). Slides showing very weak or weak immunoreactivity were considered negative (scores 0 or 1), according to Schulz et al. 9.
Parameter 2: Intensity of immunostaining of sebocytes that is, the intensity of the cytoplasmic and membranous staining color in the sebocytes.
- Grade 0: no staining.
- Grade 1: mild staining (light brown or light black).
- Grade 2: moderate staining (darker than grade 1).
- Grade 3: strong staining (very dark brown or black).
Data were analyzed on an IBM compatible computer using the statistical package for social sciences (SPSS) version 11 (SPSS Inc., Chicago, Illinois, USA). Numerical data were described in terms of means and SD. Categorical data were described in terms of number and percentages. The analytical tests used were Student’s t-test, ANOVA, and Pearson’s correlation coefficient. P value of 0.05 or less was considered significant.
Demographic and clinical data of the patients
The acne patients were 15 men (50%) and 15 women (50%). Their age ranged from 18 to 35 years, mean 21.73±4.12 years. The controls were 10 men (50%) and 10 women (50%). Their age ranged from 18 to 30 years, mean 22.35±3.88 years. No statistically significant difference in age and sex was found between patients and controls (P>0.05). The clinical criteria of the patients are shown in Table 1.
Results of examination of hematoxylin and eosin-stained sections
Examination of H&E-stained sections of controls showed normal-sized PSU with small intact sebocytes (Fig. 1a), whereas comedonal lesions showed apparently enlarged PSUs with apparently larger sebocytes filled with sebum compared with the controls (Fig. 1b). In addition to sebocytes appearing apparently larger than normal, examination of inflammatory lesions showed destroyed and ruptured sebocytes associated with the presence of aggregates of a chronic inflammatory cellular infiltrate (Fig. 1c). Analysis of morphometric results showed significantly larger PSUs and sebocytes in acne lesions compared with the controls (P<0.0001) (Table 2).
Results of immunohistochemical detection of substance P and neurokinin-1 receptor
Percentage of positive (immunoreactive) sebocytes
In contrast to controls’ skin, which showed negative staining (scores 0 or 1), all acne lesions (both comedonal and inflammatory) showed positive SP immunoreactive sebocytes (scores 2 or 3) and the difference between control skin and acne lesions was statistically significant (P<0.0001) (Table 3).
For NK-1R immunoreactivity, only two control biopsies showed positive staining (scores 2 or 3), whereas 18 control biopsies showed negative staining (scores 0 or 1). All acne lesions (both comedonal and inflammatory) showed positive NK-1R immunoreactive sebocytes (scores 2 or 3) and the difference between control skin and acne lesions was statistically significant (P<0.0001).
Intensity of immunostaining of sebocytes
The intensity of immunostaining for both SP and NK-1R in control skin varied from grade 0 to 1 (Figs 2a and 3a), whereas the intensity of immunostaining for SP and NK-1R in acne lesions varied from grade 1 to 3 (Table 4 and Figs 2b and c and 3b–d). Comparison of the intensity of immunostaining of SP and NK-1R between comedonal and inflammatory lesions showed significantly higher intensity of staining in inflammatory lesions (P<0.0001) (Table 5).
Results of immunohistochemical staining of substance P and neurokinin-1 receptor in the epidermis and inflammatory cells
The epidermis in the control skin showed focal positive staining of the basal layer for SP and NK-1R, whereas lesional epidermis, whether comedonal or inflammatory, showed diffuse positive staining.
Dermal inflammatory cell infiltrate in acne lesions, both comedonal and inflammatory, showed diffuse staining with SP and NK-1R, especially those surrounding sebaceous acini in inflammatory lesions (Fig. 2c).
There was a significant direct correlation between the expression of SP and the expression of NK-1R in inflammatory lesions (r=0.665, P=0.009), whereas comedonal lesions showed no correlation between the expression of SP and NK-1R (r=0.223, P=0.225).
The expression of SP in inflammatory and not comedonal lesions showed a positive correlation with disease severity (r=0.853, P=0.025 for inflammatory lesions, and r=0.221, P=0.231 for comedonal lesions) (Fig. 4). No significant correlation of NK-1R expression with disease severity was detected either in inflammatory or in comedonal lesions (r=0.138, P=0.807 for inflammatory lesions, and r=0.328, P=0.786 for comedonal lesions).
There was a significant positive correlation between the expression of SP in both acne lesions and the PSS of the patients (r=0.611, P=0.038 for comedonal lesions, and r=0.770, P<0.0001 for inflammatory lesions). In addition, the expression of NK-1R showed a significant positive correlation in both acne lesions with the PSS (r=0.600, P=0.015 for comedonal lesions, and r=0.870, P<0.0001 for inflammatory lesions).
Age, sex, and duration of the disease did not show any significant relation with the expression of either SP or NK-1R (P>0.05).
Our study found significantly higher expression of SP, a well-known NP, and its receptor NK-1R in sebocytes in acne lesions compared with controls, with the expression being higher in inflammatory lesions compared with comedonal lesions. SP has not been reported to be produced by sebocytes, but it is known to be secreted by nerve endings including periglandular fibers 2. Thus, it is hypothesized that cutaneous nerve fibers in acne patients secrete larger amounts of SP in comparison with controls with subsequent induction of NK-1R expression as evidenced in our study by the significant direct correlation between the expression of both SP and NK-1R and as described by Ping et al. 10 and Asadi et al. 11. Although cutaneous effects of SP are mediated by three different NK receptors: 1, 2, and 3 12,13, NK-1R showed the highest affinity for SP 14,15.
The most logical explanation for the excess production of SP in acne patients is the effect of stress. The relation between acne and stress is a well-known mutual relation 16. Stress leads to activation of the hypothalamic–pituitary–adrenal axis with the release of stress NPs 3,17, particularly SP, which is considered to be the link between the skin and the mind 12. This was confirmed in our study by the finding of a significant direct correlation between the expression of SP and NK-1R in both types of acne lesions and the PSS of the patients. Moreover, it is known that patients with severe acne are more prone to psychological problems 18,19. We found a significant direct correlation between the expression of SP in inflammatory lesions and disease severity. A similar association between SP, NK-1R, and a stress-mediated dermatosis was reported in atopic dermatitis 4,20.
Another explanation for this excess production of SP can be considered in the findings of Toyoda et al.2, who found that facial skin from acne patients contains numerous fine nerve fibers not only around but also within the sebaceous acini, in contrast to the normal facial skin. Such nerve fibers also showed immune-reactivity for SP, which is rarely observed in healthy skin specimens.
The results of our study are in agreement with the results of the early study carried out by Mostafa et al.21, who, in addition, have reported positive staining with SP of sebocytes in non lesional skin biopsies of acne patients, and with the results reported by Toyoda et al.2.
We found expression of SP and NK-1R by inflammatory cells especially in inflammatory lesions mainly around sebaceous acini. It is known that NPs show immunomodulatory activity 12, an activity that must be mediated by inflammatory cells in the skin. It is assumed that in response to psychological stress, NPs, especially SP, are released from sensory nerves and this is followed by activation of mast cells or other inflammatory cells (such as neutrophils and macrophages), which must be expressing NK-1R 2,13,15,22–27. SP is also derived by mast cell degranulation, which further substantiates its effect 25. Other immunomodulatory effects of SP include modulation of the release of proinflammatory cytokines and chemokines and upregulation of adhesion molecule expression required for trafficking of leukocytes 2,25,27,28.
The exact nature of the positively stained inflammatory cells in our study needs further studies. The above-mentioned immunomodulatory effects of SP explain the significantly higher expression that we found in inflammatory lesions compared with comedonal lesions. However, the fact that SP and NK-1R were also highly expressed in comedonal lesions indicates that SP may play a role in the initiation of acne lesions and not only being expressed in relation to inflammation.
Compared with controls, acne lesions showed significantly larger PSUs and sebocytes compared with the controls. This can be attributed to the stimulatory effects of SP on the proliferation of sebocytes and their lipogenic activity that was reported previously both in skin organ cultures and in acne lesions 5,6.
There was a difference in the SP and NK-1R epidermal staining pattern between patients and controls, being weak and restricted to the basal layer only in control skin and strong and diffuse in patients’ skin. This may be because of a difference in the quantity of SP released under physiological conditions and under the conditions of disease processes 21.
Neurogenic factors including NPs (particularly SP) are involved in the pathogenesis of acne. This provides a new insight into possible mechanisms of exacerbation of acne from a neurological point of view and highlights the importance of further research into the development of new therapeutic approaches for acne using SP and NK-1R antagonists as targets.
Conflicts of interest
There are no conflicts of interest.
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