|Year : 2016 | Volume
| Issue : 2 | Page : 396-405
Caspase-3 expression in lichen planus
Alaa H Maraeea1, Hala S El-Rebey2, Eman A Zaky1
1 Department of Dermatology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||16-Aug-2014|
|Date of Acceptance||10-Nov-2014|
|Date of Web Publication||18-Oct-2016|
Eman A Zaky
Berket El-Sabae, Menoufia, 32651
Source of Support: None, Conflict of Interest: None
The aim of this study was to examine keratinocyte apoptosis and caspase-3 expression in lichen planus (LP).
Caspase-3 expression level in LP lesions is considered a reliable marker of apoptosis, which is frequently found in LP, but the pathways leading to apoptosis are unknown.
Patients and methods:
A total of 25 skin biopsy specimens from LP lesions were used and 17 healthy volunteers were used as controls. Histopathological examination of hematoxylin and eosin-stained sections of LP was carried out for the evaluation of histopathological parameters. The expression of caspase-3 was examined immunohistochemically.
There were highly significant differences between caspase-3 immunostaining in epidermal keratinocytes in normal skin and the lesional area of LP (P= 0.006). No statistically significant correlation was found between caspase-3 expression in keratinocytes of LP cases and the histopathological parameters studied. No statistically significant correlation was found between caspase-3 expression in dermal lymphocytes and the histopathological parameters studied. A significant relation was found between various intensities of caspase-3 expression in dermal lymphocytes and the age of the individuals (P= 0.03), where the younger age group showed a strong intensity, whereas in older individuals, the mild expression predominated, which may indicate more severe disease with more apoptosis in early-onset LP.
Caspase-3 expression in epidermal keratinocytes and dermal lymphocytes may play a role in the pathogenesis of LP.
Keywords: apoptosis, caspase-3, lichen planus
|How to cite this article:|
Maraeea AH, El-Rebey HS, Zaky EA. Caspase-3 expression in lichen planus. Menoufia Med J 2016;29:396-405
| Introduction|| |
Lichen planus (LP) is a chronic inflammatory disorder involving cutaneous and mucosal surfaces, characterized by a T-cell-mediated immune response . It is characterized by violaceous, scaly, flat-topped, polygonal papules commonly involving the flexural areas of the wrists, legs, but it is uncommon in children . Caspases are evolutionarily conserved cysteine proteases; they are synthesized as inactive proenzymes and become activated following cleavage at specific cleavage sites . Caspases play important roles not only in the induction of apoptosis but also in inflammatory processes; thus, caspases can be divided into apoptotic caspases, including caspase-2, −3, −6, −7, −8, −9, and −10, and inflammatory caspases, including caspase-1, −4, and −5 .
Caspase-3 is the most downstream enzyme in the apoptosis-inducing protease pathway, and is probably the most clearly associated with cell death . It has been proposed that many characterized changes in apoptosis cell may be because of the activation of caspase-3 . Some authors supposed that caspase-3 potentially contributes toward the apoptosis of keratinocyte observed in the LP by inducing the expression of other apoptosis-related proteins and therefore caspase-3 might play a major role in the pathogenesis of LP .
The pathogenesis of LP features a complex series of interactions between inflammatory cells, chemokines, and cytokines that ultimately determine the apoptosis of basal keratinocytes, triggered by the contacts between CD8+ T cells and the basal cells .
It was suggested that apoptosis is very important in the pathogenesis of LP. Thus, in this study, the expression of caspase-3, being a member of the apoptosis-related proteins, was immunohistochemically detected in skin lesions of patients with LP to examine its role in the pathogenesis of LP.
This current study aimed to evaluate the role of caspase-3 in immunogenesis of LP.
| Patients and Methods|| |
This study was carried out on 25 patients with LP and 17 healthy volunteers as controls. The patients were selected randomly from the Outpatient Dermatology Clinic, Menoufia University Hospital, during the period between April 2010 and June 2012, whereas controls were recruited from the Plastic Surgery Department. All patients provided their formal consent to participate. The protocol was approved by the Ethical Committee of the Faculty of Medicine, Menoufia University.
The diagnosis of LP was made on the basis of patients' history and the typical clinical features that were confirmed by histopathological examination. Patients stopped medical treatment, either topical or systemic, 2 weeks before the biopsy. Each of the selected patients was subjected to the following:
- Complete assessment of history.
- Clinical examination.
- Skin biopsy: After obtaining written consent from the patients, biopsies were obtained under local anesthesia (from the lesion and normal skin). All biopsies were processed in the Pathology Department, Faculty of Medicine, Menoufia University, where they were fixed in 10% neutral-buffered formalin, dehydrated in ascending grades of ethanol, followed by xylene, and then impregnated in paraffin. Three 5-µm-thick sections from each block were taken. One was stained with hematoxylin and eosin for routine histopathological examination. The other two sections were mounted on Superfrost Plus (VWR International Ltd., Hunter Boulevard, Magna Park, Lutterworth, Leicestershire LE17 4XN, England) slides and stored at room temperature; one was stained immunohistochemically for caspase-3 and the other was used as a negative control.
Immunohistochemical staining for caspase-3
Mouse monoclonal antibody raised against caspase (CPP32) Ab-3 was used. It was received as 0.1 ml concentrated and diluted by PBS in a 1: 50 dilution (Cat #MS-1123-P0; Lab Vision, Immunohistochemistry Fremont, CA) with human tonsil as a positive control. The immunostaining technique applied in this study was the improved streptavidin – biotin amplified system. In this system, three reagents were utilized: the primary antibody specific for the antigen to be localized, the biotinylated secondary anti-immunoglobulin that is capable of binding to both the primary antibody and the streptavidin – biotin enzyme complex. Finally, the reaction was visualized by an appropriate substrate/chromogen reagent: diaminobenzidine.
Histopathological evaluation of hematoxylin and eosin-stained sections
- The sections stained with hematoxylin and eosin were examined under a light microscope to confirm the diagnosis of LP and detect any epidermal pathological changes.
- Apoptosis of basal keratinocytes: Quantitative analysis of the number of apoptotic bodies was carried out in high (×400) magnification. These apoptotic bodies, also known as civatte or colloid bodies, appear as round, shrunken homogeneous eosinophilic bodies in the stratum basale. Each (×400) high-power field was analyzed along the entire sample, and the values were recorded. To standardize the counting, the basal layer was positioned in the middle of the field. For statistical purposes, apoptotic values were classified as follows: frequent (≥median value) or infrequent (<median value) .
- Hyperkeratosis; determined subjectively as to whether present or absent.
- Hypergranulosis; this was assessed subjectively to be either mild, moderate, or severe.
- Acanthosis; the pattern was assessed subjectively as either focal or diffuse.
- Evaluation of the dermis:
The dermal inflammatory infiltrate, present as either band like or focal.
Interpretation of caspase-3 expression
Caspase-3 immunoreactivity was evaluated as negative or positive, where positive cases were considered to have diffuse brown cytoplasmic expression in more than 10% of cells. The intensity of expression was assessed subjectively as mild, moderate, and strong. The expression was assessed in epidermal keratinocytes and dermal lymphocytes.
Results were collected, tabulated, and statistically analyzed using an IBM personal computer and the statistical package SPSS (version 17; SPSS Inc., Chicago, Illinois, USA). Two types of statistics were calculated:
Descriptive statistics: for example, percentage, mean (x), and SD.
- The Fisher exact test was used to study the association between two qualitative variables.
- The Mann–Whitney U-test (nonparametric test) is a test of significance used for comparison between two groups with non-normally distributed quantitative variables.
- The χ2-test was used for comparison between qualitative variables in different groups.
- The Kruskal–Wallis test (nonparametric test) was used to test the equality of population medians among groups; it is an extension of the Mann–Whitney U-test.
- A P value of 0.05 or less was considered statistically significant.
| Results|| |
Clinicopathological data of the cases studied
This case–control study included 25 patients with LP, 16 men and nine women, ranging in age between 18 and 60 years (mean age 36.64 ± 13.24 years). Seventeen normal age-matched and sex-matched individuals were recruited as the control group. They included 10 men and seven women ranging in age between 15 and 60 years (mean age 33.0 ± 13.42 years). Most of the patients, 18 (72%), presented with the common type of LP, whereas other types of LP were found in 28%, including three (12%) patients with hypertrophic-type LP and three (12%) patients with actinic-type LP, and only one (4%) patient had combined common and actinic lesions. The duration of the disease ranged between 0.08 and 20 years, with a mean duration of 1.85 ± 4.020 years and a median duration of 0.5 years. The lesions were located either axially (face and trunk) in seven (28%) patients or on the extremities (hands and feet) in seven (28%) patients or combined axial and extremities in 11 (44%) patients. Oral affection was present in 14 (56%) patients. Itching was the main complaint in 22 (88%) patients and not recorded in the other patients. The hepatitis C virus (HCV) was present in six (24%) patients and diabetes mellitus was present in only one (4%) patient ([Table 1]).
|Table 1: Descriptive statistics of clinicopathological data among patients|
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Caspase-3 immunohistochemical results
Expression of caspase-3 in normal controls
The normal skin from the control group showed positive keratinocytes caspase-3 expression in five out of 17 (29.4%) patients. The distribution of caspase-3 expression was mainly diffuse, cytoplasmic in two (37.5%) patients, and both cytoplasmic and nuclear in three (62.5%) patients. The intensity of caspase-3 expression was mild in four (80%) patients and moderate in one (20%) patient; no patient presented with a strong intensity of caspase-3 expression ([Table 2] and [Figure 1] and [Figure 2]).
|Table 2: Comparison between cases and controls in caspase-3 expression in keratinocytes|
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|Figure 1: Normal skin showing mild nucleocytoplasmic caspase-3 expression (immunoperoxidase, ×200)|
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|Figure 2: Normal skin showing moderate nucleocytoplasmic caspase-3 expression (immunoperoxidase, ×200)|
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Expression of caspase-3 in lichen planus cases
Keratinocytes of LP specimens showed positive caspase-3 expression in 18 out of 25 (72%) patients. The intensity of expression varied from mild in eight (44.4%) patients to moderate in seven (38.9%) patients and strong in three (16.7%) patients. Twelve (66.7%) patients showed only cytoplasmic expression with a diffuse pattern, whereas six (33.3%) patients showed both cytoplasmic and nuclear expression of focal distribution ([Table 2] and [Figure 3] and [Figure 4]).
|Figure 3: A patient with lichen planus showing moderate nucleocytoplasmic caspase-3 expression (immunoperoxidase,×400)|
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|Figure 4: A patient with lichen planus showing mild cytoplasmic caspase-3 expression (immunoperoxidase, ×200)|
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Correlation between caspase-3 expression in keratinocytes and the clinicopathological parameters studied
There was no significant correlation between the parameters studied in LP cases in terms of keratinocyte expression of caspase ([Table 3]).
|Table 3: Comparison of caspase-3 expression in keratinocytes in terms of clinicopathological data|
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Correlation between caspase-3 expression in dermal lymphocytes and the clinicopathological parameters studied
No statistically significant correlation was present between caspase-3 expression in dermal lymphocytes and the parameters studied ([Table 4] and [Figure 5] and [Figure 6]).
|Table 4: Comparison of caspase-3 expression in lymphocytes in terms of clinicopathological data|
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|Figure 5: Moderate cytoplasmic caspase-3 expression in lymphocytes in a patient with lichen planus (immunoperoxidase, ×200)|
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|Figure 6: A patient with lichen planus showing moderate nucleocytoplasmic caspase-3 expression in lymphocytes (immunoperoxidase, ×200)|
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Correlation between the intensity of caspase-3 expression in dermal lymphocytes and the clinicopathological parameters studied
There was no significant relation between various intensities of caspase-3 expression in lymphocytes and the parameters studied, except for the age of the patients (P = 0.03), where the younger age group showed strong intensity but in older patients the mild expression predominated ([Table 5]).
|Table 5: Comparison of intensity of caspase-3 expression in lymphocytes in terms of clinicopathological data|
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| Discussion|| |
LP is a chronic inflammatory disorder involving cutaneous and mucosal surfaces . The pathogenesis of LP has remained unclear, but apoptotic cell death may be a potential cause for basal cell destruction in the disease ,. It is a T-cell-mediated inflammatory disease with a pathogenetic role of both CD8+ cytotoxic T cells and CD4+ helper T cells. There is a predominance of cytotoxic T cells in the dermoepidermal infiltrate responsible for keratinocyte damage. Helper CD4+ T cells were found in the perivascular dermal infiltrate, assuming that it may play a role in assisting the cytotoxic cells in the keratinocyte damage .
Caspase-3 is the most downstream enzyme in the caspase cascade, and it has been proposed that many characteristic changes in apoptotic cells may be because of the activation of caspase-3. Some authors confirm that caspase-3 is a very early and specific marker of apoptosis .
In the current study, the age of the LP patients ranged from 18 to 60 years (mean age 36.64 ± 13.24 years). According to Anbar et al. , the age range was 10–65 years; the majority of the patients were in the 21–50 year age group. Another study found that the patient ages ranged from 6 to 73 years, most being in the age range from 34 to 59 years (mean age 39.7 years) ,. Some studies have concluded that the age of LP patients ranged from 30 to 60 years . This difference may be because of small sample sizes or genetic factors.
In terms of the sex distribution of participants included in the current study, there were 16 (64%) male patients out of a total of 25 patients, with a male to female ratio of 1.7: 1 as Abdel-Latif et al. reported. Moreover, Anbaret al. reported a male: female ratio of 1.3: 1. However, some studies have reported that females are more affected with LP than males ,. Others have reported equal male to female ratios , and this difference can be explained by the small number of the cases studied or ethnicity factor.
The duration of the LP in this study ranged from 0.8 to 20 years, mean 1.85 ± 4.02 years, whereas Abdel-Latif et al.  reported a duration of LP to range from 6 months to 4 years (mean 1.6 years). Also, Nofal et al.  found that the duration of the LP in their studied cases ranged from 1 to 6 years (mean 3.54 ± 1.68 years). This indicates that LP is a chronic inflammatory disorder.
In the current study, the clinical types of the LP were 72% classic LP, and other types (28%) presented as 12% hypertrophic LP, 12% actinic LP, and 4% combined lesions. This result was in agreement with that of Aly and Shahin , who reported the predominance of the classic type (48.8%) among LP patients, followed by the hypertrophic (20%) and the actinic (31.1%) types. However, Anbar et al.  reported a predominance of actinic LP (36%), followed by classic LP (30%) and hypertrophic LP (12%). The predominance of the actinic-type LP in that study might be explained to the sunny site of the study as it was carried out in Al-Minya.
Oral mucosa affection was reported in 50% of patients with skin lesions by Xue et al.  and Aly and Shahin . In our study,56% of the total LP cases also had oral mucosal lesions. However, Kanwar and De  reported mainly skin involvement with the oral mucosa affected very rarely (2%); this may be attributed to the fact that the study was carried out on 100 children younger than 18 years old in whom LP is considered to be rare.
Infection with HCV has been speculated to be a risk factor for the development of LP . Abo-Elwafa et al.  found a highly significant increase in HCV infection among the LP group (38.6%) compared with the control group (10%). The association between HCV and LP was 24% of the total LP cases in this study. However, majority of the studies could not prove such an association ,; this difference may be because of the ethnicity factor.
In the epidermal keratinocytes of normal skin, immunostaining for caspase-3 was present in five (29%) controls; most cases (80%) had mild (weak) intensity and only 20% presented with moderate staining. All cases showed a diffuse patchy distribution of staining and no staining of lymphocytic cellular infiltrate. Also, Chen et al.  reported weak immunoreactivity of caspase-3 expression in 20% of the controls. Abdel-Latif et al.  reported positive mild immunoreactivity of caspase-3 with a homogenous distribution in basal and suprabasal layers in only 10% of the controls.
In the current work, there was a highly statistically significant difference between keratinocyte expression of caspase-3 of patients with LP compared with the controls (P=0.006). In LP specimens, the keratinocyte expression of caspase-3 was positive in 18 (72%) patients and negative in seven (28%) patients. The intensity of staining was mild in eight (44.4%) patients, moderate in seven (38.9%) patients, and strong in three (16.7%) patients. The distribution of expression was 66% diffuse pattern involving all epidermal layers and 33% focal pattern in basal and suprabasal layers. This means that basal and suprabasal layers were present in all patients with LP (100%). This indicates that proliferating basal epithelial cells may be targeted for apoptosis in LP despite being of little quantitative importance as 44% of cases showed only a mild expression. In LP keratinocytes, the site of expression was mostly cytoplasmic in 16 (76.2%) patients and less frequently combined cytoplasmic and nuclear in five (23.8%) patients.
These results are in agreement with those of Abdel-Latif et al. , who found a significant (P<0.05) increase in caspase-3 expression in LP lesions compared with the controls. Also, they found that caspase-3 was expressed more often in basal (56%) and suprabasal (48%) epithelial cells in patients with LP than in controls. Immunoreactivity for caspase-3 was found in the cytoplasm and often in the nuclei of keratinocytes and lymphocytes of LP specimens. Also, our results are in agreement with those of Chenet al.  who found that the expression of caspase-3 in normal skin was very low in two (20%) participants out of 10 controls. The expression presented as a few patchy areas of staining. Meanwhile, in LP skin lesions, caspase-3 immunoreactivity was detected in 25 (86.5%) patients with LP out of a total of 29 patients. Among these patients, 11 had strong expression, eight had moderate expression, and six had weak expression. In addition, the expression of caspase-3 was strong in basal cells and spinose cells in LP skin lesions, suggesting that the caspase cascade is active and apoptosis occurs within the proliferating compartment of LP.
These results were not in agreement with those of Mattila and Syrjänen , who reported that caspase-3 expression was detected in only 43% of cases, but 27/30 patients with positive caspase-3 expression showed intense expression in the cytoplasm of epithelial cells. This difference may be because of the small sample size or because samples were obtained from different sites.
In our study, no significant correlation was found between the LP patients studied in keratinocyte expression of caspase-3 (P > 0.05) for all the parameters studied and this is in agreement with Chen et al. , who showed that the expression levels of caspase-3 showed no relation to age and sex parameters of patients.
In this study, no significant correlation was found between the LP patients studied in terms of caspase-3 expression in lymphocytes (P > 0.05) for all the parameters studied. Also, no significant correlation was found between the clinicopathological parameters studied in patients in terms of the intensity of caspase-3 expression in lymphocytes (P > 0.05), except for age (P = 0.03), as strong intensity was observed in younger age groups. This may be because of severe inflammation in early-onset disease, but this result was not proved by previous studies.
| Conclusion|| |
Caspase-3 expression in epidermal keratinocytes and dermal lymphocytes may play a role in the pathogenesis of LP and apoptosis of epidermal keratinocytes.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]