Immunohistochemical Expression of Calmodulin in Cutaneous Lichen Planus: A Case-Control Study.

BACKGROUND: Calmodulin (CaM) is a multifunctional intermediate messenger protein that plays important role in cell motility, proliferation, and apoptosis. Therefore, it is thought to be involved in various ways in the apoptotic processes which are implicated in the pathogenesis of lichen planus.
OBJECTIVE: The aim of this study was to evaluate the immunohistochemical expression of CaM in lichen planus lesions in comparison to normal control skin to throw light on its possible role in disease pathogenesis. PATIENTS AND METHODS: This case-control study was conducted on 50 patients with lichen planus, in addition to 20 age- and sex-matched healthy individuals. Skin biopsy specimens were taken from lesional skin of lichen planus patients as well as normal skin of controls. All were examined for immunohistochemical expression of CaM antibody.
RESULTS: There was statistically significant increase of the immunohistochemical expression of CaM in lesional skin of lichen planus patients compared with normal skin of controls (Chi-square test, P < 0.001). No significant correlation could be detected between CaM expression in lesional skin and the studied clinical parameters of lichen planus patients. LIMITATIONS: Tha main limitation of this study is its small sample size.
CONCLUSION: CaM is upregulated in cutaneous lichen planus lesions suggesting a possible role in disease pathogenesis. Targeting CaM is expected to be a novel strategy for treatment of lichen planus.

Introduction

Lichen planus is a subacute to chronic, inflammatory, papulosquamous disease that affects the skin and mucous membranes with impact on the patient's quality of life. It has been classified as a complex multifactorial disease depending on genetic, environmental, and immunological interactions.[123] It is characterized by infiltrating immune cells, specifically T-lymphocytes, and natural killer and dendritic cells. Apoptosis is central to the pathogenesis of lichen planus, as evidenced by cytotoxic T-cell infiltration into the epidermis, with subsequent secretion of tumor necrosis factor-alpha (TNF-α) and CD95 L (FasL) that are implicated in triggering the cascade of basal keratinocytes (KCs) apoptosis.[456]

Calmodulin (calcium-modulated protein, CaM) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells.[7] It is an intracellular target of the secondary messenger Ca2+ and the binding of Ca2+ is required for the activation of CaM. Once bound to Ca2+, CaM acts as part of Ca2+ signal transduction pathway by modifying its interactions with various target proteins such as kinases or phosphatases.[89]

CaM has been shown to play an important role in cell cycle progression and apoptosis regulation. In cell cycle progression, the concentration of CaM progressively increases till reaches high levels at the G1/S transition and remains high during the ensuing progression of the cell cycle. In apoptosis regulation, CaM regulates apoptotic processes by both positively and negatively mediating elevated intracellular Ca2+, which can have both growth promoting and cell death-inducing consequences.[10] Therefore, it is thought to be involved in the apoptotic processes which are implicated in the pathogenesis of lichen planus.

The current work aimed to study the immunohistochemical expression of CaM in lichen planus to throw light on its possible role in the pathogenesis of this disease.

Patients and Methods

This was a case–control study conducted during the period between September 2016 and March 2017. Since we could not find any previous study on CaM expression in lichen planus lesions, to calculate the sample size, we conducted a pilot study on 50 patients with lichen planus and 20 normal skin specimens from age- and sex-matched healthy individuals to serve as a control group (case-to-control ratio was 2.5:1). Patients were selected from the Outpatient Clinic of Dermatology and Venereology Department, Tanta University Hospitals. Ethical approval was obtained from the Research Ethics Committee, Faculty of Medicine, Tanta University (code No: 549/07/17) before commencement of the study. The purpose of the study was explained and written informed consent was obtained from all participants. Their data were kept confidential by code number given to each participant. Inclusion criteria were patients with lichen planus diagnosed on the basis of clinical features and confirmed by histopathology and were willing to join the study. Exclusion criteria included patients who received any topical or systemic treatment for lichen planus in the preceding 2 months before biopsy taking, patients with any other dermatological or systemic diseases, patients with a history of drug intake, and pregnant and breastfeeding females.

All participants were subjected to careful history taking, thorough general and dermatological examinations. Detailed dermatological examination was done including examination of skin lesions, hair, nail, and mucous membrane to determine the clinical type, distribution, and extent of the lesions in all patients. Routine laboratory investigations and hepatitis-C markers were done for all participants.

Punch skin biopsy specimens (4 mm in diameter) were taken under local anesthesia and complete aseptic technique from lesional skin of lichen planus patients. In addition, normal control skin specimens were obtained from healthy individuals during plastic operations. All specimens were fixed in neutral formalin 10% and processed for paraffin blocks. 3–5 microns thickness section was cut from each block on poly-L-lysine-coated glass slides and stained immunohistochemically using rabbit monoclonal anti-CaM antibody [Cat. # EP799Y (ab45689), Abcam, United States] to detect CaM expression. The primary monoclonal anti-CaM antibody was used at a 1:2000 dilution in accordance with the manufacturer's instructions. A goat anti-rabbit immunoglobulin G (IgG) H and L (ab97051) was used as the secondary antibody at a dilution of 1/500. All slides were examined by light microscopy at the same magnification for evaluation of:

The pattern of CaM immunostaining: For each specimen, 3–5 separate fields of view were analyzed in all epidermal cell layers: basal cell layer and suprabasal layers. The expression of CaM was considered positive if immunostaining detected as nuclear, cytoplasmic, and/or membranous patterns by image analysis Leica Qwin

The intensity of CaM immunostaining was graded on a semi-quantitative scale in positive cells from 0 to +3 as follow:[11]

(0): Negative expression

(+1): Mild expression

(+2): Moderate expression

(+3): Strong expression.

The distribution of CaM immunostaining was evaluated as follow:

Basal: if immunostaining limited to basal cell layer of the epidermis

Both basal and suprabasal: if immunostaining involved both basal and suprabasal layers of the epidermis. It was either interrupted or diffuse pattern.

Statistical analysis

Data were analyzed using IBM SPSS software package version 20.0 (Armonk, NY: IBM Corp). Qualitative data were described using number and percent. Quantitative data were described using range (minimum and maximum) and mean ± standard deviation. Comparison between different groups regarding categorical variables was tested using Chi-square test. For normally distributed data, comparison between two independent populations was done using independent t-test. For abnormally distributed data, Kruskal–Wallis test was used. A significant difference was considered at P < 0.05.

Results

Clinical results

The clinical features of all participants enrolled in this study were summarized in Table 1.

Table 1

Distribution of demographic data among two groups

	Control (n=20)	LP (n=50)
Gender, n (%)
Male	10 (50.0)	20 (40.0)
Female	10 (50.0)	30 (60.0)
Age (year)
Minimum–maximum	26.0–45.0	30.0–75.0
Mean±SD	39.40±5.20	43.56±10.65
Median	39.50	43.0
Duration (year)
Minimum–maximum		0.01–1.0
Mean±SD		0.28±0.31
Median		0.17
Hepatitis C virus, n (%)
Negative		26 (52.0)
Positive		24 (48.0)
Clinical type, n (%)
Classical LP		8 (16.0)
Actinic LP		22 (44.0)
Hypertrophic LP		6 (12.0)
Atrophic LP		8 (16.0)
Generalized with oral		6 (12.0)

LP: Lichen planus, SD: Standard deviation

Pathological results

The results of immunohistochemical expression of CaM were as follow:

In normal control skin specimens, CaM expression was observed in the epidermis as positive nuclear immunostaining limited to the basal cell layer only. The intensity of its expression was mild in all the 20 specimens (100%) [Figure 1]

Figure 1

Control skin specimen showed mild (+1) nuclear expression of calmodulin in the basal cell layer of the epidermis (Immunohistochemistry, ×400)

In all the 50 specimens of lichen planus (100%), CaM expression was observed as positive nuclear and cytoplasmic immunostaining of the epidermal KCs involving both basal cell layer and suprabasal layers. The intensity of its expression in the epidermis was moderate in 12 specimens (24%) and strong in 38 specimens (76%). In addition, nonspecific expression of CaM was detected in the dermal inflammatory cell infiltrate [Figures 2-4]

Figure 2

(a) Lichen planus specimen showed moderate (+2) interrupted pattern of calmodulin nuclear expression in both the basal and suprabasal layers, with nonspecific expression in dermal inflammatory cells infiltrate (Immunohistochemistry, ×100). (b) High-power view of the previous section (Immunohistochemistry, ×400)

Figure 4

(a) Lichen planus specimen showed strong (+3) diffuse nuclear and cytoplasmic expression of calmodulin in all epidermal layers, with nonspecific expression in dermal inflammatory cells infiltrate (Immunohistochemistry, × 100). (b) High-power view of the previous section (Immunohistochemistry, ×400)

There was statistically significant increase of CaM expression in lesional skin specimens of lichen planus patients in comparison to normal control skin specimens (P < 0.001) [Table 2]

Table 2

Comparison between the studied groups according to intensity of calmodulin expression

Intensity of calmodulin	Control (n=20), n (%)	LP (n=50), n (%)	χ2	P
Negative (0)	0	0 (0.0)	78.871	<0.001
Mild (+1)	20 (100.0)	0 (0.0)
Moderate (+2)	0 (0.0)	12 (24.0)
Strong (+3)	0 (0.0)	38 (76.0)

χ2: Chi-square test for comparison between the studied groups, using χ2 or Fisher’s exact test, LP: Lichen planus

No significant correlation could be detected between intensity of CaM expression in lesional skin of lichen planus patients and the studied clinical parameters regarding gender, age, duration, onset (acute or chronic), distribution (localized or generalized), clinical types of the disease, association with oral lesions, or HCV infection.

(a) Lichen planus specimen showed moderate (+2) diffuse nuclear and cytoplasmic expression of calmodulin in all epidermal layers, with nonspecific expression in dermal inflammatory cells infiltrate (Immunohistochemistry, ×100). (b) High-power view of the previous section (Immunohistochemistry, ×400)

Discussion

The current work evaluated the immunohistochemical expression of CaM in lesional skin of lichen planus patients in comparison to normal skin to throw some light on its possible role in disease pathogenesis. To the best of our knowledge, no previous study has been done to evaluate CaM expression in lichen planus.

In the present study, CaM expression was observed in all lichen planus specimens (100%) as positive nuclear and cytoplasmic immunostaining of the epidermal KCs involving both basal and suprabasal cell layers. However, in normal skin of control specimens, just mild CaM expression could be observed limited to basal cell layer only. There was statistically significant increase of CaM expression in lichen planus lesions in comparison to normal skin allowing us to suggest that it might participate in the pathogenesis of lichen planus. However, the exact cause of CaM overexpression in lichen planus and its mechanism of action in disease pathogenesis are still unknown.

As CaM is the main intracellular Ca2+ signal mediator protein that plays an important role in cell viability and proliferation, it is not surprising that its interaction with target proteins and their modulation have major impact on cell death and apoptosis. In addition, because elevated intracellular Ca2+ can have both growth promoting and cell death-inducing consequences, it is possible to suggest that CaM can regulate apoptotic processes both positively and negatively.[10] It is involved in various ways in different types of apoptotic processes.[12] One of the possible mechanisms of CaM role in apoptotic pathway includes the activation of transcriptional programs that functionally involves the CaM-binding protein neurogranin. Neurogranin, which is transcriptionally induced by interleukin 2 (IL-2), increases the intracellular Ca2+ concentration leading to cell death by sequestering CaM and stabilizing its Ca2+-free form, thereby increasing intracellular Ca2+.[13]

The importance of CaM in apoptotic pathways is also highlighted by the evolutionary conservation of CaM-dependent cell death pathways. One example is its participation in an apoptosis pathway in Drosophila, where Reaper, which has similarity to vertebrate Fas and the TNF-α receptor, has been shown to activate a Ca2+/CaM-dependent signaling pathway finally leading to caspase activation and death.[1415]

Moreover, among viruses that are suspected of having an association with oral lichen planus are Epstein–Barr virus (EBV) and human immunodeficiency virus (HIV).[16] A major effect of HIV infection is the apoptotic death of T-cells known to be frequently mediated by Ca2+ signaling. It was found that CaM may be involved in HIV-triggered and Fas-dependent T-cell apoptosis.[1718]

Contini et al.[19] demonstrated that HLA-Class I molecules trigger apoptosis in activated CD8+ EBV-specific cytotoxic T-lymphocytes by FasL/Fas interaction. The apoptotic effect of human leukocyte antigen-I on CD8+ EBV-specific cytotoxic T-lymphocytes was found to be dependent among other factors on the CaMK-II-signaling pathway. This is followed by nuclear translocation of the transcription factor together with nuclear factor kappa-B that was responsible for inducing FasL expression followed by T-cell apoptosis.

In addition, the efficacy of immunosuppressive therapy, such as, cyclosporine and tacrolimus in the treatment of lichen planus may be due to reduction of CaM level. Cyclosporine binds to the cytosolic protein, cyclophilin of immunocompetent lymphocytes, especially T-lymphocytes. This complex of cyclosporine and cyclophilin inhibits calcineurin, which under normal circumstances induces the transcription of IL-2. They also inhibit lymphokine production and IL release, leading to a reduced function of effector T-cells.[20]

The results of the current study point out to interesting different pathways in the pathogenesis of lichen planus. Based on the fact that, apoptosis is central to the pathogenesis of lichen planus, as evidenced by cytotoxic T-cell infiltration into the epidermis with subsequent basal KCs apoptosis,[4] it is possible to suggest that CaM may participate in the pathogenesis of lichen planus through promotion of KC apoptosis as follow:

CaM seems to promote cell apoptosis through activation of IL-2 and TNF-α

CaM seems to promote cell apoptosis through activation of HIV or EBV which are implicated in the pathogenesis of oral lichen planus.

The efficacy of calcineurin inhibitors, which act as CaM inhibitors, in the treatment of lichen planus, adds further support to this suggestion.

CaM level was previously evaluated in a range of inflammatory skin diseases including psoriasis,[21222324] atopic dermatitis,[23] and seborrheic dermatitis[23] in comparison to normal skin using CaM 125I radioimmunoassay kit for measuring immunologically reactive CaM. The mean values of CaM levels were significantly elevated only in psoriasis and seborrheic dermatitis lesions and not in atopic dermatitis. Those data in addition to the results of the current work suggested that increased CaM expression in lichen planus lesions observed in this study was not something specific to lichen planus as it was previously detected in other inflammatory skin diseases that are associated with KC hyperproliferation and/or impairment of differentiation such as psoriasis and seborrheic dermatitis.

Limitation of our work is that it was a case–control study conducted with a small sample size. Moreover, it was not designed to evaluate CaM expression in lichen planus before and after treatment with CaM inhibitors (e.g., calcineurin inhibitors). Thus, further prospective studies are needed on larger scale population to throw more light on the possible role of CaM in the pathogenesis of lichen planus.

Conclusion

CaM expression is upregulated in skin lesions of lichen planus suggesting a possible role in disease pathogenesis. Furthermore, targeting CaM expression in lichen planus patients in the future studies may open new insights for effective therapeutic approaches for lichen planus.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.