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Effect of PUVA therapy on melanocytes and keratinocytes in non-segmental vitiligo: histopathological, immuno-histochemical and ultrastructural study
Article first published online: 28 DEC 2011
© 2012 John Wiley & Sons A/S
Photodermatology, Photoimmunology & Photomedicine
Volume 28, Issue 1, pages 17–25, Febuary 2012
How to Cite
Anbar, T. S., El-Sawy, A. E., Attia, S. K., Barakat, M. T., Moftah, N. H., El-Ammawy, T. S., Abdel-Rahman, A. T. and El-Tonsy, M. H. (2012), Effect of PUVA therapy on melanocytes and keratinocytes in non-segmental vitiligo: histopathological, immuno-histochemical and ultrastructural study. Photodermatology, Photoimmunology & Photomedicine, 28: 17–25. doi: 10.1111/j.1600-0781.2011.00631.x
- Issue published online: 28 DEC 2011
- Article first published online: 28 DEC 2011
- Manuscript Accepted: 17 AUG 2011
Background and Aims
Psoralen ultraviolet A (PUVA) is an important modality in treating vitiligo. Its effect on melanocytes and keratinocytes is not sufficiently studied. In this work, we investigated 30 cases of non-segmental vitiligo regarding the changes of melanocytes and keratinocytes in both vitiliginous and nearby areas before and after PUVA therapy.
Three skin biopsies were obtained from each patient from the vitiliginous, marginal and perilesional areas before and after 12 months of PUVA. Biopsies were examined histologically using haematoxylin and eosin, Masson–Fontana stains and 3,4-dihydroxyphenylalanine (DOPA) reaction and histochemically using human melanoma black-45 (HMB-45) antibody while ultrastructural examination was performed on six patients. Control biopsies were taken from five healthy volunteers.
In 10% of pretreated biopsies from the centre of vitiligo lesions, scanty melanocytes were detected histologically and ultrastructurally, while they did not stain with DOPA or HMB-45 antibody suggesting that these melanocytes were inactive. Moreover, degenerative changes were detected by electron microscopy in both melanocytes and keratinocytes in all areas. After PUVA therapy, obvious improvement of the histopathological changes occurred with significant increase in active melanocytes. The degeneration of melanocytes and keratinocytes was also reduced at the ultrastructural level.
Vitiligo affects both melanocytes and keratinocytes causing degenerative changes. These changes were present in both the leucodermic and the apparently normal perilesional skin. PUVA increases the number of active epidermal melanocytes in the three tested areas and recovers the melanocyte and keratinocyte degeneration.
Vitiligo is an acquired idiopathic pigmentary disorder of skin and hair characterized by well-circumscribed asymptomatic white macules . There is no universally effective medical or surgical modality for vitiligo therapy; however, there are a number of active therapeutic approaches that are known to be effective . Phototherapy, including narrow band ultraviolet B (NB-UVB) and psoralen ultraviolet A (PUVA), is considered a successful modality among those approaches .
Many researchers studied the clinical effect of PUVA on vitiligo; however, only two studied the PUVA-induced repigmented areas regarding the histopathological changes in melanocytes (MCs) with no comparison between before and after therapy [4, 5]. In addition, there were no comments on keratinocyte (KC) changes. Even the only report dealing with histopathological, immuno-histochemical and ultrastructural changes in vitiligo patients before and after phototherapy used a combination of NB-UVB and pseudocatalase . Thus, the role of NB-UVB alone in these changes is still unclear.
In this work, we studied the effect of PUVA on vitiligo MCs and KCs using 3,4-dihydroxyphenylalanine (DOPA) reaction, immuno-histochemical stain [human melanoma black-45 (HMB-45)] and electron microscope.
Patients and methods
The present study was approved by Al-Minya University Committee concerned with the approval of the researches from the scientific and ethical points of views. The start date of the study was December 2007 and the end date was July 2010, so the duration of the study was 2 years and 7 months. The study had been conducted on 30 cases of vitiligo who received PUVA according to Gupta and Anderson's protocol  after signing a written consent to be included in the study. The methoxypsoraline used was ultra-micronized 8-methoxypsoralen at a dose of 0.4 mg/kg of body weight/session on a full stomach 1 hour before ultraviolet A (UVA) exposure (ultra-melandinine capsules; 10-mg capsules, Memphis Pharmaceutical Co., Cairo, Egypt). Patients received PUVA sessions twice weekly using a UVA compact irradiation cabin (GP-42, Cosmedico Medizintechnik, Villingen-Schwenningen, Germany).
From each patient, 5-mm punch skin biopsies were obtained, using 2% lignocaine local anaesthesia, before the start of therapy and after repigmentation.
The biopsies were obtained from three sites – vitiliginous (V), marginal (M) and perilesional (P) – which are 1–5 cm peripheral to M area.
Control biopsies were taken from five healthy volunteers. The controls were subjected to all the investigations done for the 30 cases of vitiligo.
Each biopsy was divided into three parts:
- The first part was fixed in 10% neutral buffered formalin and was routinely processed. The resulting sections were mounted on glass slides and subjected to the following:
- Histopathological evaluation using haematoxylin and eosin (H&E) stain.
- Detection of melanin by the use of Masson–Fontana stain according to Masson.
- Immuno-histochemical examination for evaluation of HMB-45 expression. We stained sections according to the manufacturer instructions with monoclonal mouse anti-human HMB-45 protein (code no: 364S207, NeoMarkers, Fremont, CA, USA). A ready-to-use detection system, UltraVision detection system, anti-polyvalent, horseradish peroxidase/diaminobenzidine, was used for the procedure (code no: TP-015-HD, Lab Vision Corporation, Fremont, CA, USA).
- The second part was immediately mounted in tissue Tek II and then frozen in supercooled isopentane pre-cooled by immersion in liquid nitrogen for the DOPA reaction. Detection of active MC was determined by the use of DOPA stain . Light microscope with built-on camera (Optiphot 1, Nikon, Tokyo, Japan) was used to examine and to photograph these sections.
- The third part was prepared for electron microscopic (EM) examination as described by de Los Reyes and Mora. Interpretation of the sections was done using JEOL 100 C-X transmission electron microscope (JEOL, Tokyo, Japan). Six cases together with the five controls were examined. The former included the three cases with vacuolated cells in semi-thin sections together with another three randomly chosen cases. Screening of the biopsies for the presence of the histopathological and immuno-histochemical findings was done and regardless of the degrees of these findings the biopsies were considered either negative or positive, e.g. number of patients showing basal clear cells, degenerated KCs, DOPA positive cells, etc. (Table 1).
|Before PUVA||After PUVA||P-value|
|H&E||Basal clear cells||3 (10%)||30 (100%)||30 (100%)||27 (90%)||30 (100%)||30 (100%)||< 0.001|
|Melanin in basal KCs||Few traces 1 (3.33%)||Abundant 25 (83.33%)||Abundant 25 (83.33%)||Abundant 27 (90%)||Abundant 30 (100%)||Abundant 30 (100%)||< 0.001||< 0.05||< 0.05|
|Degenerated KCs||11 (36.67%)||14 (46.67%)||13 (43.33%)||4 (13.33%)||9 (30%)||7 (23.33%)||< 0.05||> 0.05||> 0.05|
|MF stain||Melanin in basal KCs||4 (13.33%)||30 (100%)||30 (100%)||27 (90%)||30 (100%)||30 (100%)||< 0.001|
|DOPA||Reactive MCs in basal layer||0 (0%)||24 (80%)||27 (90%)||26 (86.67%)||26 (86.67%)||29 (96.67%)||< 0.001||> 0.05||> 0.05|
|HMB-45||Reactive MCs||0 (0%)||30 (100%)||30 (100%)||27 (90%)||30 (100%)||30 (100%)||< 0.001|
Further analysis of the HMB-45 stained biopsies was done quantitatively to determine the difference in the number of the active MCs before and after PUVA therapy (Table 2).
|Sites of lesion||MC number before PUVA||MC number after PUVA||P-value|
|Mean ± SD||Range||Mean ± SD||Range|
|V||0 ± 0||(0–0)||1.01 ± 0.73||(0–2.1)||P < 0.001|
|M||0.49 ± 0.19||(0.1–0.8)||1.05 ± 0.56||(0.3–2)||P < 0.001|
|P||1.03 ± 0.28||(0.5–1.6)||1.26 ± 0.36||(0.6–1.9)||P < 0.05|
It is worthy to note that the patients continued treatment until repigmentation of all lesions for a maximum of 1 year.
To test the significance of differences between the data of the study groups, we used the Student's t-test. P-value (the probability of chance) was considered statistically significant when it was less than 0.05 and highly significant if it was less than 0.001.
The age of the patients at the time of the examination ranged from 17 to 52 years old, with a mean ± standard deviation (SD) of 30.93 ± 11.85 years. The duration of the disease prior to PUVA ranged from 0.5 to 30 years, with a mean ± SD of 9.18 ± 7.82 years. The maintenance dose of UVA ranged from 2 to 3.5 J/cm2, with a mean of 2.67 ± 0.40 J/cm2.
In 10% of pretreated biopsies, few MCs were detected on the V area by using H&E stain and were further confirmed by EM examination, while they did not stain with DOPA or HMB-45 suggesting that these MCs were inactive cells.
The M and P areas of V patients were histologically normal regarding the morphology of MCs. Meanwhile, the EM examination showed that MCs, which were present in pretreated V, M and P biopsies, were degenerated.
These degenerative changes, which were absent in the control group, were in the form of vacuolization of the melanocytic cytoplasm, pyknosis of nuclei and peripheral margination of chromatin.
Statistical studies, using independent t-test, revealed a highly significant decrease in the number of HMB-45-positive MCs in pretreated M than pretreated P biopsies (P < 0.001). In 28 out of 30 (93.3%) pretreated M biopsies, stained by both DOPA and HMB-45, MCs had retracted dendrites. In P biopsies, MCs showed normal dendrites as that seen in normal healthy control biopsies.
Degeneration of KCs, observed by the electron microscope, was in the form of dilatation of the rough endoplasmic reticulum (RER) and peripheral margination of the fragmented tonofilaments resulting in the visualization of an electron-lucid peri-nuclear area. Cytoplasmic vacuoles without limiting membranes were apparent.
The nuclear changes, seen in most degenerated KCs, were pyknosis, peripheral margination of the nuclear chromatin and absent nucleoli.
Extracellular granular material (EGM), deposited between KCs, was found in the epidermis and was identifiable only with the electron microscope. It occupied dilated intercellular spaces between basal cell layer and the two rows of KCs above it. Neither cytoplasmic constituents, such as mitochondria and RER, nor tonofilaments and Langerhans cell (LC) granules had been observed. EGM was absent from the epidermis of healthy volunteers.
After 12 months of PUVA therapy, obvious improvement of the histopathological changes occurred in both KCs and MCs. By H&E, DOPA and HMB-45, there was a highly statistically significant increase in the percentage of presence of MCs in post-treated V biopsies than in pretreated V biopsies (P < 0.001).
By light microscopic examination, statistical data using test of proportion revealed a significant decrease in the percentage of KC degeneration in post-treated V biopsies than in pretreated V biopsies (P < 0.05). In M and P areas, there was no statistically significant decrease in the percentage of KC degeneration in post-treated biopsies than in pretreated biopsies (P > 0.05).
There is a long-standing controversy over whether MCs in vitiligo lesions are actually lost or still present but functionally dormant or inactivated . The work of Tobin et al.  supported the second view by demonstrating clear cells in H&E stained sections that were negative for MC immuno-histochemical markers.
In the present study, similar findings were obtained from biopsies of V area before therapy where clear cells, with negative staining with DOPA and HMB-45 marker, were present in three out of 30 biopsies (10%).
The EM examination of the three biopsies supported the presence of inactive MCs where they were observed just below basal KCs. This situation was being similar to those of basal MCs of normal epidermis described by Ackerman et al. . However, we considered them as inactive MCs as they were free from melanosomes. They were clearly distinguishable from LCs by their lack of characteristic Bierbeck granules, indented nuclei and plentiful cytoplasm containing lysosomes, and from KCs by the absence of desmosomal junctions or tonofilaments. These clear MCs were in a state of degeneration showing vacuolization of the cytoplasm as well as nuclear changes in the form of pyknosis with peripheral margination of the nuclear chromatin.
MCs in M and P areas of all cases were apparently normal by H&E and positive for HMB-45. However, EM examination showed evident degenerative changes with a few mature melanosomes. On applying the t-test to compare MC number in pretreated M and P areas, there was a highly significant decrease in the number of MCs in pretreated M to P biopsies (P < 0.001). This decrease may indicate histological progression of the disease on moving towards the centre of the lesion. Another support of this progression was the presence of normal dendrites, as that seen in normal healthy controls, in biopsies from the P areas as compared with M areas that showed short dendrites.
After 12 months of PUVA therapy, there was a highly significant increase in the percentage of existence of MCs in post-treated than pretreated V biopsies (P < 0.001) by using H&E, DOPA and HMB-45.
These cells had large nuclei, the cytoplasm contained all stages of melanosomes and the dendrites were long and contained completely melanized melanosomes in the supra-basal layers. These findings are similar to the results of Ortonne et al. , who reported that the MCs of post-PUVA repigmented areas were hyperactive and the melanosomes were larger than those of the surrounding skin. However, their study did not compare the findings with the pretreated lesions.
By immuno-histochemical staining, MCs appeared larger in post-treated biopsies than in pretreated biopsies with increased density of reaction and dendricity in all M and P biopsies. This observation is in accordance with Tsamali et al. , who showed that dendrites of MCs are longer and the transfer of melanosomes into the KCs is more likely after ultraviolet radiation.
From all the previous results, PUVA therapy resulted in an increase in the number of epidermal HMB-45-positive MCs in all three sites: V, M and P areas.
Some authors only described the cytoplasmic changes in degenerated KCs, without any nuclear changes [6, 14, 15]. In our study, similar cytoplasmic changes were detected and it is worthy to note that evident nuclear changes were also present. These KC nuclear changes were in the form of pyknosis, peripheral margination of chromatin and absence of nucleoli. This absence may give another proof that these KCs were inactive as the nucleoli are the sites of synthesis of ribosomal ribonucleic acid . This observation may be a part of the disease. Another explanation is that it is due to random failure of the cutting edge of the knife in passing through the nucleolus during sectioning of the biopsy. The nucleoli reappearance after PUVA is in favour of the first possibility.
EGM, deposited between KCs, was found almost exclusively in the epidermis and was identifiable only with the electron microscope. This material was previously noted by Moellmann et al. and Panuncio and Vignale[14, 15] who considered it as cytoplasmic debris liberated from degenerated KCs. After PUVA, the deposition of this material was notably decreased.
This improvement after PUVA therapy is similar to that reported by Tobin et al.  after topical application of a NB-UVB-activated pseudocatalase. The authors observed complete recovery of certain morphological changes that were seen by EM such as vacuolation, granulation and dilatation of RER and disappearance of EGM.
Vitiligo is a disease that affects the MCs and the KCs in both the leucodermic and the apparently normal P skin. The extents of the degenerative changes that occur in vitiligo determine whether or not the disease will manifest clinically.
PUVA therapy increases the number and activity of epidermal MCs in all areas and decreases the degenerative changes in both MCs and KCs. The reversal of degeneration in both leucodermic and apparently normal skin after PUVA points towards the role of this modality in both repigmentation and protection against further depigmentation.
The authors are grateful to Dr Khaled El-Houshy, Assistant Clinical Professor, Wayne State University, Detroit, MI, USA, for his comments in preparing this manuscript.
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