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Apoptotic cell clearance in Systemic Lupus Erythematosus (SLE)Reefman, Esther

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CCHHAAPPTTEERR 33

Skin sensitivity for UVB irradiation in Systemic Lupus Erythematosus is not related to the level of

apoptosis induction of keratinocytes

Esther Reefman1, Hilde Kuiper2, Marcel F. Jonkman3, Pieter C. Limburg1,2, Cees G.M. Kallenberg1 & Marc Bijl1

Departments of Rheumatology & Clinical Immunology1, Pathology & Laboratory Medicine2 and Dermatology3, University Medical Center Groningen,

University of Groningen, Groningen, The Netherlands

Rheumatology (Oxford). 2006 May;45(5):538-44.

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ABSTRACT Objective: Accumulation of apoptotic cells has been suggested to be involved in the pathogenesis of Systemic Lupus Erythematosus (SLE). As sunlight exposure is one of the factors that can trigger disease activity, we hypothesized that UV light may induce increased numbers of apoptotic cells in SLE. Methods: Fourteen SLE patients and 16 controls were irradiated with UVB to determine their minimal erythemal dose (MED). Subsequently, skin was irradiated with 1MED and 2MED, respectively, and after 24 hrs skin biopsies were analyzed (immuno)histologically for the number of apoptotic cells and presence of pyknotic nuclear debris. Results: MED in SLE patients was significantly decreased and the presence of a decreased MED was associated with a history of butterfly rash. Decreased MED was not related to other skin related ACR criteria nor to autoantibody specificities. No differences were detected in the numbers of apoptotic keratinocytes between patients and controls nor in the amount of pyknotic nuclear debris following 1 and 2 MED irradiation, respectively. Absolute UVB doses were correlated with the number of apoptotic keratinocytes; dose-responses did not differ significantly between patients and controls. Conclusions: Increased sensitivity of SLE patients for UVB, although associated with a history of malar rash, is not related to increased induction of apoptosis or increased levels of secondary necrosis in the skin. Thus, compared to controls, UVB-induced apoptosis is not increased in SLE patients under physiological conditions.

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INTRODUCTION Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease

characterized by the presence of autoantibodies to nuclear and cytoplasmic antigens in conjunction with a wide range of clinical manifestations. Photosensitivity is one of the characteristics of SLE affecting 10-50% of patients (1). Most, but not all, cutaneous lupus lesions occur in light-exposed areas and can be triggered by sunlight exposure. Sunlight exposure even can induce systemic disease activity. The processes that induce cutaneous and systemic inflammatory lesions have not been elucidated, but in recent years apoptotic cells have been suggested to be one of the major factors involved.

The precise role of apoptotic cells in development and progression of autoimmunity is unknown. Increased production and/or inefficient clearance of apoptotic cells by phagocytes could result in accumulation of apoptotic cells, as seen in SLE patients (2-4). Furthermore, increased numbers of apoptotic keratinocytes have been detected in cutaneous LE lesions (5). During the apoptotic process intracellular constituents are excessively presented to the immune system due to cell-surface expression of intracellular constituents and/or posttranslational alteration of cellular proteins during apoptosis (6-9). Binding of autoantibodies specific for these antigens may change the clearance of apoptotic cells. In skin of SLE patients deposition of IgG is often found at the dermal-epidermal junction, the so called lupus-band, or in the epidermis (10;11). In addition, autoantibodies specific for SSA/Ro and SSB/La are associated with cutaneous LE (12;13).

Ultraviolet (UV) irradiation, especially UVB, is a potent inducer of apoptosis. The increased sensitivity for sunlight seen in a subpopulation of SLE patients may reflect increased susceptibility of keratinocytes to become apoptotic after exposure to UV light. During apoptosis cells first shrink and their nuclei condense, subsequently they disintegrate into well-enclosed apoptotic bodies. Apoptotic keratinocytes may be detected in skin biopsies after exposure to UV by hematoxylin-eosin staining as cells characterized by eosinophilic cytoplasm and pyknotic nuclei, also known as sunburn cells (SBC) (14). In vitro, apoptosis finally leads to plasma membrane permeabilization, known as secondary necrosis. However, it is believed that this will not occur under physiological circumstances in vivo due to rapid clearance of apoptotic cells by macrophages or surrounding cells (15).

In the present study we tested the hypothesis that UV light induces increased numbers of apoptotic cells in SLE. First, we assessed the sensitivity of SLE skin for UVB by determining the minimal erythemal dose (MED). Secondly, we analyzed whether photosensitivity is related to the induction of apoptosis in the skin of SLE patients. MATERIALS & METHODS Patients and controls

Consecutive patients were asked to participate in the study. Patients eligible for the study had to fulfill at least four American College of Rheumatology (ACR) criteria for SLE and to be in an inactive phase of the disease defined as Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) ≤ 4 (16) and no active cutaneous disease. Fourteen patients (age 45.1±13.1 years (mean±SD), 1 male/13 female) were included. Table 1 shows patient characteristics and immunosuppressive medication

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used at the time of the study. Skin related ACR criteria were based on patient history. Autoantibodies to double stranded DNA (dsDNA) were determined by FARR assay and reactivity to SSA, SSB, nRNP and Sm was determined by counter immuno-electrophoresis. Sixteen healthy volunteers (age 37.6±17.0 years (mean±SD), 3 male/ 13 female) were included as controls. Patients and controls of whom the buttock skin had been exposed to sunlight or other sources of UVB over the past 6 months were excluded from the study. Skin types were determined based on the Fitzpatrick skin typing chart (17). Skin type distribution (apart from two type 5 or higher scores in non-caucasian patients) were similar in patients (2 type2, 10 type 3, 1 type 5, 1 type 6 ) and controls (2 type2, 14 type 3). The local ethical committee approved the study. Irradiation protocol

UVB irradiation was performed using the Waldman 800 “sky” light with TL-12 lamps (Philips, Eindhoven, The Netherlands) at a distance of 15 cm from an unexposed area of the buttock skin. A Diffey grid (18) was used to irradiate the skin with 10 different doses (0.026-0.200 J/cm2) during one exposure. After 24 hrs the MED was determined by two independent observers, with more than 90% agreement. In case of disagreement the mean of the two separate values was taken. The reproducibility of MED assessment was determined by a second irradiation in 10 subjects, 5 healthy controls and 5 patients. After determination of the MED, subjects were irradiated with 1 MED and 2 MED on the other buttock, respectively. After 24 hours, 4 mm skin biopsies were taken from non-irradiated skin and from the two areas of skin irradiated with 1 MED and 2MED, respectively. Biopsies were fixed in formaldehyde. Chemicals and antibodies

Diaminobenzidin (DAB) solution contains 25mg DAB, 50mg imidazol in 50ml PBS filtrated before use, and 50ul of 30% H202 added just before incubation. For hematoxylin staining Mayer’s Hemalum solution (Merck, Darmstadt, Germany) was used and Kaiser’s glycerol gelatin (Merck) was used as a mounting medium. Rabbit antibody to cleaved caspase-3 antibodies (#9661S) was purchased at Cell Signaling Technology, Beverly MA, USA. Secondary antibodies, goat-anti-rabbit IgG-Horse Radish Peroxidase (HRP) and rabbit-anti-goat IgG-HRP, were obtained from DakoCytomation (Glostrup, Denmark). Immunohistochemistry

Skin sections (4µm) on 3-amino-propyltriethoxysilane (APES)-coated glass slides were used for all experiments. Sections were deparaffinized by subsequent incubations in xylene (10min), 100% ethanol (5min) and 96% ethanol (2min), twice, followed by ethanol 70% (2min) and distilled water. Hematoxylin-eosin staining was performed according to standard protocol using the linear stainer from Medite (Burgdorf, Germany). For cleaved-caspase-3 staining, slides were boiled for 10 min in 1mM EDTA buffer (pH 8.0) and rinsed with PBS. Endogenous peroxidase was blocked by incubation in 0.37 % H202 in PBS for 30min. Slides were incubated with anti-cleaved caspase-3 antibodies (diluted 1:75 in 1% BSA/PBS) for 1 hour at room temperature (RT). Subsequently, slides were washed in PBS (3 times) and incubated

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for 30 min at RT with goat-anti-rabbit IgG labeled with horse radish peroxidase (HRP) (1:50 in 1% BSA/PBS), then washed again (3 times) in PBS followed by incubation with rabbit- anti-goat-IgG-HRP for another 30 min at RT. After washing in PBS (3 times), slides were incubated in DAB solution for 15-20 min, and washed subsequently with distilled water (5 times). Slides where then counterstained with hematoxylin for 1 min, washed in distilled water (5 times), and dehydrated in 96% ethanol and subsequently in 100% ethanol, and then mounted. Scoring procedure

Using Olympus Soft Pro software (Tokyo, Japan), the surface area of the epidermis was determined by manually drawing a line around this area and calculating the total surface (mm2). Subsequently, the numbers of SBC and pyknotic nuclei were scored by calculating their numbers in 3 sequential hematoxylin-eosin stained sections. A pyknotic nucleus was defined as one whole pyknotic nucleus or a group of pyknotic nuclear fragments (as indicated in Fig 3A, with black arrows and white arrows, respectively). The numbers of SBC or pyknotic nuclei per mm2 were determined by dividing the numbers counted by the epidermal surface area and calculating the mean value of the 3 sections. Cleaved caspase-3 positive cells were scored accordingly.

Table 1: Patient characteristics, use of medication and autoantibody specificity at the time of the study. F:female, M: male, mg/day: milligram per day. Positive score for skin related ACR criteria was based on patient history. Autoantibody specificities directed against dsDNA: double stranded DNA (dsDNA), SSA, SSB, nonhistone ribonuclear protein (nRNP) and/or Sm complex (Sm).

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Statistics Differences in MED, numbers of apoptotic cells and debris between groups

were determined using Mann-Whitney test. Correlations between MED values and medication, and between numbers of SBC and amount of pyknotic nuclear debris were analyzed by Spearman rank correlation test. To study associations between increased sensitivity and ACR criteria, medication use or autoantibodies present in serum a Chi-square test was performed. To analyze differences in correlation between patients and controls slopes of linear regression lines were compared (GraphPad Software, San Diego, USA). To analyze the relationship between absolute doses of UVB and numbers of apoptotic cells two tests were performed. First, Spearman rank correlation test to determine whether a correlation existed; secondly, a curve fit was made using data from patients and controls, followed by a sign test on rank differences, in order to analyze whether patients and controls were equally distributed around the calculated dose-response curve (SPSS 12.0.1., Chicago, USA). RESULTS Sensitivity of SLE patients for a single dose of UVB

Minimal erythemal dose (MED) of patients and controls were determined as an indicator of sensitivity for a single dose of UVB. To determine the MED, ten different doses of UVB were applied within one exposure using a Diffey grid. This device has been reported to be comparable in accuracy to conventional multi-exposure testing (19). To assess the reproducibility of this method MED was determined at two consecutive days in 10 subjects. Reproducibility ranged from 0 to 22% (median 3%). MED of SLE patients (0.090±0.036 J/cm2 (mean±SD)) was lower compared to controls (0.112±0.036 J/cm2), p=0.012. MED values ranged from 0.05 to 0.18 J/cm2 in patients and from 0.085 to 0.20 J/cm2 in controls (Fig 1). Six out of the 14 patients (43%) had MED values lower than any of the controls and were defined as sensitive patients. We assessed whether age, disease duration, use of medication, particular autoantibodies or history of skin related ACR criteria (Table 1) were associated with

Figure 1: Determination of minimal erythemal dose (MED) in 14 SLE patients and 16 controls. MED of SLE patients is significantly lower than in the control group. MED is expressed as Joule/cm2 (J/cm2). P-value was determined by two-tailed Mann-Whitney test. Horizontal line denotes the mean.

the presence of increased sensitivity. No correlations could be found between age and MED values in patients (r=0.03, p=0.92) nor between MED values and disease duration in the patients (r=-0.16, p=0.61). As shown in Table 2 no association was found between decreased MED and use of medication. Also, medication doses did not

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correlate with MED values (corticosteroids r=0.20, p=0.50; hydroxychloroquine r=0.30; p=0.30; azathioprine r=0.23, p=0.45). Furthermore, no association was found with antibody specificities. Butterfly (malar) rash was associated with increased sensitivity for UVB but not discoid lupus or photosensitivity by history (Table 2).

Table 2: Presence of autoantibodies and history of skin related ACR criteria in patients with and without increased sensitivity to UVB

Sensitive patients had a lower MED than any of the controls (MED<0.085J/cm2). Number of patients are given that are positive for the respective autoantibody specificities, the ACR-criteria, and the medications indicated. *: significant p-value (p<0.05 by Chi-square test).

Apoptosis induction in skin after two different UVB doses Apoptosis induction was studied in previously sun-protected buttock skin 24

hours after exposure to 1 and 2 MED of UVB, respectively. Apoptotic keratinocytes could be detected in H&E stained sections by their eosinophilic cytoplasm and pyknotic nuclei (Fig 2A), as well as by staining for cleaved caspase 3 (Fig 2B). As expected, hardly any apoptotic cells were detected in unexposed skin in patients and controls, both defined as SBC in H&E staining or by the presence of cleaved caspase-3 positive cells (Fig 2C, D). After exposure to 1 MED, numbers of apoptotic cells increased significantly in patients and controls; 26.9±28.1 (mean±SD) SBC/mm2 versus 15.3±12.7 SBC/mm2, respectively. Exposure to 2 MED led to a 3 to 9 fold increase of apoptotic cells compared to 1 MED in both groups, resulting in 116.1±79.4 SBC/mm2 in patients and 97.8±63.6 SBC/mm2 in skin of controls. Significant differences in the number of apoptotic cells could not be detected between patients and controls by either H&E detection or cleaved caspase 3 staining at any of the UVB doses used. The two methods for the detection of apoptotic keratinocytes were highly correlated (r=0.89, p<0.0001) indicating that the data reliably represent the number of apoptotic cells induced (Fig 2E).

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Figure 2: Apoptosis induction in SLE patients and controls 24 hrs after UVB irradiation. A) Representative hematoxylin-eosin (H&E) staining of skin irradiated with 2MED at 100x magnification. B) Representative immunohistochemical staining for cleaved caspase-3, visualized using DAB. Arrows indicate apoptotic cells. C) Numbers of Sunburn cells (SBC) detected in the epidermis of H&E stained sections (SBC/mm2). D) Numbers of cleaved caspase-3 positive cells detected in the epidermis (cleaved Casp3+/mm2). E) Relationship between number of SBC and cleaved caspase-3 positive cells per mm2 of skin in SLE patients and controls (●: patients, ○: controls).

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Induction of pyknotic nuclear debris after 2 different UVB doses Besides the formation of SBC, pyknotic nuclear debris could be seen in the

epidermal layer by H&E staining (Fig 3A). We determined the amount of pyknotic nuclear debris in unexposed and exposed skin by counting the numbers of these whole or fragmented pyknotic nuclei present between the keratinocytes. Hardly any of these nuclei could be detected in unexposed skin. Numbers increased after exposure to increasing doses of UVB (Fig 3B). No differences in numbers of these nuclei could be detected at any dose of UVB between SLE patients and controls. A high correlation was found between the formation of SBC and the occurrence of nuclear debris (patients r=0.88/p<0.0001, controls r=0.89/p<0.0001). The correlation was linear and did not differ between patients and controls (Fig 3C).

Figure 3: Pyknotic nuclear debris present in skin of SLE patients and controls 24hrs after UVB irradiation. A) Two representative hematoxylin-eosin (H&E) stained sections showing SBC (triangle arrows) and nuclear debris (black arrow: indicates one intact pyknotic nucleus, white arrow indicates pyknotic fragmented nuclei) in skin biopsies after irradiation with UVB. Magnification 400x. B) Numbers of pyknotic nuclei found in unexposed skin and in 1MED and 2MED exposed skin, respectively. C) Relationship between formation of SBC and numbers of pyknotic nuclei found. (●: patients, ○: controls). Patients, r= 0.88/p<0,0001 and controls, r=0.89/p<0.0001, ns.

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Relationship between absolute UVB dose and induction of apoptosis Absolute UVB values have been shown to correlate more strongly with DNA damage and apoptosis than the standardized MED (20). As shown before, SLE patients had significantly lower MED values. As a consequence, absolute doses of UVB received by the patient group were lower compared to controls. However, the same numbers of apoptotic cells were induced in patients and controls after exposure to one and two MED doses, respectively. Next, we investigated the correlation between absolute doses of UVB and numbers of apoptotic cells induced, comparing patients and controls. A significant correlation was found both for patients and controls (r=0.56/p=0.002 and r=0.60/p<0.001, respectively) (Fig 4). The relationship between numbers of apoptotic cells and absolute doses of UVB was not linear. To compare the dose-response in patients vs. controls we performed a curve fit after which the distribution of the patient and control values was compared in a sign test on rank differences. This test showed that patients and controls are distributed equally along this curve (x2=1.45/p=0.23) indicating that the dose-response did not differ significantly between patients and controls. Furthermore, we compared the number of SBC in sensitive patients (as defined earlier) with that in less sensitive patients or controls. Sensitive patients had significantly lower numbers of SBC in their skin after 2 MED UVB compared to less sensitive patients (p=0.01, data not shown). Taken together, this indicates that apoptosis is not increased in lupus patients when they receive the same absolute UVB dose compared to controls.

Figure 4: Relationship between SBC induction and absolute UVB doses. Number of SBC/mm2 in the skin after 24hrs is depicted on the y-axis while absolute UVB dose (J/cm2) received is expressed on the x-axis. Spearman’s correlation test shows a significant correlation between numbers of SBC and absolute doses of UVB (r=0.56/ p=0.002 for patients and r=0.60/p<0.001 for controls). (▲: sensitive patients, ●:other patients, ○: controls).

DISCUSSION In this study we observed that SLE patients have a lower MED for UVB compared to controls. This was associated with a history of butterfly rash but not with discoid lesions or photosensitivity.

Because inter-individual variation in skin types leads to differences in sensitivity to UV light between individuals, the minimal dose needed to induce erythema (MED) has been introduced to standardize the sensitivity of subjects for UV. Discrepancies between previous studies investigating sensitivity of SLE patients to UV light might reflect differences in patient characteristics or use of medication (21-23). Erythema can be influenced by topical administration of medication such as corticosteroids (24), however oral corticosteroids up to 80 mg daily were reported not

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to decrease erythema (25). None of the patients in this study was using topical corticosteroids or high doses of corticosteroids (Table 1). Also, use of other (immunosuppressive) drugs was not associated with decreased MED. Therefore, the lower MED detected in our patient group reflects, in all likelihood, increased sensitivity to a single dose of UVB.

Single or multiple exposures with moderate doses of UVB have been reported to induce skin lesions in SLE patients (23;26;27). Although UVA exposure can also induce skin lesions in SLE patients considerably higher doses are needed (28). Furthermore, UVA is weakly absorbed by biomolecules, like DNA or proteins and, therefore, a weaker inducer of apoptosis. In skin hardly any apoptotic keratinocytes can be detected after UVA exposure (29;30). In contrast, UVB is known to be a potent inducer of apoptosis (31;32). In this study we wanted to investigate induction of apoptotic cells in relation to the increased UV sensitivity as seen in SLE patients. Therefore, UVB was chosen in this study as UV source for irradiation.

We determined whether the increased sensitivity to UVB in part of the patients was associated with autoantibody specificities or skin related ACR criteria. Association has been reported between autoantibody specificities for SSA and SSB and cutaneous LE, either induced naturally or by provocative photo-testing (12;13;26). Other reports, however, have not found this association (27). Also in our study no association was found between increased sensitivity and any autoantibody specificity, although the presence of such an association can not be excluded due to the limited number of patients studied. However, our data indicate an association between increased UVB sensitivity and a history of butterfly rash. Butterfly (or malar) rash is a common feature in SLE presents as a photosensitive eruption, although sun exposure is not always required (1;33). The increased sensitivity for a single dose of UVB might reflect the susceptibility of these patients to develop a butterfly rash. Accumulation of apoptotic cells due to an increased rate of apoptosis, decreased elimination of apoptotic cells or a combination of both has been hypothesized to be an important factor in the development of inflammatory lesions as seen in the skin in SLE patients (34;35). In LE-lesions, increased numbers of apoptotic cells (36), increased Fas antigen expression and decreased Bcl-2 expression (5) have been reported. We investigated the induction of apoptosis before development of lesions, which might give a better indication whether or not apoptosis is involved in the initiation of LE-lesions. We showed that apoptosis induction is not increased in the skin of SLE patients after irradiation with UVB as compared to controls. Two methods were used for detection of apoptotic cells in biopsies, the morphological detection in H&E stained sections and a specific staining for the cleaved form of caspase-3. Detection of SBC in H&E has been used extensively for many years (31;37). The presence of cleaved caspase-3 is an early marker for apoptosis, as its cleavage initiates all the morphological changes (38). Although these two methods show positive results in different, partly overlapping time periods, we demonstrated that these methods are well correlated. Terminal deoxynucleotidyl transferase-mediated in situ end labeling (TUNEL), although historically well known to be used in the field of apoptosis detection, was not used as several reports have stated that the TUNEL technique is not specific for apoptosis and will also stain necrotic (39-42) and highly proliferating cells (43).

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By H&E staining pyknotic nuclear debris was detected 24 hrs after UVB irradiation. As suggested by the strong and linear correlation between SBC and nuclear debris the progression of the apoptotic process into post-apoptotic events like secondary necrosis, probably, was responsible for the occurrence of nuclear debris. Therefore, levels of nuclear debris detected after 24hrs could be a reflection of apoptosis occurring in the earlier phases after UVB irradiation. Other studies have shown that the relative contribution of apoptosis induced up to 12 hrs after irradiation to the total amount of apoptosis that is seen later on, is very low (37;44;45). The contribution of early apoptotic cell induction in our study seems only one fifth or less of the total amount of apoptosis occurring over a 24 hrs period, as can be deduced from figures 2C and 3B. In accordance with the data on apoptotic keratinocytes, the amount of nuclear debris present in the skin before and after UVB exposure did not differ between patients and controls at any dose. Neither did we find differences between patients and controls in the linear relationship between formation of SBC and nuclear debris. This indicates that the level of apoptosis in the earlier phases and the rate at which secondary necrosis occurs is comparable between SLE patients and controls. Further studies, however, are needed to determine the number of apoptotic cells present at earlier and, especially, later time points after irradiation.

Furthermore, despite the lower absolute dose of UVB received by patients compared to controls in the present study, no differences in the relationship between induction of apoptosis and absolute doses of UVB were detected between patients and controls. Apoptosis induction is, therefore, comparable between SLE patients and healthy controls under physiological conditions.

In conclusion, we suggest that not the number of apoptotic cells that are induced, but the clearance of these cells might be involved in the pathogenesis of light induced skin lesions in SLE patients. Studies are underway to test the latter hypothesis.

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