Evaluation of apoptosis in skin biopsies of patients of borderline leprosy and lepra type 1 reaction.

BACKGROUND: The role of apoptosis is not clear in leprosy and lepra reactions.
OBJECTIVES: To evaluate frequency of apoptosis in skin lesions of borderline leprosy and Type 1 lepra reaction.
METHODS: Sixty patients with borderline leprosy (30 with clinically diagnosed Type 1 reaction (T1R) (Group I) and 30 without clinical evidence of reaction (Group II)) were analyzed in this prospective study. Apoptosis was detected by two different methods for comparison, that is, histopathologic examination (HPE) and deoxyribonucleic acid (DNA) fragmentation and electrophoresis. Quantification of apoptotic bodies/10 high power fields (HPF) was also done.
RESULTS: Out of 30 cases, apoptosis was detected in 29 cases in Group I and 24 cases in Group II by HPE (P = 0.103), whereas, with the use of DNA electrophoresis it was detected in 24 cases in Group I and 18 cases in Group II (P = 0.091). On quantitative estimation it was found that number of apoptotic bodies are higher in Group I in comparison to Group II (2.77 vs 1.99), which is statistically significant.
CONCLUSIONS: There was moderate agreement (κ = 0.47) between the two methods of apoptosis detection. Apoptosis was seen more in patients with T1R both qualitatively (statistically nonsignificant) and quantitatively (statistically significant). Clinical significance of this novel finding is that apoptosis can be used as one of the variables for diagnosis of T1R to increase detection rate.

What was known?

Apoptosis is one mechanism of cell loss, which is an active, self-destructive cellular process with no accompanying inflammatory component. Although importance of apoptosis in tuberculosis has been shown, its significance has not been established yet in leprosy and leprosy reactions.

Introduction

Leprosy also known as Hansen's disease is a slowly progressive infection caused by Mycobacterium leprae (M. leprae) that mainly affects skin and peripheral nerves resulting in disabling deformities. Although India has achieved the target of leprosy elimination of less than one case per 10,000 population since 2005,[1] but still India continues to record the highest number of new leprosy cases in the world followed by Brazil and Indonesia.[2]

Two major clinical types of leprosy reactions occur; Type 1 reaction (T1R) which includes both upgrading and downgrading reactions and erythema nodosum leprosum-Type 2 (T2R). Out of these two types, T1Rs are caused by spontaneous change in cell-mediated immunity to mycobacterial antigen.[3] But these reactions are usually underdiagnosed. For example, in the study done by Lockwood et al.,[4] only 32-62% of clinically diagnosed reactions were given a histological diagnosis, and diagnosing these reactions correctly and in early stage is important for patients because they then may need treatment with steroids for 4-9 months to prevent severe disabilities and impairment due to nerve injury.

In leprosy, majority of skin lesions heal without much fibrosis; while on the other hand, other granulomatous diseases of the skin usually heal with fibrosis, for example, the scarring left behind by lupus vulgaris or scrofuloderma. This is because in leprosy one mechanism of cell loss is believed to be apoptosis, which is an active, self-destructive cellular process with no accompanying inflammatory component. It may explain the relative lack of fibrosis in leprosy.

It has already been shown that in tuberculosis, Mycobacterium tuberculosis induced activation accelerates neutrophil's apoptosis.[5] But unlike in tuberculosis, the significance of apoptosis in leprosy remains unclear, though few studies have shown apoptosis to be present in leprosy lesions.[678]

Since lepra reactions may be associated with changes in immune status, the number and density of apoptosis is likely to vary in these states also. Apoptosis may also have a role in nerve damage in leprosy.[9] However, sufficient information on apoptosis in various situations, such as during reactions, in different portions of the leprosy spectrum of disease (indirectly indicating immune status), and the effects of treatment, is not available.

Hence, the present study was undertaken to determine the frequency of apoptosis in skin lesions of borderline leprosy and leprosy T1R.

Materials and Methods

The present study was a cross sectional observation study conducted on patients with borderline leprosy recruited from the leprosy clinic at the Department of Dermatology and STD, Guru Teg Bahadur Hospital. Patients were enrolled in two groups depending on inclusion and exclusion criteria.

In Group I, patients of leprosy with clinical T1R; while in Group II, borderline leprosy patients without lepra reactions were recruited.

Inclusion criteria

Group I: Either of the following was taken as a clinical evidence of T1R: (a) Sudden appearance (within last 2 weeks) of new erythematous skin lesion. (b) Skin lesions becoming erythematous after a period of quiescence. 2) Group II: Patients of borderline leprosy without any clinical or histological evidence of lepra reactions as defined above.

Exclusion criteria

Patients with polar forms of leprosy (tuberculoid and lepromatous leprosy) and T2R were excluded from the study.

The institutional research ethics committee approved the study. Informed consent was taken from all the patients enrolled in the study. A detailed, clinical history was taken along with physical examination and histopathologic examination (HPE) was done to establish the diagnosis of leprosy and T1R.

Skin biopsy

A 4-mm deep punch biopsy was taken under local anesthesia from the edge of one representative clinically active skin lesion from each patient. The biopsies were fixed in 10% buffered formalin and routinely processed and embedded in paraffin. Two sections each of 4 μm thick were cut. Out of those one was stained with hematoxylin and eosin (H and E) and one with Fite's stain to detect Acid-fast bacilli (AFB).

Method of H and E staining

H and E was done using standard procedures using Harris's hematoxylin.

From the tissue specimens, apoptosis was detected by two different methods for comparison, that is, HPE and by deoxyribonucleic acid (DNA) fragmentation and electrophoresis.

HPE

A set of two slides, one H and E and one AFB for each case, was taken. Basic characteristics for diagnosis of T1R taken were, as explained by Ridley, an influx of mononuclear phagocytes with epithelioid differentiation, infiltration of lymphocytes, and edema inside and around granulomas leading to distortion of surrounding tissue and nerves. Apoptotic bodies seen in H and E sections of skin biopsies were identified by the following features: Nuclear condensation, round to ovoid bodies, eosinophilia of the cytoplasm, and karyorrhexis/karyolysis.

The number of apoptotic bodies per 10 high power fields (HPF)/sample was recorded and the mean no. of apoptotic bodies for each case was calculated. The fields were within the granulomas, in biopsies where the granulomas were not prominent; the fields chosen were among the inflammatory infiltrates.

DNA Fragmentation and electrophoresis

Six to ten, 10-μm thick sections were taken from skin biopsies in two to three slides for DNA fragmentation and electrophoresis.

Method of DNA extraction and electrophoresis

DNA was isolated from all the tissue sections of skin biopsies by the method of Palmiter et al.,[10] using routinely deparaffinized slides (10 μm sections). Briefly, the tissue was scraped and suspended in 500 μl of lysis buffer containing 1% sodium dodecyl sulfate (SDS) and 0.01% protienase K in Tris-ethylenediaminetetraacetic acid (EDTA) (TE) buffer (pH 8.0). Incubation was done at 55°C overnight.

Phenol-chloroform method was used for DNA extraction.

Quality Assessment of extracted DNA

Spectrophotometric analysis: 5 μl of dissolved DNA and 995 μl of TE buffer. Optical density (OD) was taken at 260 and 280 nm. Ratio of OD 260/280 nm (1.7-2.0) was calculated.

Agarose gel electrophoresis-Extracted DNA with the bromophenol blue dye was loaded into the well in the 2% agarose gel in Tris-acetate EDTA buffer. Electrophoresis was carried out at 80 V for 1 h. Gel was then stained with ethidium bromide for 10 min and visualized under ultraviolet (UV) light after washing and observed for “ladder pattern” of fragmented DNA specific to apoptosis. The pictures were captured using a gel documentation system.

Statistics

Statistics was done by using Statistical Package for Social Sciences (SPSS) version 20 considering ≤ 0.05 as statistically significant and ≤ 0.001 as statistically highly significant.

Chi-square test was used to calculate the difference of apoptosis in T1R (Group I) compared to non-reactional Group II

Cohen's kappa coefficient was calculated to evaluate the degree of agreement between the two methods of apoptosis detection-HPE and by DNA fragmentation and electrophoresis

Student's t-test was used to calculate the statistical significance of mean of number of apoptotic bodies seen on HPE in T1R (Group I) compared to non-reactional group (Group II). P < 0.05 was taken as significant.

Results

A total of 94 patients were recruited initially, 50 patients in Group I and 44 patients in Group II. Out of 50 patients of Group I (borderline leprosy patients who were clinically in reaction), 10 cases were excluded from evaluation because one was in T2R, two were LL, two were indeterminate leprosy histologically, and five cases were unsatisfactory for assessment. Thus, a total of 40 cases were available for assessment in Group I.

On the other hand, 44 patients of borderline leprosy without clinical or histological evidence of T1R were enrolled in this group. Four cases were excluded from evaluation because one had unsatisfactory tissue for assessment, two cases were indeterminate leprosy, and one was LL, hence excluded. Thus, a total of 40 cases were available for assessment in Group II.

While evaluation of apoptosis by DNA fragmentation and electrophoresis method, DNA could not be successfully extracted in 10 cases of Group I due to small tissue sections of skin biopsies. Therefore random 30 cases, using random number generator, from Group II were taken for analysis purpose. Finally, a total of 60 patients were evaluated for apoptosis.

Macrophage apoptosis by HPE [Figure 1] was seen in 29/30 (96.7%) cases in Group I; whereas, 24/30 (80%) cases in Group II. While apoptosis by DNA electrophoresis [Figure 2] was seen in 24/30 (80%) cases in Group I and 18/30 (60%) cases in Group II.

Figure 1

Photomicrograph of apoptotic body (arrow) seen in granuloma under oil immersion (H and E, ×1000)

Figure 2

Agarose gel electrophoresis in Tris-acetate-ethylenediamine-tetra-acetic acid (TAE) buffer

Qualitatively, via both methods, apoptosis was detected more in Group I in comparison to Group II [Figure 3], but this was not statistically significant. For HPE method, P value was 0.103 and for DNA fragmentation and electrophoresis method, P value was 0.091 [Table 1].

Figure 3

Comparison between histopathologic examination (HPE) and deoxyribonucleic acid (DNA) electrophoresis in detection of apoptosis in Group I (Type 1 reaction (T1R)) and II (non-T1R)

Table 1

Results of apoptosis detection by HPE and DNA electrophoresis with P values

There was moderate agreement (κ = 0.47) in between these two methods (HPE and DNA electrophoresis) of apoptosis detection [Table 2].

Table 2

Comparison between the two methods of apoptosis detection (HPE and DNA electrophoresis)

Sensitivity, specificity, and positive and negative predictive values of HPE and DNA fragmentation and electrophoresis for detection of apoptosis in T1R patients were calculated [Table 3]. Clinical diagnosis of leprosy Type 1 reaction was taken as gold standard for this study.

Table 3

Comparison of sensitivity, specificity, and positive and negative predictive values of two methods of apoptosis detection (HPE and DNA electrophoresis)

Quantification of apoptosis

The mean of the number of apoptotic bodies seen in Group I was 2.77 ± 1.7 and in Group II was 1.19 ± 0.93. The difference in the mean number of apoptotic bodies seen in Group I and II was statistically significant (P < 0.001) [Table 4].

Table 4

Mean of number of apoptotic bodies/10 HPF (HPE)

To assess, whether mean number of apoptotic bodies seen on HPE was more in patients in whom apoptosis was detected by DNA fragmentation and electrophoresis, P value was calculated and found to be statistically insignificant (0.091) [Table 5]. Apoptosis detection by DNA electrophoresis does not depend on number of apoptotic bodies seen on histopathology. For patients in whom apoptosis was detected by DNA fragmentation and electrophoresis, independent sample T test was used to assess whether mean number of apoptotic bodies seen on histopathological examination was more. P value was statistically insignificant.

Table 5

Comparison of mean number of apoptotic bodies seen on HPE with detection of apoptosis via DNA Electrophoresis

Discussion

It is well-known that granulomatous diseases of the skin heal with fibrosis, for example, the scarring left behind by lupus vulgaris or scrofuloderma, while on the other hand fibrosis is not seen in leprosy lesions usually. It has been postulated that this relative lack of fibrosis in leprosy lesions may be due to apoptosis, that is, an active, self-destructive, cellular process, which is considered an important part of the repertoire available to the cell to respond to deleterious stimuli from within and without.[11] In apoptosis there is lack of accompanying inflammatory component.

There are few studies on the role of apoptosis in leprosy.[891213] Niang et al.[12] had studied proportions of apoptotic lymphocytes on blood mononuclear cells in leprosy patients. They had compared these values in leprosy patients and healthy controls. A highly significant increase (P = 0.01) in the level of spontaneous apoptosis in leprosy patients was found as compared to controls, suggesting a notable impact of the M. leprae infection. Hence, apoptosis seems to be an active phenomenon in leprosy. The present study was done on skin biopsies of borderline leprosy patients because lesional apoptosis would be more specific for disease than study on blood samples which can be confounded by other systemic factors.

Walsh et al.,[8] in his study analyzed sections of paraffin-embedded, untreated leprosy lesions from 21 patients by an indirect immunofluorescent terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay and showed that apoptosis characterized both PB and MB leprosy lesions and may be more frequent in PB disease. However, sufficient information on apoptosis in various situations such as during lepra reactions was not available.

Oliviera et al.,[9] showed apoptosis may also have a role in nerve damage in leprosy. They had investigated the possibility that human Schwann cells are susceptible to cell death through the activation of Toll-like receptor 2 (TLR2), a pattern recognition receptor of the innate immune system. They conclude that the ability of M. leprae ligands to induce the apoptosis of Schwann cells through TLR2 provides a mechanism by which activation of the innate immune response contributes to nerve injury in leprosy.

In the present study, apoptosis was detected using two methods, that is, HPE and DNA fragmentation and electrophoresis, as used by Ajith et al.[13] Qualitative detection of apoptosis by both methods showed that apoptosis was in more number of cases of Group I than Group II, which was statistically not significant. This result of the present study was in agreement with Ajith et al.,[13] who also did not find any statistically significant increase in apoptosis in T1R compared to borderline non-reactional group.

The novel finding of this study was a highly significant difference in quantity of apoptosis seen in T1R patients, which has not yet been reported by any study to the best of our knowledge. This is contrary to the finding of Ajith et al.,[13] who found increase in apoptosis bodies in T2R and not in T1R.

While comparing these two methods of apoptosis detection, the HPE method was found to be a better method as it provides both qualitative and quantitative evaluation of apoptosis. However, the HPE method for apoptosis detection is subject to observer variability and hence is not ideal. Incorrect interpretation is one of the major limitations when read by an observer not experienced in apoptosis.[79] DNA fragmentation and electrophoresis is not subject to observer variation and the result are more reliable and probably of greater value than HPE, but since it was a qualitative test, quantification of apoptosis is not possible. Using DNA fragmentation and electrophoresis, apoptosis could be detected in less number of cases.

On the other hand, apoptosis detection by DNA fragmentation and electrophoresis does not depend on the number of apoptotic bodies seen on HPE as shown by P value of 0.091. In other words, we can say that quantity of apoptotic bodies does not correlate with apoptosis detection via DNA fragmentation and electrophoresis [Table 5].

The trends in the detection of apoptosis between HPE and DNA fragmentation and electrophoresis were similar as shown in the present study. Relation between these two methods of apoptosis detection was done using Cohen's kappa coefficient with κ = 0.47, which indicates moderate agreement.

One of the shortcomings of the present study was that only a single clinically active skin lesion was biopsied in each patient and in spite of being meticulously chosen, it may not be fully representative of lesions elsewhere on the body in all the cases.

Conclusions

Apoptosis is a method of cell destruction and its significance has not been established yet in leprosy and leprosy reactions. In the present study, there was moderate agreement (κ = 0.47) between the two methods of apoptosis detection. Apoptosis had been detected more frequently in patients with T1R in comparison to patients without reaction, qualitatively (statistically nonsignificant) as well as quantitatively (statistically significant). Therefore, we hypothesize that apoptosis can be used as one of the variables for diagnosis of T1R to increase detection rate which will result in early therapy with oral steroids and prevention of nerve damage.

What is new?

This study has elucidated that apoptosis is seen qualitatively and quantitatively more in lepra type 1 reaction and has proposed its use as a variable in diagnosis of type 1 reaction.