The Correlation between Neutrophil CD64, Interleukin-17 (Il-17),

Interleukin-10 (Il-10) in Skin Tissue and Neutrophil CD64, Il-17 and

Il-10 in Blood Circulation in Erythema Nodosum Leprosum (ENL)

Patients

I. Gusti Nyoman Darmaputra

, Luh Mas Rusyati

, Wibi Riawan

, Anang Endaryanto

, Cita Rosita

Sigit Prakoeswa

Department of Dermatology and Venereology Universitas Udayana, Sanglah Hospital, Denpasar, Indonesia

Department of Biochemistry-Biomolecular Faculty of Medicine, Universitas Brawijaya, Dr.Saiful Anwar Teaching

Hospital Malang, Indonesia

Department of Pediatrics Faculty of Medicine Universitas Airlangga, Dr. Soetomo Teaching Hospital Surabaya, Indonesia

Department of Dermatology and Venereologi Faculty of Medicine Universitas Airlangga, Dr. Soetomo Teaching Hospital,

Surabaya Indonesia

Keywords: ENL, neutrophil CD64, IL-17, IL-10, local immunity

Abstract: Erythema nodosum leprosum (ENL) remain major health problem globally. Current pathogenesis of ENL not

only involving immune complex, but also neutrophil and cellular immune response. Neutrophil CD64,

interleukin-17 (IL-17) and interleukin-10 (IL-10) are mediators that suggested to be involved in regulation of

ENL. This study aimed to determine the correlation between neutrophil CD64, IL-17 and IL-10 in skin tissue

with neutrophil CD64, IL-17 and IL-10 in blood circulation. This was an analytical observational study with

a cross-sectional design which involved 30 patients of multibacillary (MB) type of leprosy patients which

underwent ENL. Neutrophil CD64 levels in blood circulation were measured by flow cytometry, while IL-10

and IL-17 levels in the blood circulation with ELISA. Biopsy was performed prior to the examination of

neutrophil CD64, IL-17 and IL-10 from skin tissue with immunohistochemistry (IHC). The obtained data

were analyzed descriptively and with correlation analysis. The mean of neutrophil CD64, IL-17 and IL-10 in

skin tissue was 19.10 ± 5.371, 11.47 ± 5.029 and 6.73 ± 3.039, respectively, while the mean of neutrophil CD

64, IL-17 and IL-10 in circulation was 66.807 ± 14.794, 59.55 ± 36.397 and 186.30 ± 272.974, respectively.

No significant correlation was found between IL-17, neutrophil CD64 and IL-10 in skin tissue with IL-17,

neutrophil CD64 and IL-10 in blood circulation with significance level of 0.511, 0.186 and 0.401,

respectively. There was no correlation between dysregulation of local immunity with systemic immunity in

ENL.

1 INTRODUCTION

Erythema nodosum leprosum (ENL) is one type of

leprosy reactions. Current world health organization

(WHO) multidrug therapy (MDT) had not been able

to eliminate the incidence of leprosy reactions yet.

The prevalence of ENL cases varies greatly depends

on geographical variations. ENL cases in Asia were

high which varied from 19% to 26% among all

borderline lepromatous (BL) and lepromatous

leprosy (LL) patients (Kahawita et al., 2008).

To date, pathogenesis of ENL not only involving

immune complex. Neutrophil and cellular immune

response were found to be greatly involved in the

pathogenesis of ENL. Neutrophil is the main

inflammatory cells infiltrate in ENL. The

accumulation of neutrophil in skin tissue caused by

the increase of E-selectin expression in the

endothelium due to interleukin-17 (IL-17) and

interferon γ (IFN-γ) stimulation. A study by Schmitz

found that the active neutrophil cells were

characterized by increased expression of Fc gamma

receptor I (FcγRI) or cluster of differentiation 64

(CD64). CD64-expressing neutrophils were found to

be increased either in blood circulation or skin tissue

286

Darmaputra, I., Rusyati, L., Riawan, W., Endaryanto, A. and Prakoeswa, C.

The Correlation between Neutrophil CD64, Interleukin-17 (Il-17), Interleukin-10 (Il-10) in Skin Tissue and Neutrophil CD64, Il-17 and Il-10 in Blood Circulation in Erythema Nodosum Leprosum

(ENL) Patients.

DOI: 10.5220/0008155802860290

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 286-290

ISBN: 978-989-758-494-7

of ENL patients and related to the severity of ENL

(Schmitz et al., 2016).

Another molecular factor which found in ENL

and involved in nerve damage regulation due to

leprosy is interleukin-10 (IL-10). Lipid droplet (LD),

found in lepromatous type of leprosy patients, is an

active catalyst place for the synthesis of

prostaglandin-E2 (PGE-2) which promote the

production of IL-10. Either PGE-2 or IL-10 involved

in suppressing the inflammation in the nerve. LD in

Schwann cells (SC) indirectly acts as protective factor

to maintain SC from damage caused by nerve

inflammation (Mattos et al., 2011; Mattos et al.,

2010).

Previous study reported that neutrophil CD64

increased in blood circulation or skin tissue of ENL

patients and related to the severity of ENL (Schmitz

et al., 2016).

Either IL-17 or IL-10 also involved in

the pathogenesis of ENL. Nerve damage, which

occurred in ENL, is assumed to be affected by the

dysregulation of systemic and local immunity. Thus,

this study aimed to analyze the correlation between

local mediators (neutrophil CD64, IL-17 and IL-10 in

skin tissue) with systemic mediators (neutrophil

CD64, IL-17 and IL-10 in circulation) in ENL.

2 METHODS

This was an analytical observational study with cross-

sectional design conducted at Dr. Soetomo General

Hospital, Surabaya, Indonesia, during July to

September 2017. This study involved 30 patients with

multibacillary (MB) type of leprosy who suffered

ENL leprosy reaction, based on calculation with

sample size formula. Consecutive sampling was used

to obtain the samples. The inclusion criteria were

patients with MB type of leprosy with ENL which

defined clinically and bacteriologically, aged

between 18 to 60 years and willing to participate in

this research by signing the informed consent.

Whereas patients with a pregnant condition or poor

general condition, history of peripheral nerve

lesion/trauma, and having a history of oral

thalidomide treatment in the last two days were

excluded.

2.1 Measurement of Neutrophil CD64,

IL-10 and IL-17 in Circulation and

IL-17, Neutrophil CD64 and IL-10

in Skin Tissue

The blood sample was taken for examination of

neutrophil CD64, IL-10 and IL-17 levels in

circulation. Neutrophil CD64 measured by flow

cytometry while ELISA was used for IL-10 and IL-

17 measurement.

The sample of skin tissue was taken from foot

region with punch biopsy. The procedure of the

biopsy described as follows; disinfection was

performed with 70% of alcohol on the foot region of

the patients. Subsequently, local anesthesia with 0.25

ml of lidocaine was done. On that area, biopsy with

punch method was performed with diameter of 4 mm.

The obtained skin tissue was put in the eppendorf

containing 10% formalin. The wounded area cleaned

with 0.9%NaCl, applied with a topical antibiotic and

covered with sterile gauze. The samples then

processed and immunohistochemistry (IHC) method

was used to examine IL-17, neutrophil CD64 and IL-

10 expressions (by counting on 20 fields of view in

1000 times magnification by microscope, with results

of ratio-scale data). Measurement of IL-10 in skin

tissue in this study was specified to IL-10 expressed

by Schwann cells (SC), through staining of S-100

protein as a marker of SC.

2.2 Data Analysis

Data were analyzed with Statistical Package for the

Social Sciences (SPSS) for Windows version 22.0.

The univariate/descriptive analysis was conducted to

describe the subject characteristics and research

variables. The numerical data presented in mean and

standard deviation (SD), while the categorical data

with relative frequency or amount and percentage.

The result of the descriptive analysis presented in

single distribution table. Spearman or Pearson

correlation analysis was performed to determine the

correlation between variables in blood circulation and

skin tissue. A p-value of less than 0.05 was

considered significant.

The Correlation between Neutrophil CD64, Interleukin-17 (Il-17), Interleukin-10 (Il-10) in Skin Tissue and Neutrophil CD64, Il-17 and

Il-10 in Blood Circulation in Erythema Nodosum Leprosum (ENL) Patients

287

3 RESULT

3.1 Characteristics of Study Subjects

Characteristic of study participants are age, sex,

occupation, type of leprosy, type of ENL reaction and

the severity of ENL. Variables in skin tissue

(neutrophil CD64, IL-17 and IL-10) and blood

circulation (IL-17, neutrophil CD64, IL-10) are

presented in Table 1.

3.2 Immunohistochemistry (IHC)

Results

The results of IHC examination from skin tissue are

depicted in figure 1.

Table 1. Characteristics of ENL patients at Dr. Soetomo General Hospital, Surabaya.

Number. Variables Respondents (N = 30)

1. Age, n (%)

11-20 years

21-30 years

31-40 years

41-50 years

51-60 years

3 (10.0%)

8 (26.7%)

9 (30.0%)

7 (23.3%)

3 (10.0%)

2. Sex, n (%)

Male

Female

6 (20.0%)

24 (80.0%)

3. Occupation

Housewife

Students/college students

Government employees

Private sector

3 (10.0%)

2 (6.7%)

22 (73.3%)

3. Type of Leprosy, n (%)

18 (60.0%)

12 (40.0%)

4. Type of ENL reaction, n (%)

Acute

Chronic

0 (0.0%)

30 (100.0%)

5. The Severity of ENL, n (%)

Mild

Moderate

Severe

9 (30.0%)

18 (60.0%)

3 (10.0%)

6. Variables in skin tissue, Mean ± SD (CI)

Neutrophil CD64

IL-17

IL-10

19.10 ± 5.371 (17.09–21.11)

11.47 ± 5.029 (9.59–13.34)

6.73 ± 3.039 (5.60–7.87)

7. Variables in circulation, Mean ± SD (CI)

Neutrophil CD64

IL-17

IL-10

66.807 ± 14.794 (61.283–72.331)

59.55 ± 36.397 (45.962–73.144)

186.30 ± 272.974 (84.373–288.233)

Normal distribution of data with Kolmogorov Smirnov test of 1 sample;

Normal distribution of neutrophil CD64

data but not for IL-17 and IL-10 with Kolmogorov Smirnov test of 1 sample. CI: Confidence interval.

Figure 1: Skin tissue biopsy with IHC staining using: A. Anti-human IL-17 monoclonal antibody; B. Anti-human CD64

monoclonal antibody; C. Anti-human IL-10 and anti-S-100 monoclonal antibody (black arrows = positive reactions) (1000x

magnification).

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Table 2: The correlation between variables in skin tissue with variables in circulation.

Variables r Significance

IL-17 in skin tissue

IL-17 in circulation

0.125 0.511

Neutrophil CD64 in skin tissue

Neutrophil CD64 in circulation

0.248 0.186

IL-10 in skin tissue

IL-10 in circulation

0.159 0.401

Spearman;

Pearson

3.3 The Correlation of IL-17,

Neutrophil CD64 and IL-10 in Skin

Tissue with IL-17, Neutrophil

CD64 and IL-10 in Blood

Circulation

Correlation analysis between variables in skin tissue

(IL-17, neutrophil CD64 and IL-10) with variables in

circulation (IL-17, neutrophil CD64 and IL-10) was

performed. There is no significant correlation

between IL-17, neutrophil CD64 and IL-10 in skin

tissue with IL-17, neutrophil CD64 and IL-10 in

blood circulation with significance level of 0.511,

0.186 and 0.401, respectively (table 2).

4 DISCUSSION

This study found that neutrophil CD64 in skin tissue

did not correlate with neutrophil CD64 in blood

circulation. Few CD64 was expressed on the surface

of neutrophil (average of 1400 receptors per cell) in

inactive neutrophil. The increased expression of

CD64 on the surface of neutrophil as a marker of

active neutrophil occurs in four until six hours after

stimulation of IFN- or granulocyte-macrophage

colony-stimulating factor (GM-CSF) and other

kinetic factors, e.g., lipopolysaccharide (Nuutila,

2010; Allen et al., 2002). CD64 surface receptors

were not only expressed on neutrophil cells surface

but also on monocytes. In this study, CD64 expressed

by neutrophil in blood circulation was distinguished

from CD64 expressed by monocytes through flow

cytometry examination with anti-CD163 to mark out

monocytes, thus only CD64 expressed by neutrophil

was calculated.

Neutrophil CD64 in circulation entered skin tissue

because of increased expression of E-selectin on

endothelium. However, this study found no

significant correlation between neutrophil CD64 in

skin tissue with neutrophil CD64 in circulation with

significance value of 0.186 (< 0.05).

IL-17 is an inflammatory cytokine in skin tissue

which may promote the production of IL-6, IL-8 and

GM-CSF from non-immune cells, e.g., fibroblast and

epithelial cells via activation of nuclear factor-

kappaB (NF-kB) transcription factor (Stettner et al.,

2014). In general, IL-17 is involved in inducing pro-

inflammatory chemokines, hematopoietic cytokines,

acute phase response genes and anti-microbial

substances. The current study found no significant

correlation between IL-17 in skin tissue with IL-17 in

blood circulation with significance value of 0.511 (>

0.05).

There is no study concerning the relationship

between IL-17 in circulation with IL-17 in skin tissue

of ENL patients. One study by Li et al. on Guillain-

Barré syndrome patients reported a significant

correlation was found between IL-17 in circulation

with in cerebrospinal fluid. This may be due to IL-17

in circulation is able to penetrate the blood-brain

barrier, hence its levels in cerebrospinal fluid also

increased. The different result which obtained in this

study may be due to IL-17 in circulation is unable to

enter skin tissue in large quantities.

IL-10 is a cytokine with multiple and pleiotropic

effect in the immunoregulatory process. This study

found no significant correlation between IL-10 in skin

tissue with IL-10 in circulation with a significance

value of 0.401 (> 0.05). It has been suggested that this

occur due to IL-10 in blood circulation is produced by

monocytes and other cells, e.g., lymphocytes and

mast cells. In this study, the levels of IL-10 in blood

circulation measured by ELISA which detects IL-10

levels regardless of the cells that generate it, while the

expression of IL-10 in skin tissue was specific to IL-

10 which expressed by SC, through S-100 staining.

It has been assumed that aberration which occurs

in ENL, e.g., nerve damage is related to dysregulation

of systemic and local immunity, hence in this study

the local mediators in skin tissue and systemic

mediators in blood circulation were examined. This

study obtained novel findings that there was no

The Correlation between Neutrophil CD64, Interleukin-17 (Il-17), Interleukin-10 (Il-10) in Skin Tissue and Neutrophil CD64, Il-17 and

Il-10 in Blood Circulation in Erythema Nodosum Leprosum (ENL) Patients

289

correlation between dysregulation of local immunity

with systemic immunity.

5 CONCLUSION

There was no correlation between dysregulation of

local immunity with systemic immunity in ENL.

Nerve damage in ENL is dominated by local

processes. Further research is required to investigate

local immune responses and to explore local

intervention for management of neurological damage

in ENL.

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