Difference of Histamine Expression between Multibacillary Leprosy

with and without Erythema Nodosum Leprosum (ENL) Reaction

Dwi Septiana

, Puguh Riyanto

, Asih Budiastuti

, Dhiana Ernawati

, Soejoto

, E. S. Indrayanti

Department of Dermatology and Venereology

Faculty of Medicine Diponegoro University / Dr. Kariadi General Hospital Semarang

Jl. Dr. Sutomo No. 16 Semarang-Indonesia.

Keywords Multibacillary leprosy, erythema nodosum leprosum, histamine expression.

Abstract Leprosy is a chronic infectious disease that poses a problem for public health because it causes disability. One

of the causes for this disability is leprosy reaction. Erythema Nodosum Leprosum (ENL) is a type 2 leprosy

reactions, which were more frequent in borderline lepromatous (BL), and lepromatous (LL) type of leprosy

receiving multi drug therapy (MDT) for multibacillary leprosy (MB). Histamine, primarily H4 receptor-

mediated, plays role in inflammation by increasing vascular permeability and muscle contraction resulting in

leukocytes migration and leakage of plasma containing antibodies and tissue complements. This cascade

finally results in tissue inflammation. To our knowledge, there is no previous study analysing histamine

expression difference between MB leprosy with and without ENL reaction. The purpose of the research was

to understand the difference of histamine expression in MB leprosy patients with and without ENL reactions

who received MDT for at least 6 months. The method of the research was an observational study with cross-

sectional approach. Study samples were 32 people divided into 2 groups, 16 samples in without ENL reactions

group and 16 samples in with ENL reactions group. Histamine expression was measured based on Allred

scoring system. The result from Difference test for histamine expression between two study groups using

Mann Whitney Test showed p < 0.05 (p=0.030 ). The study found that mean histamine expression of ENL

MB patients was higher than non-ENL MB patients.

1 INTRODUCTION

Leprosy is a chronic infectious disease caused by

Mycobacterium leprae (M. leprae) that were initially

attacked peripheral nerves, then attacked the skin and

other tissues, but excepted the central nervous system

(Kosasih, A., Wisnu., I.M., and Daili, E.S., 2011 and

Lee, D.J., Rea, T.H., Modlin, R.L., 2012). Leprosy

reaction is an acute episode in chronic course of

leprosy. It is an immune reaction, an antigen-antibody

reaction. Since they often result in disfigurements,

leprosy reactions have to be treated carefully.

Leprosy reactions consisted of two different types,

specifically type 1 or reversal reaction (RR) and type

2 or erythema nodosum leprosum (ENL) (Odom,

R.B., James, W.D., and Berger, T.G., 2010;

Lockwood, D.N.J., 2010; and Silva, M.R. and Castro,

M.C.R.D, 2012).

Coomb and Gell suggested that type 2 reaction is

a type III hypersensitivity reaction. The remains of

deceased bacilli after multi-drug therapy (MDT)

result in antigen (Odom, R.B., James, W.D., and

Berger, T.G., 2010; Lockwood, D.N.J., 2010; nd

Daili, E.S., 2011 and Lee, D.J., Rea, T.H., Modlin,

R.L., 2012). The antigen involves in reactions with

IgM, IgG, and C3 complement, forming immune

complexes that spread through blood circulation and

attach in the end organs. This cascade results in

complement system activation, producing

anaphylatoxin and promoting degranulation of mast

cell to release histamine. Histamine release will cause

inflammation, primarily after activation of histamine

h4 receptor (Frenzel, I. and Hermine, O., 2013).

After phagocytosis, the macrophage-eaten M

leprae can produce Hsp70 during the stressful

phagolisosom creation. Antileprosy drugs that given

during the course of the therapy, add more pressure to

bacilli inside the macrophages. Hsp70 accumulation

inside the macrophages invites the cytotoxic

lymphocytes to lyse the macrophages and activates

Septiana, D., Riyanto, P., Budiastuti, A., Ernawati, D., , S. and Indrayanti, E.

Difference of Histamine Expression between Multibacillary Leprosy with and without Erythema Nodosum Leprosum (ENL) Reaction.

DOI: 10.5220/0008153001590162

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 159-162

ISBN: 978-989-758-494-7

159

Table 1: The difference of mean histamine expression between leprosy patient with and without ENL reaction.

Note: b. Mann-Whitney Test

pro-inflammatory cytokines, such as TNF. After the

lysis, released Hsp70 attach to the TLR4 receptors on

the surface of mast cells. The next process is

activation of cell mast by the TLR4, as indicated by

the histamine formation, TNF, IL-6, and IL-8 then

finally promote the inflammation (Mortaz, E., et al,

2007).

Immunohistochemistry is a method to detect the

existence of specific antigen in a tissue based on

antibody (Ab) and antigen (Ag) binding. ENL is an

inflammatory reaction in tissue. Histamine-bound

histamine h4 receptors will be captured by histamine

h4 receptor antibodies and create histamine

expression on immunohistochemistry technique

(Trufelli, D.C., et al, 2010; Lin, Fan and Shi, Jianhui,

2015; and Park, C.S., Roh, J., and Kim, S.W., 2016).

Previous study have address the role of mast cell

in leprosy reaction have been conducted. They found

higher density of mast cell in leprosy reactions

(Antunes, S.L.G., et al, 2003). Therefore, the

objective of the study was to evaluate the histamine

expression difference between MB leprosy with and

without ENL reaction.

2 METHODS

This cross-sectional observational study was

conducted in outpatient setting of leprosy clinic at

Donorojo Leprosy Hospital, Jepara. The subjects

were patients with multibacillary (MB) infection and

aged 20-65 years old. To be included, the patients

must have been received MDT for more than 6

months. During study period (March 2017- June

2017), 32 patients were included, 16 MB patients

without ENL reaction and 16 MB patients with ENL

reaction. The skin tissue samples were processed and

analysed by immunohistochemistry method in

anatomical pathology laboratory of Medical Faculty,

Gadjah Mada University, Yogyakarta.

Immunohistochemical examination and measurement

of histamine expression can be seen using a light

microscope at 10 fields of view using 400x

magnification. Each field of view will be

photographed and then analyzed using a computer

(image raster software) to calculate the percentage

and intensity of cells that bind histamine h4 receptor

antibodies, which then according to the percentage

and the intensity of the staining will be incorporated

into the semi-quantitative system by using the Allred

scoring system

3 RESULT

In non-ENL group, the number of female and male

patients was equal, the mean age was 42.19 + 2.22

years, mean leukocyte count was 7806.25 + 2094.54

/mm

, mean MDT duration was 8.5 + 2.22 months. In

ENL group, all of the patients were males, the mean

age was 32.63 + 11.93 years, mean leukocyte count

was 8587.5 + 1872.92 /mm

, mean MDT duration

was 8.96 + 2.024 months. The mean histamine

expression in non-ENL and ENL group were 6.31 +

1.35 and 7.31 + 0,48, respectively.

The present study found that mean histamine

expression of ENL MB patients was higher than non-

ENL MB patients. The Mann-Whitney test for mean

histamine expression found significant difference (p

< 0 .05) with p = 0.030.

4 DISCUSSION

In this study we obtained that male leprosy patients

were more than female where in multibacillary

leprosy group without ENL reactions the number of

men was eight persons (50%) and 16 persons (20%)

in multibacillary leprosy group with ENL reaction.

Gender generally is not a risk factor of ENL.

Sample distribution by age was found to be

statistically significant. Erythema Nodosum

Leprosum based on age group does not represent a

particular group risk for developing ENL.

Non ENL Reaction ENL Reaction P

Mean±SD Median

(Min-maks)

Mean±SD Median

(Min-maks)

Histamine

expression

6,31 ± 1,35 6(4 – 8) 7,31±0,48 7 (7-8) 0,030

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Figure 1: The study found that mean histamine expression of ENL MB patients was higher than non-ENL MB patients.

Table 2: Characteristic subjects of the study.

Non Reaksi ENL Reaksi ENL P

N(%) Mean±SD Median

(Min-maks)

N(%) Mean±SD Median

(Min-maks)

Gender male

8(50) 16(100)

0,002

female

8(50) 0(0)

Age

42,19±11,2 40(23-59) 32,63±11,93 31,5(20-59) 0,011

leukocytes

7806,25±20

95,54

7750(3700-

10700)

8587,5±187

2,92

9100(4400-

10900)

0,275

MDT

Duration

Job

student

0(0)

8,5±2,22

8(6-12)

2(12,5)

8,96±2,024

8(6-12)

0,688

non job

farmer

3(18,8)

1(6,3)

7(43,8)

0(0)

Labor

Retirement

0(0)

1(6,3)

0,053

driver

fisherman

Housewife

Private

employe

Embankment

farmer

0(0)

1(6,3)

7(43,8)

4(25)

0(0)

1(6,3)

0(0)

3(18,8)

1(6,3

Note: a.Uji chi square ; b. Mann Whitney test ; c. Uji t independen

Leprosy is known to occur in all ages ranging

from infancy to old age (3 weeks to over 70 years).

But the most is happened at a young and productive

ages. ENL occurs at any time during leprosy episodes

and most often after 6 months treatment

(Triamchaisri, S., et al, 2015).

Subjects of the study were limited by the number

of leukocytes <11.000 / mmk to exclude

inflammatory and infectious diseases in which

histamine expression increased in the presence of

inflammatory and infectious conditions (Zampeli, E.

and Tiligada, E., 2009). The mean difference of

leukocyte counts of multibacillary leprosy study

subjects with ENL and without ENL was found to be

insignificant ( p = 0.275) showed that the leukocyte

rates of sample was homogeneous.

MDT duration therapy of multibacillary leprosy

group with ENL and without ENL was found to be

insignificant (p = 0,688). Erythema Nodosum

Leprosum can occur at any time during leprosy

course but most often occurs after 6 months of MDT

treatment, since many leprosy bacteria are destroyed,

that means many antigens are released and react with

antibodies, thus activates the complement system and

results in ENL (Odom, R.B., James, W.D., and

Berger, T.G., 2010; Lockwood, D.N.J., 2010; Lee,

Difference of Histamine Expression between Multibacillary Leprosy with and without Erythema Nodosum Leprosum (ENL) Reaction

161

D.J., Rea, T.H., Modlin, R.L., 2012; and Silva, M.R.

and Castro, M.C.R.D, 2012).

Histamine expression value in MB group with

ENL reaction was higher than MB group without

reaction with significant differences (p = 0.030) by

Mann-Whitney test. Histamine plays a role in

immunoinflamatory response particularly in those

with H4 receptor (mast cells, eosinophils, T cells and

dendritic cells) that activates immune cell maturation,

increases cytokine production and increases cellular

chemotactic response (Gutzmer, R., et al, 2011 and

Harvima, I.T. and Nilsson, G., 2011). Histamine

increases in inflammatory states such as ENL.

Histamine expression in MB leprosy group with ENL

reaction was higher than MB leprosy group without

ENL reaction.

5 CONCLUSION

There was a significant difference in histamine

expression between MB leprosy with and without

ENL reaction where the mean of histamine

expression in MB leprosy group with ENL reaction

was higher than leprosy group without ENL reaction.

ACKNOWLEDGEMENT

This study was supported by Faculty of Medicine

Diponegoro University / Dr. Kariadi General Hospital

Semarang, leprosy clinic at Donorojo Leprosy

Hospital, Jepara and anatomical pathology laboratory

of Medical Faculty, Gadjah Mada University,

Yogyakarta.

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