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Serum cytokine profile in leprosy and its correlation with clinico-histopathological profile NEHA KAWATRA MADAN, KIRAN AGARWAL & RAM CHANDER Departments of Pathology and Dermatology, Lady Hardinge Medical College and Smt. Sucheta Kriplani Hospital, New Delhi, India Accepted for publication 19 September 2011 Summary Objectives: To analyse the serum levels of cytokines in leprosy patients, to correlate them with clinico-histopathological profile, and to study the effect of standard multidrug therapy on serum cytokine levels. Design: Serum immunoassays of TNF-a, IFN-g, IL-1b and IL-10 were performed by ELISA in 61 newly diagnosed cases of leprosy before starting therapy and during reactional episodes. Of these, cytokine assays could be performed in 17 cases after completion of therapy. Results: Levels of all the studied cytokines were significantly raised in cases compared to controls (P , 0·05). Levels of TNF-a and IFN-g were significantly raised in paucibacillary cases whereas all the studied cytokines were raised in multibacillary cases with rise in IL-1b and IL-10 being statistically significant (P , 0·001). No significant difference was however noted between TT and BT type; and BB, BL and LL types. All the studied cytokines were raised in reactional cases as compared to non-reactional cases. Comparing Type 2 reaction (T2R) and Type 1 reaction (T1R) cases, levels of IFN-g, IL-1b and IL-10 were higher in T2R cases but only IL-10 was found to be statistically significant (P ¼ 0·05) while TNF-a was higher in T1R cases. Post therapy serum levels of all the studied cytokines were significantly lower than pretherapy levels (P , 0·05) and were comparable to controls. Among the paucibacillary cases, levels of all the cytokines were seen to decrease after 6 months of standard multidrug therapy. In the multibacillary cases, mean levels of the cytokines were found to decrease after 1 year of therapy except IFN-g. Conclusion: Serum cytokine estimation may have a significant role in classifying various forms of leprosy and can be used to monitor therapy. Introduction Leprosy is a chronic infectious granulomatous disease caused by M. leprae which is an obligate intracellular pathogen. 1 Cases of leprosy are highly concentrated in Correspondence to: Neha Kawatra Madan, Department of Pathology, Lady Hardinge Medical College, New Delhi, India (Tel: þ 91-9953236682; þ 91-124-2320532; e-mail: [email protected]) Lepr Rev (2011) 82, 371–382 0305-7518/11/064053+12 $1.00 q Lepra 371

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Serum cytokine profile in leprosy and its correlation

with clinico-histopathological profile

NEHA KAWATRA MADAN, KIRAN AGARWAL &

RAM CHANDER

Departments of Pathology and Dermatology, Lady Hardinge Medical

College and Smt. Sucheta Kriplani Hospital, New Delhi, India

Accepted for publication 19 September 2011

Summary

Objectives: To analyse the serum levels of cytokines in leprosy patients, to correlate

them with clinico-histopathological profile, and to study the effect of standard

multidrug therapy on serum cytokine levels.

Design: Serum immunoassays of TNF-a, IFN-g, IL-1b and IL-10 were performedby ELISA in 61 newly diagnosed cases of leprosy before starting therapy and during

reactional episodes. Of these, cytokine assays could be performed in 17 cases after

completion of therapy.

Results: Levels of all the studied cytokines were significantly raised in cases

compared to controls (P , 005). Levels of TNF-a and IFN-gwere significantly raisedin paucibacillary cases whereas all the studied cytokines were raised in multibacillary

cases with rise in IL-1b and IL-10 being statistically significant (P , 0001).

No significant difference was however noted between TT and BT type; and BB, BL

and LL types. All the studied cytokines were raised in reactional cases as compared to

non-reactional cases. Comparing Type 2 reaction (T2R) and Type 1 reaction (T1R)

cases, levels of IFN-g, IL-1b and IL-10 were higher in T2R cases but only IL-10 wasfound to be statistically significant (P 005) while TNF-a was higher in T1R cases.Post therapy serum levels of all the studied cytokines were significantly lower

than pretherapy levels (P , 005) and were comparable to controls. Among the

paucibacillary cases, levels of all the cytokines were seen to decrease after 6 months

of standard multidrug therapy. In the multibacillary cases, mean levels of the

cytokines were found to decrease after 1 year of therapy except IFN-g.

Conclusion: Serum cytokine estimation may have a significant role in classifying

various forms of leprosy and can be used to monitor therapy.

Introduction

Leprosy is a chronic infectious granulomatous disease caused by M. leprae which

is an obligate intracellular pathogen.1 Cases of leprosy are highly concentrated in

Correspondence to: Neha Kawatra Madan, Department of Pathology, Lady Hardinge Medical College,New Delhi, India (Tel: 91-9953236682; 91-124-2320532; e-mail: [email protected])

Lepr Rev (2011) 82, 371382

0305-7518/11/064053+12 $1.00 q Lepra 371

10 countries- India, Brazil, Myanmar, Nepal, Mozambique, Madagascar, Angola, Central

African Republic, Democratic Republic of Congo and United Republic of Tanzania.

India alone represents 64% of the prevalence and 78% of new case detection worldwide.2

Leprosy is classified according to the degree and type of host immune response. Patients

with tuberculoid leprosy have a strong cell mediated immune (CMI) response to M. leprae,

manifested by positive skin tests (Mitsuda reaction) and marked lymphocyte proliferative

responses to M. leprae in vitro. In contrast, patients with lepromatous leprosy have a

depressed CMI response toM. leprae, characterised by negative skin tests and diminished or

absent lymphocyte proliferation.3 The polar groups [Tuberculoid (TT) and Lepromatous

(LL)] are stable, but within the central groups [Borderline tuberculoid (BT), Borderline

borderline (BB), Borderline lepromatous (BL)], downgrading towards the lepromatous

pole, and upgrading (Reversal reactions) towards the tuberculoid pole may occur. The

reactional episodes are due to an increase in the CMI and are characterised clinically by

the leprosy lesions becoming inflamed and erythematous, with swelling and destruction of

the peripheral nerves.4,5

Cytokines are low molecular weight glycoproteins produced by immune as well as

non-immune cells which act as molecular signals for communication between cells of the

immune system.6 Defects in cytokine production or their expression at the target tissue sites

are associated with pathologic states, therefore, delivery of an exogenous cytokine can lead to

clinical benefit.

Even though there have been exciting new developments in the field of immunology,

there are still many queries related to the immunopathology of leprosy. There is paucity

of documented studies on the assessment of serum cytokine levels in leprosy patients by

ELISA which is easily available especially in the developing world and has a fair sensitivity

and specificity. Literature reveals controversial results on the levels of serum cytokines in

various forms of leprosy. Moreover, the effect of multidrug therapy on the cytokine levels

has not been substantiated well in the past. With this background in mind, the aim of the

present study was to analyse serum cytokine profiles of untreated leprosy patients, compare

them with healthy controls, co-relate the patterns with the clinico-histopathological picture

and to see the effect of Multidrug Therapy (MDT) on the levels of serum cytokines.

Materials and Methods

A total of 61 untreated cases of leprosy attending the Dermatology Out Patient Department

(OPD) of Smt. Sucheta Kriplani Hospital were enrolled for this study. After a written consent,

a detailed history was obtained and a thorough physical examination was done. The patients

were classified according to Ridley-Joplings five subgroups (TT, BT, BB, BL, LL)7 and

a sixth subgroup Pure Neuritic (PN) which was described by the Consensus Classification

of Indian Association of Leprologists (1982).8 Slit smear examination for lepra bacilli and

skin biopsies were performed in all cases except the Pure neuritic ones.

The cases comprised 38 paucibacillary (5 TT, 26 BT, 7 PN) and 13 multibacillary (1 BB,

6 BL, 6 LL); 10 patients presented with reactions (six of Type 1 and four of Type 2) and 2 LL

patients developed Type 2 reaction during the course of our study.

All patients received the standard WHO-MDT regimen for leprosy [Rifampicin and

Dapsone for 6 months in paucibacillary; Rifampicin, Dapsone and Clofazimine for 12 months

N. Kawatra Madan et al.372

(BI # 4)/24 months (BI $ 5) in multibacillary] and patients were followed-up to look for

onset of any lepra reaction and to monitor the effect of therapy.

Patients who were already on multidrug therapy or steroids or having any other systemic

illness were excluded from the study. Thirty age and sex matched healthy controls were also

included. Blood was collected in a plain vial, centrifuged immediately, and sera were stored

at 2708C prior to assay. At least one cytokine belonging to each of the Th1, Th2 and

macrophage cytokine subsets were chosen for reasons of feasibility. Cytokine assay for

TNF-a, IFN-g, IL-1b and IL-10 was done by ELISA kit method (Diaclone, France).

Post-therapy cytokine assays were performed in 17 cases within 1 week of completion of

therapy [11 PB (after 6 months) and 6 MB (after 12 months)]. Out of 17 follow up cases, skin

biopsies could be performed at the completion of therapy in 10 cases (all PB).

The concentration of individual cytokines in pg/ml in each patient was used for data

analysis. Students t-test and chi square test were employed for comparison between two

groups. P values # 005 were considered statistically significant. The correlation coefficient

(r) was applied between the serum cytokine levels.

The study was approved by the institutions Ethics Committee and the universitys

Review Board and the funding for cytokine kits was provided by the Department of

Pathology, Lady Hardinge Medical College.

Results

The majority of cases (5245%) were in the age group of 2040 years with male to female

ratio of 3:1. Mean age of affliction was 30 years. BT was the predominant form of leprosy

comprising 26 (426%) cases. Nerve involvement in the form of thickened nerves/neuritis was

found in 58 (951%) cases at the time of sera collection. Ten patients (144%) had abnormal

facies (madarosis, infiltration or lagophthalmos) out of which two patients (both LL) had

leonine facies. Four patients (66%) had deformities (claw hand, thenar atrophy or foot drop).

CLINICO-HISTOPATHOLOGICAL CORRELATION

The maximum cases were of Borderline Tuberculoid Leprosy (426%). Only two cases out of

61 (i.e. 33%) showed discordance in clinical and histopathological findings; 100% of the

paucibacillary patients had a BI between 0 and 2 and 70% of the multibacillary patients

Table 1. Comparison of cytokine levels of cases with controls

CASES (N 61) CONTROLS (N 30)

CYTOKINESMEAN(pg/ml)

STD DEV(pg/ml)

MEAN(pg/ml)

STD DEV(pg/ml)

P value(Students t-test)

TNF-a 7017 5084 1946 670 003IFN-g 10001 10745 2579 1190 ,0001IL-1b 9397 8098 1576 611 ,0001IL-10 6308 5948 1590 230 01

Serum cytokine in leprosy 373

had a BI between 3 and 6 . The rest (30%) of the patients had a BI of 02 but displayedmore than six skin lesions and were placed in the multibacillary group.

CYTOKINE ANALYSIS

Levels of all the studied cytokines were significantly raised in cases rather than controls

(P , 005) (Table 1).In the patient group (including all subtypes), mean serum levels of TNF-a, IFN-g, IL-1b

and IL-10 were 7017 ^ 5084 pg/ml, 10001 ^ 10745 pg/ml, 9397 ^ 8098 pg/ml and

6308 ^ 5948 pg/ml respectively.

Between the paucibacillary (PB) and multibacillary (MB) cases, higher levels of TNF-aand IFN-g were found in PB patients whereas higher levels of IL-1b and IL-10 were foundin MB cases (P , 005) (Figure 1).

No significant difference was noted among the cytokine levels between cases of TT and

BT leprosy; and between BL and LL cases.

All the studied cytokines were raised in reactional cases as compared to non-reactional

cases (Figure 2).

When cases with T1R were compared with non-reactional PB cases, it was found that

levels of TNF-a and IL-1b were significantly higher in T1R than in PB cases (P , 0001).IFN-g was raised in T1R cases as compared to PB cases however was not statisticallysignificant (Table 2).

140Paucibacillary cases

Multibacillary cases

120

100

80

60

40

20

0MeanTNF-

MeanIFN-

MeanIL-1

Cytokines

Levelsinpg/ml

MeanIL-10

Figure 1. Comparison of cytokine levels in paucibacillary and multibacillary cases.

N. Kawatra Madan et al.374

When cases with T2R were compared to non-reactional multibacillary cases, it was found

that all of the studied cytokines were raised in T2R than in non-reactional multibacillary cases

but only IFN-g levels were statistically significant (P , 005) (Table 3).Between T2R and T1R patients, levels of IFN-g, IL-1b and IL-10 were higher in T2R

cases than T1R cases but only IL-10 was found to be statistically significant (P 005) whileTNF-a was higher in T1R cases (Table 4).

The bacillary index (BI) showed a positive correlation with levels of TNF-a and IFN-gand a negative correlation with levels of IL-1b and IL-10 (Figure 3).

Positive correlation was found between TNF-a and IFN-g levels and between IL-1b andIL-10 levels (Figure 4A and 4B).

EFFECT OF THERAPY ON CYTOKINE LEVELS

Post-therapy serum levels of all the studied cytokines in the paucibacillary group were

significantly lower than the pre-therapy levels (P , 005) and were comparable to controls (6months post-therapy) (Figure 5).

The difference in the mean levels of TNF-a and IFN-g, before and after MDT was foundto be significant (P , 001).

250

200

150

Levelsinpg/ml

100

Reactional casesNon reactional cases

50

0MeanTNF-

MeanIFN-

MeanIL-1

Cytokines

MeanIL-10

Figure 2. Cytokine levels in reactional vs non-reactional cases.

Serum cytokine in leprosy 375

In the multibacillary cases, mean levels of TNF-a, IL-1b and IL-10 were found todecrease after 1 year of treatment but did not come down to the level of healthy controls.

However, two multibacillary cases showed increase in IFN-g levels (these two cases wentinto T2R during the study period).

HISTOPATHOLOGY POST THERAPY

Post therapy skin biopsies of the 10 PB cases, taken within a month of completion of

therapy, were found to be normal with the disappearance of the inflammatory granulomas.

Discussion

Leprosy provides an ideal model to address the role of T-cell subsets in human diseases.9

Cytokines play important roles in both protection and immunopathology of leprosy and are

considered important components of lepra reactions. M. leprae is not toxic to its host cells,

and the pathologic process reflects the immune response of the patient to the organisms.

At the tuberculoid pole, patients exhibit a strong CMI response to M. leprae leading to

control of bacillary replication. At the lepromatous pole, patients exhibit a defective CMI

response to M. leprae thereby causing a high bacillary load.

In the present study, serum levels of TNF-a, IFN-g, IL-1b and IL-10 were raised in allleprosy cases, both paucibacillary and multibacillary. TNF-a and IFN-g were raised inpaucibacillary leprosy, whereas all the studied cytokines were raised in multibacillary leprosy

cases with rise in IL-1b and IL-10 being statistically significant (P , 005).

Table 2. Cytokine levels in paucibacillary cases compared to cases with type 1 reaction

PB (N 31) PB with T1R (N 6)

CYTOKINESMEAN(pg/ml)

STD DEV(pg/ml)

MEAN(pg/ml)

STD DEV(pg/ml)

P value(Students t-test)

TNF-a 6166 2855 11823 5486 0001IFN-g 7707 6090 16661 13729 01IL-1b 2471 1968 7849 3402 0003IL-10 1909 1487 2196 689 07

Table 3. Cytokine levels in multibacillary cases compared to cases with type 2 reaction

MB (N 13) MB with T2R (N 4)

CYTOKINESMEAN(pg/ml)

STD DEV(pg/ml)

MEAN(pg/ml)

STD DEV(pg/ml)

P value(Students t-test)

TNF-a 5532 7678 10545 5678 02IFN-g 5517 6242 35231 14020 002IL-1b 13195 9873 13578 6342 09IL-10 6030 2996 15726 8806 01

N. Kawatra Madan et al.376

It has been hypothesised that IFN-g and TNF-a play a role in immunoprotection andimmunopathology, whereas IL-10 and IL-1b are immunosuppressive.1012 Our results areconsistent with this hypothesis. IFN-g activates antimicrobial mechanisms in macrophagesby inducing Nitric oxide synthase, leading to the production of Nitric oxide, a powerful

microbicidal molecule.6,13 TNF-a is necessary for granuloma formation containingbactericidal macrophages that play an essential role in preventing the extension and

dissemination of mycobacterial infection.6,1416 IL-10 is a potent inhibitor of IFN-gproduction17,18 and to some extent of TNF-a. It suppresses the macrophage mediated

Table 4. Cytokine levels in type 1 vs. type 2 reactional cases

T1R (N 6) T2R (N 6)

CYTOKINESMEAN(pg/ml)

STD DEV(pg/ml)

MEAN(pg/ml)

STD DEV(pg/ml)

P value(Students t-test)

TNF-a 11823 5486 10545 5678 07IFN-g 16661 13729 35231 14020 007IL-1b 7849 3402 13578 6342 009IL-10 2196 689 15726 8806 005

400

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IL1

TNF-

IL10

IFN-

200

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4 6 0 2

Bacillary index

4 6

0 2

Bacillary index

4 60 2

Bacillary index

4 6

250

200

150

100

50

0

Figure 3. Scatter plots showing correlation between serum cytokines and bacillary index.

Serum cytokine in leprosy 377

600(A)

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Figure 4. (A) Scatter plots showing correlation between serum cytokines. (B) Scatter plots showing correlationbetween serum cytokines.

N. Kawatra Madan et al.378

destruction of intracellular pathogens, leading to increased bacillary load.19 Raised levels of

IL-1b in LL patients can be explained in view of enhanced B-cell responses and antibodyproduction in LL patients.20

The results of present study were similar to those shown by Moubasher et al.12

Belgaumkar et al.21 showed increased levels of IFN-g in tuberculoid leprosy, while Sarnoet al.22 demonstrated increased levels of TNF-a. On the contrary, Parida et al.23 showed thatlepromatous leprosy is associated with a rise in TNF-a and IL-1b whereas tuberculoidleprosy is associated with low serum TNF-a and IL-1b. Similarly, an increase in TNF-a wasseen in lepromatous leprosy by Pisa et al.24Watson et al.25 and Jayapal et al.26 observed that

the quantity of IL-1b produced by LL/BL patients before therapy was significantly less whencompared to healthy controls.

Leprosy reactions are major causes of hospitalisation and disability of patients with

leprosy. Type 1 reaction or reversal reaction which occurs in patients with borderline forms

of disease represents delayed type hypersensitivity reaction with increased cell-mediated

immune reactivity to antigens of M. leprae that can rapidly produce nerve damage. It is

associated with infiltration of cytokine secreting CD4 lymphocytes in skin lesions and nerves

resulting in edema and painful inflammation. High levels of cytokines indicate that this

reaction represents a state of immune hypersensitivity and exaggerated CMI response that can

lead to clearing of bacilli and concomitant tissue damage.27

120Before MDT

After MDT

100

80

60

40

20

0MeanTNF-

MeanIFN-

MeanIL-1

Cytokines

Levelsinpg/ml

MeanIL-10

Figure 5. Cytokine levels before and after mdt in paucibacillary cases.

Serum cytokine in leprosy 379

Type 2 reaction is a systemic inflammatory response to the deposition of immune

complexes that are responsible for vasculitis, arthritis, panniculitis and nerve injury.

Transient IFN-g production by Th2 cells is operative in ENL as opposed to a more

established production of IFN-g by Th1 clones in Reversal Reaction.28Moreover, TNF-a and

IL-1b are proinflammatory cytokines that play a role in chronic inflammatory

pathologies.2931 These two cytokines are responsible for the vasculitic lesions characteristic

of leprosy reactions.

In our study we found that all the studied cytokines were raised in reactional cases as

compared to non-reactional leprosy as was also shown by Moubasher et al.12 Rise in the

levels of IFN-g and TNF-a has been demonstrated by Iyer et al.,32 Bhattacharya et al.,33

Parida et al.,23 and Esquenazi et al.34 in reactional leprosy. In the present study, on comparing

T2R and T1R, TNF-a was found to be higher in T1R whereas higher levels of IFN-g, IL-1b

and IL-10 were seen in T2R cases and rise in IL-10 was statistically significant. This contrasts

with the findings of Sarno et al.22 and Barnes et al.35 who reported higher TNF values in T2R

cases as compared to T1R cases. During our study period, the two patients who went into T2R

were showing initial TNF-a, IL-1b and IL-10 levels . 100 pg/ml.Raised TNF-a in T1R indicates a state of high immunity and plays an important role in

granulomas becoming more epithelioid and activated, with concomitant tissue damage. IL-10

plays a role in the pathogenesis of T2R. Its high levels enhance B-cell responses and augment

antibody formation potentiating the formation of immune complexes. The results of the

present study suggest that a significant rise in TNF-a and IL-10 may predict the occurrence of

T1R and T2R respectively, whereas rise in IL-1b and IFN-g may predict the occurrence of

both T1R and T2R. Thus serial estimation of cytokine expression may allow early indication

of occurrence and evolution of reactional inflammation in leprosy. Further studies are

however required to establish this.

The effect of multidrug therapy of leprosy on the serum cytokine levels has not been

widely reported in the past. Most of the researchers have observed the effect of steroid

treatment in reactional leprosy only.3638 In this study, we found that serum levels of all the

studied cytokines in paucibacillary cases significantly decreased after 6 months of MDT and

came down to the level of control subjects. In the multibacillary cases, mean serum levels

of all the cytokines were found to decrease after 1 year of MDT except IFN-g. Two

multibacillary cases showed increase in IFN-g levels after treatment suggesting conversion to

a higher immunity state. In the study of Moubasher et al.39 the serum levels of the studied

cytokines were significantly reduced after 1 year of treatment in paucibacillary leprosy

patients. After 1 year of MDT (but not 6 months) paucibacillary patients showed a significant

reduction in all the studied serum cytokines to levels comparable with those of healthy

controls. Multibacillary patients also showed a significant reduction in all studied serum

cytokines, but the levels were higher than those of healthy controls after one year of therapy.

On the other hand, Jayapal et al26 found no change in the quantity of IL-1b produced by

LL/BL patients after 6 months of MDT.

Cytokines are produced in response to antigenic stimulation of the immune system.

If these antigens are removed, there will be a decrease in the levels of cytokines.40Multidrug

therapy decreases the bacterial load in leprosy patients and the decrease in antigenic

stimulation of immune system possibly causes reduction in serum cytokine levels. Moreover

dapsone and clofazimine have anti-inflammatory effects which can explain the reduction of

cytokine levels particularly the proinflammatory cytokines like TNF-a and IL-1b.

N. Kawatra Madan et al.380

Conclusion

This study concluded that serum cytokine estimation may have a significant role in

classifying various forms of leprosy. It can help in predicting the course of the disease and

monitoring therapy. Furthermore, the increased cytokines suggest a potential target for

therapeutic modulation. Additional studies may determine the sensitivity and specificity of

these cytokines as leprosy disease markers.

Estimation of cytokines like TNF-a, IFN-g, IL-1b and IL-10 can help predict thedevelopment of reactional states in leprosy. More research is needed in this regard as

revelation of the cytokine patterns during reactions, might lead to development of specific

targeted immunotherapy which can prevent such episodes, thus saving the patient from

physical and psychological trauma.

Acknowledgements

We are grateful to the patients and staff of Leprosy Clinic, Lady Hardinge Medical College

and Smt. Sucheta Kriplani Hospital, New Delhi, India for their cooperation.

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