serum cytokine profile in leprosy and its correlation with clinico ... · serum cytokine profile...
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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 , 0·05). 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 , 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 wasfound 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: þ91-9953236682; þ 91-124-2320532; e-mail: [email protected])
Lepr Rev (2011) 82, 371–382
0305-7518/11/064053+12 $1.00 q Lepra 371
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-Jopling’s 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. Student’s t-test and chi square test were employed for comparison between two
groups. P values # 0·05 were considered statistically significant. The correlation coefficient
(r) was applied between the serum cytokine levels.
The study was approved by the institution’s Ethics Committee and the university’s
Review Board and the funding for cytokine kits was provided by the Department of
Pathology, Lady Hardinge Medical College.
Results
The majority of cases (52·45%) were in the age group of 20–40 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 (42·6%) cases. Nerve involvement in the form of thickened nerves/neuritis was
found in 58 (95·1%) cases at the time of sera collection. Ten patients (14·4%) had abnormal
facies (madarosis, infiltration or lagophthalmos) out of which two patients (both LL) had
leonine facies. Four patients (6·6%) had deformities (claw hand, thenar atrophy or foot drop).
CLINICO-HISTOPATHOLOGICAL CORRELATION
The maximum cases were of Borderline Tuberculoid Leprosy (42·6%). Only two cases out of
61 (i.e. 3·3%) showed discordance in clinical and histopathological findings; 100% of the
paucibacillary patients had a BI between 0 and 2þ and 70% of the multibacillary patients
Table 1. Comparison of cytokine levels of cases with controls
CASES (N ¼ 61) CONTROLS (N ¼ 30)
CYTOKINESMEAN(pg/ml)
STD DEV(pg/ml)
MEAN(pg/ml)
STD DEV(pg/ml)
P value(Student’s t-test)
TNF-a 70·17 50·84 19·46 6·70 0·03IFN-g 100·01 107·45 25·79 11·90 ,0·001IL-1b 93·97 80·98 15·76 6·11 ,0·001IL-10 63·08 59·48 15·90 2·30 0·1
Serum cytokine in leprosy 373
had a BI between 3þ and 6þ . The rest (30%) of the patients had a BI of 0–2 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 , 0·05) (Table 1).
In the patient group (including all subtypes), mean serum levels of TNF-a, IFN-g, IL-1band IL-10 were 70·17 ^ 50·84 pg/ml, 100·01 ^ 107·45 pg/ml, 93·97 ^ 80·98 pg/ml and
63·08 ^ 59·48 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 found
in MB cases (P , 0·05) (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 , 0·001).
IFN-g was raised in T1R cases as compared to PB cases however was not statistically
significant (Table 2).
140Paucibacillary cases
Multibacillary cases
120
100
80
60
40
20
0MeanTNF-α
MeanIFN-γ
MeanIL-1β
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 , 0·05) (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 ¼ 0·05) while
TNF-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 , 0·05) and were comparable to controls (6
months 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 , 0·01).
250
200
150
Levelsinpg/ml
100
Reactional casesNon reactional cases
50
0MeanTNF-α
MeanIFN-γ
MeanIL-1β
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 to
decrease 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 all
leprosy cases, both paucibacillary and multibacillary. TNF-a and IFN-g were raised in
paucibacillary leprosy, whereas all the studied cytokines were raised in multibacillary leprosy
cases with rise in IL-1b and IL-10 being statistically significant (P , 0·05).
Table 2. Cytokine levels in paucibacillary cases compared to cases with type 1 reaction
PB (N ¼ 31) PB with T1R (N ¼ 6)
CYTOKINESMEAN(pg/ml)
STD DEV(pg/ml)
MEAN(pg/ml)
STD DEV(pg/ml)
P value(Student’s t-test)
TNF-a 61·66 28·55 118·23 54·86 0·001IFN-g 77·07 60·90 166·61 137·29 0·1IL-1b 24·71 19·68 78·49 34·02 0·003IL-10 19·09 14·87 21·96 6·89 0·7
Table 3. Cytokine levels in multibacillary cases compared to cases with type 2 reaction
MB (N ¼ 13) MB with T2R (N ¼ 4)
CYTOKINESMEAN(pg/ml)
STD DEV(pg/ml)
MEAN(pg/ml)
STD DEV(pg/ml)
P value(Student’s t-test)
TNF-a 55·32 76·78 105·45 56·78 0·2IFN-g 55·17 62·42 352·31 140·20 0·02IL-1b 131·95 98·73 135·78 63·42 0·9IL-10 60·30 29·96 157·26 88·06 0·1
N. Kawatra Madan et al.376
It has been hypothesised that IFN-g and TNF-a play a role in immunoprotection and
immunopathology, whereas IL-10 and IL-1b are immunosuppressive.10–12 Our results are
consistent 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 containing
bactericidal macrophages that play an essential role in preventing the extension and
dissemination of mycobacterial infection.6,14–16 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 ¼ 6) T2R (N ¼ 6)
CYTOKINESMEAN(pg/ml)
STD DEV(pg/ml)
MEAN(pg/ml)
STD DEV(pg/ml)
P value(Student’s t-test)
TNF-a 118·23 54·86 105·45 56·78 0·7IFN-g 166·61 137·29 352·31 140·20 0·07IL-1b 78·49 34·02 135·78 63·42 0·09IL-10 21·96 6·89 157·26 88·06 0·05
400
350
300
250
IL1
TNF-α
IL10
IFN-γ
200
150
100
50
0
300
250
200
150
100
50
00
100
200
300
400
500
0 2
Bacillary index
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)
(B)
500
400
300
200
100
0
600
500
400
300
200
100
0
00 50 100 150
TNF-α
200 250 300
50
100
150
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250
300
00 50 100 150
IL1
IL10
200 250 300 350 400
50
100
150
200
250
300
350
400
0 100 200 300
TNF α
IFN-γ
TNF-α
TNF-γ
00
50
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100 200 300
IL10
0 1000 200 400
IFN-γ600 200 300
IL10
IL1
0
50
100
150
200
250
300
350
400
IL1
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 antibody
production 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 Sarno
et 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 tuberculoid
leprosy 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-α
MeanIFN-γ
MeanIL-1β
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.29–31 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.36–38 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 the
development 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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