Interleukin-13 in Iranian patients with visceral leishmaniasis: relationship to other Th2 and Th1 cytokines

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Interleukin-13 in Iranian patients Th2 and Th, cytokines

(2001)95,85-88

with visceral leishmaniasis:

relationship

to other

’ Tab& University of Medical Sciences, Departmenr Zohreh Babaloo’, Paul M. Kaye2 and Mohammad B. Eelami3 of Immunology, Tab&, Iran; ‘Depament of Infectious and Tropical Diseases, ton&n School of Hygiene and Tropical Medicine, Keppel Street, London, UK; 3Tehran University of Medical Sciences, Departnaent of Pathobiologv, Tehran, Iran Abstract The role ofinterleukin (IL)-13, a Th, cytokine sharing many of the features of IL4, has not previously been examined in patienrs with visceral leishmaniasis (VL). We examined sera from Iranian patients with VL caused by Leishmania infanturn. Serum IL-l 3 was detected in 50% (22144) of patients with active primary disease. In comparison, IL-10 was detected in 79.5% (35/44), interferon y (IFNy) in 385% (17/44),, and IL4 in only 5% (2144) of these patients. With few exceptions all 3 cytokines were undetectable after chnical recovery following antimony therapy. Five of 7 patients (71%) who failed antimony therapy and had relapsing disease had similar levels of IL-10 to patients with active primary disease. However, with only 1 exception, IL-13, IFNy and IL-4 were not detected in such patients. These data suggest that relapsing disease may result from defective cellular immunity, unrelated to immunosuppression mediated by ILlO. Kevwords: visceral leishmaniasis, L&Amandu irrfanncm, cytokines, interleukin-4, inrerleukin-10, intedeukin-13, in&feron y, Iran Introduction Infection with Leishmania &novani and L. infanturn (= L. chagasz) leads to either subclinical, granulomatous infection or life threatening clinical disease (reviewed by BAKER et al., 1999). The factors which govern the outcome of infection remain to be fully defined, but it is generally accepted that interferon y (IFNy) is required for protection, and that the production and action of IFNy can be inhibited by various Th, cytokines (REED & SCOTT, 1993; MIRALLES etaE., 1994). In humanvisceral leishmaniasis (VL), both Thl and Thz cytokines are detectable in the serum of patients with acute primary disease (CILLARI et al., 1995; SUNDAR et al., 1997; DE MEDEIROS et aE., 1998). Expansion of L. donovani specific T cells, producing IL-4, IL10 and/or IFNy (KEMP et al., 1999) and the accumulation of messenger ribonucleic acid (mRNA) for IL-10 and IFNy (KARP et al., 1993; -NNEY es al., 1998) have also been reDorted. Nevertheless, the role of Th, cvtokines during *remains uncertain. some studies h&
This research was approved by the ethical committees of the Iranian Ministry of Health and Medical Education and the London School of Hygiene and Tropical Medicine. Informed consent was obtained from patients and controls and/or their parents or guardians. Address for correspondence: Dr Zohreh Babaloo, No. 3, Golshahr, Foroughy, Baliasr 5 1576, Tab&, Iran; e-mail [email protected]

We have evaluated serum IL-1 3, along with other Thz and Th, cvtokines. in different stages of VL caused bv L. infant&. ?his is, tb our knowledge, the first report oithe presence of IL-13 in the serum of patients with VL. Our data indicate that IL1 3, but not IL4, is readily detectable in active primary cases and that, as for IL10 and IFNy, serum IL-13 returns to control levels after successful treatment. Importantly, although IL-13 and IFNy frequently coexist with IL-10 in individuals with primary active disease, IL- 10, in the absence of IL-l 3 and IFNy, characterizes patients who fail to respond to therapy and have relapsing disease. Materials and Methods This study was conducted in Meshkin-shahr, Iran. Five mL of venous blood were obtained from 85 individuals, as follows. Group A, 44 patients (29 males, 15 females; age 1- 11 years, mean 2.25 * O-3 years) with active primary VL and before the initiation of therapy. Group B, 20 patients (9 males, 11 females; age l-l 1 years, mean 3.9 f 0.5 years) who had recovered after completing a successful course of pentavalent antimony therapy (20 mg Sbikg body weighti24 h, intramuscularly for 21 d). Group C, 7 patients (all male; age 2-5 years, mean 4.14 f O-45 years) whose disease was resistant to routine antimonial therapy and who had had 2-7 relapses. Group D, 14 healthy controls (9 males, 5 females; age l-l 1 years, mean 4.4 i 0.87 years) from the same endemic area who were seronegative for antileishmanial antibodies and had no history of L&hmania infection. Six patients were included in both groups A and B (i.e., pre and post chemotherapy samples were obtained), whereas 1 patient, who was apparently cured but relapsed 3.5 months later, becoming refractory to further treatment, was included in groups A, B, and C. Serum from 3 mL ofblood was stored frozen at -20°C immediately after collection and subsequently divided into aliquots and scored at -80°C. The remaining 2 mL of blood were used for flow cytometric analysis and routine haematology (Table). Diagnosis of visceral leishmaniasis was based on the characteristic symptoms of active VL, including hepatosplenomegaly, irregular fever, anaemia, leucopenia, thrombocytopenia and high &es (> 113200) of antiteishmanial antibodies. Detection of amastigotes in bone marrow aspirates confirmed the diagnosis of VL in patients with relapsing disease. Cytokine assays IL-13, IL-lo, IL-4 and IFNy levels were determined by a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) using commercial kits (Cyto-

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ZOHREH

Table.

Haematological

data of the visceral

White blood cells (log/L) Neutrophils

(%)

Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%)

Red blood cells ( 10la/L) Haemoglobin (g/dL) Haematocrit (%) Platelets (1 Og/L) Direct agglutination test’

lefshmaniasis

patients

ET/IL.

studied”

Clinical groupsb

A (n = 44)

B (ta = 20)

5.98 f 2.65 21.27 f 11.99 66.95 f 16.38 3.94 f 2.31 o-99 + l-02 0.84 !c O-40 8-49 ic l-48 26.57 f 4-14

1O-01 f 2.78 44.65 f 9.73 40.18 f 8.58 4.06 i 1.27 4-79 f 2.88 0.98 f O-29 4.88 f 0.34 11.91 ~5l-25 36.36 i 3.48

179.62 zt 84.57

290.10 f 92.12

3.76 i O-78

48990 f 42490

BABALOO

35620 f 41439

c (n = 7)

5.15 f 25.02 f 56.35 f 5.37 f O-52 f 0.72 f 3.59 f 8.58 f 26.80 f 207.33 f 80914 ic

2.66 17.52 17.82 2.33 0.46 0.29 065 2.20 5.54 81.96 39223

D (n = 14)

9.17 f 2.75 41.59 f 10.03 40.78 f 10.21 4.83 * 1.98 5.25 + 3.10 1.35 * 0.49 4.71 * o-20 12.00 f 1.02 35.95 ic 2.92 291.27 zk 52.39 -

‘Values were obtained using aTecbnicon Hl automatic counter; differential cell counts were verified by microscopy. Data are expressed as mean f SD. bGroupA: acute primary diseasebefore chemotherapy; group B: after completion of successfulantimony therapy; group C: relapsing disease;group D, endemic controls. ‘Reciprocal titre.

screer?, Biosource, Watford, UK) and following the manufacturer’s protocol. 100 pL volumes of serum were analysed and the assaywas calibrated against the recombinant standards provided. Lower limits of detection in these assays were: IL-l 3, l-2 pg/mL; L-10, 1 pg/mL; IG4, 0.2 pg/mL; IFWy, 1 pg/mL. Values below these limits were assigned as zero for the purpose of data calculation. Andeishmanial

antiBody &es

Antibody responses were evaluated by the direct agglutination test (DAT). Briefly, blood samples were spotted on Whatman no.4 filter paper and dried; 6 mm diameter circles (equivalent to 10 pL of blood) were punched out and left overnight in 0.5 mL of citrate saline containing 1% fetal calf sertmr and O-1M p-2 mercaptoethanol. The DAT antigen was prepared from logphase promastigotes of an Iranian strain of L. infanturn (L. infanturn LON 49) at Tehtan University of Medical Sciences by World Health Organization standard methods (WHO, 1996). DATs were performed with sequential dilutions of serum in a solution containing 0.9% NaCl, 0.78% 0-2 mercaptoethanol and 0.1% fetal calf serum. Data analysis

Statistical significance between cytokine levels was assessed by Student’s t test for unpaired ot paired samples, as appropriate. Fisher’s exact test or x2 was used to compare rates of detection (defined aspercentage detected). Values of P < 0.05 were considered to be significant. Results Serum cytokines in Iranian patients before and a&r antimonial therapy

We first analysed the Thi and Tha type cytokines IFNy, IL4 and IL1 0, to determine how these patients compared to others infected with L. infanturn and studied elsewhere (CILLARI et al., 1995; DE MEDEIROS et al., 1998) (Figure). None of the 14 control individuals (group D) had detectable serum IL-10 or IL4, whereas 3 (21%) had low levels of IFNy. Among the 44 patients with active primary disease (group A), IL-10 was detectedin35 (79.5%) andIFNyin 17 (38.6%);43% ofthe patients with detectable IL10 also had detectable IFNy (33% of the 12 males over 2 years old). Conversely, 88% of patients with IFNy also had detectable IL- 10 in their serum. In contrast, IL-4 was detected in only 2 of the 44 patients and then at the borderline of sensitivity of the assay. In group B, 20 patients whose serum cytokine levels were studied after clinical recovery, K-10, IFNy

and IL4 were all below the level of detection. Hence, this patient group displayed a similar cytokine profile to that reported for L. infanturn in other geographical areas. IL-1 3 was detected at very low levels in only 2 of the 14 endemic controls (group D) (1.29 f 1.62 ng/mW. However, in marked c&trait to &r inability todetect IL4, IL13 was readilv detected in 50% of the acute VL patients (group A) (20.1 4 18.7 pg/mL; P < 0.02 vs. controls). 82% ofpatients with Ur13 also haddetectable ILlOand51%ofthosewithILlOalsohadIL13(42% of the 12 males over 2 years old). Furthermore, IL-13 and IFNy co-existed in 40% of the patients in whom we detected at least 1 of these cytokines. The presence or absenceof IL-l 3, and the absolute concentration of this cytokine, were not correlated with either ageor sexwithin this patient group (data not shown). After chemotherapy andclinicalrecovery, the level of serum IL13 was below detectable levels in all but 3 cases.These individuals had very high serum concentrations of IL13 (45.57, 143.8 and 127.70 pg/mL, after 8*5,15 and 3 months of clinical recovery). DAT titres were also still high (> l/25600), but no other serum cytokine was detected (Figure). Of the serum samplesobtained f?om groups A and B, 6 were obtained from the same patients both before, and immediately after, their course of antimony. All 6 had detectable IL10 levels before treatment. which were reduced by between 52% and 98% after treatment (P < 0.05). Only 3 of the 6 had detectable IFNy and K-13, and in each case this was reduced to below the assaydetection limit.

Cytokine profires in patients with relapsing VL

Although many patients responded well to antimony treatment, as in other geographical areas, treatment failures did occur. Seven patients (all male, over 2 years old) who were resistant to antimony therapy and had relapsing diseasewere studied. IL-1 0 was detectable in 5 (71*4%), at levels almost identical to those seen in patients with active primary disease (54-l f 43 vs. 54.5 f 42 PrrimL). In contrast to IL-lo, neither IL-4 nor IFNy was detected in these patients and only 1 had detectable IL-13 112.3 &mL: P= 0.085 vs. nrimarv cases),Of the patients wixIL- lb, none had IFN{ in the& serum and only 1 (20%) had ILi3. This 1 case with serum IL13 had previously been seen during acute primary disease, at which time we detected ILlO, IL 13 and IFNy. Nevertheless, in this group as a whole, there appeared to be a disproportionally high number of patients with serum IL.-lo, in the absence of other detectable Th, and Th, cytokines.

IN VISCERAL

INTERLFUKIN-13

87

LEISHMANIASIS

250

-__-

---

. _~~~.-

I--

_.__-_

50 r----.

.

40 3 $

30

f

20

.

. .

l 10

7

.

l

l

*

. 2 ! -

l l

l

.

I

1 0

4

0C

A

B

C

A

B

C

D

Figure. Cycokine responsesin patients with visceral leishmaniasis. Serum levels of interleans [IL) 4,13 and 10 and interferon y (IFNy) in patients with acute primary disease and before chemotherapy (A, n = 44), after completion of successful antimony therapy (B; n 5 ZO),and with relapsing disease(C; n = 7), and in endemic controls (D; n = 14). Cyrokine levels were determined by enzyme-linked immunosorhent assay;each point represents one individual. Median IL 10 levels were 4 1 pgknL in group A and 24 pgimL in group C. Median IL-13 level in group A was 0.37 pg/mL. Medisnvalues for all other cytokines in each group were zero.

Discussion This study demonstrated the presence of serum IL-1 3, IL IO and IFNy during the acute phase of primary VL in Iranian patients with L. infanturn. In contrast, IL-4 was undetectable in these patients. Absence of serum IL-4 was also noted by CILLAM et al. (1995), whereas this cytokine was detected in 84% ofIndian VL cases infected with L. &~ozM.% (see SUNDAR et al., 1997). Discrepancies in the rate of detection of IL4 have been noted before (see discussion by SLJNDAR et al., 1997) and may reflect (i) differences in host response to these visceralizing species, (ii) an underlying immunological difference between the patient groups examined, e.g. in their genetic predisposition towards IL-4 production or in their underlying levels of pathogen exposure, or (iii) differential production of soluble IL-4R during L. donovani and L. infanturn infection (SANG et al., 1999). Nevertheless, the high detection rate for IL-13, in the absence of detectable IL-4, suggested that IL-1 3 may be a good surrogate marker of ThZ celt activity in viva in VL. Although It13 shares many activities with IL-4, distinct functions for the 2 cytokines have been proposed. Notably, ILrI3 is the major Thz-type cytokine driving type I and type III collagen mRNA production and hepatic fibrosis in mice with schistosomiasis (CBIARAMONTFZ et d., 1999). This observation has led to the suggestion that IL-13 inhibitors may have therapeutic potential for preventing fibrosis in chronic infectious diseases. Collagen deposition has been described in both murine and human VL (GUTIERREZ et al., 1984; BRYCESON, 1996), but the value of clinical intervention in this process is uncertain. In our study, 3 of 20 recovered patients had serum IL-13, the level of which in 2 cases

greatly exceeded that seen in active primary disease. In contrast, they had no detectable IL-10 or IFNy. We believe that the most likely explanation for these results was the presence of a concurrent asymptomatic helminthic infection in the individuals. Supporting this possibility, the patient with the highest IL-13 level (143.8 pg/mL) also had a more than 4-fold elevation of his eosinophil count (data not shown). In Indian VL, treatment failure is associated with the maintenance of serum IL-10 and an increased IL4:IFNy ratio, compared to that seen in primary cases (SUNDAR et al., 1997). Although we confirmed the maintenance of IL-10 levels in primary vs. relapsing cases, with only 1 exception relapsing patients in our study had no detectable serum IFNy, IL4 or IL-13. Furthermore, even among active cases, a subset of patients had detectable IL10 but no IL-13 or IFNy. Thus, IL-10 appeared to be independently regulated compared to other Th, (IFNy) and Th, (Z-13, IL-4) cytokines in these patients. We are considering 2 possible mechanisms to explain these data. First, regulatory T cells selectively producing IL-10 may predominate in patients with relapsing disease (&kSEhUN et al., 1999). Second, cytokine production by T cells (Thl, Thz, Trcg) may be globally impaired, perhaps due to underlying defects in antigen presentation (MURPHY et al., 1998). The lamer implies that T cells are not a major contributor to serum IL-10 levels, although it does not preclude IL 10 production by T cells during restimulation in vitro (KEMP et al., 1999). It is noteworthy that the cellular source of IL10 remains to be established even in acute primary VL. Studies are now in progress to distinguish between these 2 mechanisms.

88

ZOHREH

In summary, we have shown for the first time that IL13 is present in serum from patients with active primary VL and that, like IL- 10 and IFNy, senun levels of IL 13 return to control levels after successful treatment. Furthermore, in patients resistant to treatment, elevated IL13 does not appear to be associated with treatment failure. Rather, the discordant expression of IL1 0 in the absence of other Th, and Th, cytokines characterizes both a subset of active casesand a majority of relapsing patients. Further prospective studies will be required to define any further relationships between these 2 groups. Acknowledgements This work was supported by grants from the Iranian Ministry of Health and a World Health Organization TDR Training Fellowship to Zohreh Babatoo. Paul Kaye is supported by grants from the WeIlcome Trust and the Medical Research Council of Great Britain. References Asseman, C., Mauze, S., Leach, M. W., Coffman, R. L. & Powrie, F. (1999). An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. Journal of Experimental Medicine, 190,995- 1004. Baker, R., Chiodini, P. & Kaye, P. M. (1999). Leishmaniasis. In: T%eGranulomatousD&&er~, Geraint James,D. & Zumla, A2(ee;y) Cambridge: Cambridge University Press, pp. Bryceson, A. D. M. (1996). Leishmaniasis. In: Manson’s TropicalDtieuses,Cook, G. C. (editor), 20thedition, London: W. B. Saunders, pp. 1213-1245. Chiaramonte, M. G., Donaldson, D. D., Cheever, A. W. & Wynn, T. A. (1999). An IL1 3 inhibitor blocks the development of hepatic fibrosis during a T-helper type P-dominated inflammatory response. Journal of Clinical Investigatin, 104, 777-785. Cillari, E., Vitale, G., Arcoleo, F., D’Agostino, P., Mocciaro, C., Gambino, G., Malta, R., Stassi, G., Giordano, C., Milano, S. & Mansueto, S. (1995). In viva and in vim cytokine profiles and mononuclear cell subsets in Sicilian patients with active visceral leishmaniasis. Cytokine, 7, 740-745. De Medeiros, I. M., Castelo, A. & Salomao, R. (1998). Presence of circulating levels of interferon-gamma, interleukin-10 and tumor necrosis factor-alpha in patients with visceral leishmaniasis. Revisra do Institute de Medicina Tropical de SGo Paula, 40,31-34. Finkefman, F. D., Wynn, T. A., Donaldson, D. D. & Urban, J. F. (1999). The role of IL 13 in hehninth-induced inflammation and protective immunity against nematode infections. Current Opinionin Immunology, 11,420-426. Gutierrez, Y., Maksem, J. A. & Reiner, N. E. (1984). Pathologic changes in murine leishmaniasis (Letihnaandadolaovandwith special reference to the dynamics of granuloma formation in the liver. AmericanJournralofParhulogy, 114,222-230.

BABALOO

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Karp, C. L., el-Safi, S. H., Wynn, T. A., Satti, M. M., Kordofani, A. M., Hashim, F. A., Hag-Ali, M., Neva, F. A., Nutman, T. B. & Sacks,D. L. (1993).Inviwo cytokineprofiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma [see comments]. Journnl of Clinical Investigarion, 91, 1644- 1648. Kemp? K., Kemp, M., Kharazmi, A., Ismail, A., Kurtzbals, J. A., Hvild, L. & Theander, T.G. (1999). Leishmania-specific T cells expressing interferon-gamma (IFN-gamma) and IL-10 upon activation are expanded in individuals cured of visceral leishmaniasis. Clinical and Experimental Immunology, 116, 500-504. Kenney, R. T., Sacks, D. L., Gam, A. A., Murray, H. W. & Sundar, S. (1998). Splenic cytokine responsesin Indian kalaazar before and after treatment. Journal of Infectious Diseases, 177,815-818. Kropf, P., Schopf, L. R., Chung, C. L., Xu, D., Liew, F. Y., Sypek, J. P. At Mullet-, I. (1999). Expression ofTh2 cytokines and the stable Th2 marker STZLin the absenceofIL4 during Leidzmania major infection. European Journal of Immunology 29.3621-3628. McKenzie, G. J., Emson, C. L., Bell, S. E., Anderson, S., Fallon, I?., Zurawski, G., Murray, R., Grencis, R. & McKenzie, A. N. (1998). Impaired development of Th2 cells in IL 13-deficient mice. Immunity, 9,423-432. Mitalles, G. D., Stoeckle, M. Y., McDermott, D. F., Finkelman, F. D. & Murray, H. W. (1994). T‘hl and Th2 cellassociated cytokines in experimental visceral leishmaniasis. Infection and Immunity, 62, 105S- 1063. Murphy, M. L., Cotterell, S. E., Gorak, I?.M., Engwerda, C. R. & Kaye, P. M. (1998). Blockade of Cm4 enhances host resistance to the intracellular pathogen, Leisshmania donovani. Joumalofimmunolo~, 161,4153-4160. Reed, S. G. & Scott, I?. (1993). T-cell and cytokine responsesin leishmaniasis. Current Opinion in Imnaunobgy, 5, 524-53 1. Sang, D. K., Ouma, J. H., John, C. C., Whalen, C. C., King, C. L., Mahmoud, A. A. & Heinzel, F. P. (1999). Increased levels of soluble interleukin-4 receptor in the sera of patients with visceral leishmaniasis. Journal of Infectious Diseases, 179, 743-746. Satoskar, A., Bluethmann, H. &rAlexander, J. (1995). Disruption ofthe murine interleukin-4 gene inhibits diseaseprogression during I&hmania mexicana infection but does not increase control of Leishmania donovani infection. Infection and Immuniv, 63,4894-4899. Sundar, S., Reed, S. G., Sharma, S., Mehroua, A. &Murray, H. W. (1997). Circulating T helper 1 (Thl) cell- and Th2 cellassociated cytokines in Indian patients with visceral leishmaniasis. American Journal of Tropkd Medicine and Hy&ne, 56, 522-525. WHO (1996). Manual on visceral lkshmaniasis control. Geneva: World Health Organization, mimeographed document WHO/‘LEISH/96.40. Received 2% March publication l%&ly

2000; revised I I July 2000; nccepdfor 2000

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