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, IFN- INDUCING CYTOKINES, AND IFN- INDUCIBLE CXC CHEMOKINES IN VISCERAL LEISHMANIASIS
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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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plays an important role during immune responses against leishmaniasis. Production of IFN- is regulated by interleukin (IL)-12, IL-18, and IL-15. Interferon--inducible protein (IP)-10 and monokine induced by IFN- (Mig) are CXC chemokines, the production of which, at least in part, is IFN- dependent. A follow-up study of individuals infected with Leishmania donovani was undertaken in an area of Ethiopia endemic for visceral leishmaniasis (VL). Plasma levels of IFN-, IL-12p40, IL-18, IL-15, IP-10, and Mig were markedly elevated in symptomatic VL patients (n = 70) compared with individuals with asymptomatic Leishmania infections (n = 39), malaria patients (n = 13), and healthy controls from the endemic area (n = 12). A significant decrease of IFN- and all mediators was observed after treatment of VL patients (n = 33). These data show that increased plasma levels of IFN-, as well as the mediators involved in the production and the activity of this cytokine, are characteristic of active VL in humans, and may play an important immunopathogenic role. The data also suggest that in patients with VL, the production of type 1 cytokines is not depressed, but there appears to be an unresponsiveness to the stimuli of type 1 cytokines. The underlying causes of immunologic unresponsiveness remain a subject of further investigation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) is an important Th1 cytokine crucial for the control of intracellular infections.1 The production of IFN- is controlled by a number of monocyte/macrophage-derived cytokines, among which, interleukin-12 (IL-12) is a potent inducer of IFN- production when used as single stimulus.2 While IL-18 is known to synergistically enhance the effect of IL-12 on IFN- release,3 IL-15 has also been implicated as an important co-stimulus for IFN- production.4 Interferon--inducible protein-10 (IP-10) and monokine induced by IFN- (Mig) are members of the CXC chemokine superfamily, which induce T cell chemotaxis5 by interacting with CXCR3 preferentially expressed on memory/ activated Th1 cells.6 Many cell types, including neutrophils, when activated by IFN-, produce IP-10 and Mig,7 which in turn increase production of IFN-.
In visceral leishmaniasis (VL), development of disease or control of the infection depend on the effectiveness of IFN--induced innate and adaptive cellular immune responses, which affect intracellular killing by activated macrophages.8 Exposure of humans to Leishmania donovani or L. infantum may lead to either subclinical granulomatous infection or to overt VL that may become life threatening. Subclinical infections are believed to be asymptomatic in nature,9 mostly resulting in naturally acquired immunity.
It is generally accepted that IFN- is needed for control and protection of Leishmania infections. In genetically predisposed murine models of L. major infection, distinct patterns of cytokine production by CD4+ T-cells, i.e., IL-4 /IL-10 and IFN-/IL-2, respectively, were associated with susceptibility and resistance.10,11 The existence of such a distinct immunologic spectrum in human VL remains unclear, partly due to the conflicting reports on plasma cytokine profiles, especially IFN-. Previous investigations have shown that in the plasma of VL patients, IFN-12–14 and other cytokines such as tumor necrosis factor-ß, IL-1, IL-6, and IL-10 are increased.12–16 Conversely, decreased plasma concentrations of IFN- in Indian17 and Brazilian18 VL patients have been reported.
Investigations that examined the plasma concentrations of IFN--inducing cytokines (IL-12, IL-15, IL-18) and IFN--inducible CXC chemokines (IP-10, Mig) in patients with active and asymptomatic VL are lacking. Here we present data on circulating levels of IFN-, IL-12p40, IL12p70, IL-15, IL-18, IP-10, and Mig in VL patients before and after treatment, and also during asymptomatic VL, in comparison with patients with malaria and local healthy controls in an area of Ethiopia endemic for VL.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All individuals were given a physical examination. Fresh and preserved fecal specimens were examined for intestinal parasites, using direct stool examination in the field, and for-molether concentration methods in the laboratory. Blood was examined for hematocrit (packed cell volume [PCV]), white blood cell (WBC) and differential counts, and hemo-parasites. Plasma was stored at –20°C in the field and subsequently at –70°C until tested.
Leishmanin prepared from L. infantum ZLON49, a gift of Dr. Marina Gramiccia (Istituto Superiore di Sanita, Laboratorio di Parassitologia, Rome, Italy) was administered intradermally on the volar surface of the lower arm. Induration size was measured 48–72 hours later, as previously described,19 and an induration 
5.0 mm was considered positive.
Specific antibodies to Leishmania were measured using the direct agglutination test (DAT). The DAT was performed as previously described20 with serum dilutions from 1:100 (numerically scaled at 0) to 1:102,400 (scaled at 10). The cut-off titer was 1:1,600 (scaled at 4), as previously established for the area.21
Plasma levels of cytokines (IFN-, IL-12, IL-15, IL-18) and monokines (IP-10, Mig) were measured using commercially available enzyme-linked immunosorbent assays with the following detection limits: IFN- = 5.0 pg/mL (Diaclone Research, Besancon, France), IL-12p40 = 11.0 pg/mL, IL-12p70 = 3.2 pg/mL, IL-18 = 31.1 pg/mL, IL-15 = 8.2 pg/mL, IP-10 = 20.0 pg/mL (all from R & D Systems, Abingdon, United Kingdom), and Mig = 8.0 pg/mL (Pharmingen, San Diego, CA).
Groupwise comparison of average values of cytokines/ monokines was performed using nonparametric statistics (Mann-Whitney U test or the Wilcoxon two-sample test ([Kruskal-Wallis test for two groups]). The DAT titers were transformed into simple numeric integers ranging from -1 to 11. Epi-Info version 6.03 (Centers for Disease Control and Prevention, Atlanta, GA and World Health Organization, Geneva, Switzerland) and SPSS version 7.5 for Windows (SPSS Inc., Chicago, IL) were used for the analysis of data.
All individuals included in this report provided informed consent to participate in the study. Examination of patients and sample collection was performed as per the national guidelines and procedures of Ethiopia. The study was reviewed and approved by the Institutional Ethical Clearance Committee (Institute of Pathobiology) and the National Ethical Clearance Committee (Ethiopian Science and Technology Commission).
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RESULTS |
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. The difference in age distribution between group 3 and the other groups, except group 5, was statistically significant (
2 > 7.0, P < 0.05). In VL patients (group 1), leukopenia and anemia were characteristic, and the lowest of all groups (2 > 4.0, P < 0.05; Table 1) while PCV in VL and malaria patients was significantly lower than in healthy controls (P < 0.05). However, anemia was common in all study groups, reflecting the widespread malnutrition in the study area.
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. Differences in the rates of parasitic infections between groups were not significant (P > 0.05), with the exception of helminthiases between groups 3 and 4 (2 = 4.18, P = 0.04; Table 2) and groups 3 and 5 (P < 0.05). Most of the parasitic infections were due to Ascaris lumbricoides and Giardia intestinalis (Table 2). Patients with VL characteristically had prolonged fever (91.4%), splenomegaly (97.1%), lymphadenopathy (52.9%), wasting (75.7%), and a negative LST result (100%).
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levels of 69 VL patients was significantly (2 = 32.7, P < 0.01) increased, with a median concentration of 118 pg/mL (interquartile [IQ] range = 75.2-197.0 pg/mL) compared with the level in 30 treated VL patients (mean = 31.0 pg/mL, IQ range = 38.1–50.2 pg/mL). The mean (IQ range) levels in the other groups were 36.0 mg/mL (27.7–56.0 pg/mL) (2 = 45.3, P < 0.01) in 37 asymptomatic cases; 36.0 mg/mL (38.5–79.7 pg/mL) (2 = 9.6, P < 0.01) in 13 malaria patients, and 31.7 mg/mL (39.8–62.5 pg/mL) (2 = 14.0, P < 0.05) in the control group (Figure 1a). These data are consistent with those of previous reports, which found elevated plasma concentrations of IFN- in patients with active VL.12–14 We add to these data that plasma levels of IFN- decrease after treatment, and are undetectable in the vast majority of patients with asymptomatic infection (Figure 1). Moreover, we report for the first time that the plasma concentrations of the IFN- inducing cytokines IL-12, IL-18 and IL-15, and of the IFN- inducible CXC chemokines Mig and IP-10 are increased in VL patients and decrease after therapy for Leishmania.
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), even though the levels were measurable in a few individuals in group 2 and in one individual in group 3. The median and IQ range concentrations of IL-12p70 were below the detection limit in all groups.
The median (IQ range) concentrations of IL-15 in 63 VL patients were 69.0 pg/mL (14.0–209.0 pg/mL). In groups 2, 3, 4, and 5, the median concentrations were below the detection limit, even though a few individuals had detectable levels (Figure 1c).
The median (IQ range) concentrations of IL-18 in 63 untreated VL patients were 1,172.3 pg/mL (304.9–1,822.2 pg/mL), while in malaria patients they were 22.3 pg/mL (below the detection limit to 809.1 pg/mL). The median concentrations in groups 2, 3, and 5 were below detection limit (Figure 1d).
The median (IQ range) plasma concentrations of IP-10 were 857.4 pg/mL (432.0–1,628.2 pg/mL) in VL patients before treatment, 380.0 pg/mL (172.0–744.0 pg/mL) after treatment, from below the detection limit to 308.7 pg/mL in group 3, from below the detection limit to 585.4 pg/mL in group 4, and 103.3 pg/ml (below the detection limit to 329.5 pg/mL) in group 5 (Figure 1e).
The median (IQ range) plasma concentrations of Mig were 11,110.2 pg/mL (9,986.7– 12,022.5 pg/mL) in group 1, 7,673.5 pg/mL (5,670.1–10,163.7 pg/mL) in group 2, 5649.0 pg/mL (4,108.1–8,753.4 pg/mL) in group 3, 5,198.2 pg/mL (838.8–8,879.8 pg/mL) in group 4, and 7,407.5 pg/mL (6,388.6–7,972.7 pg/mL) in group 5 (Figure 1f).
For all cytokines except IL-12p70, the levels in VL patients were significantly higher than in all other groups. It must be emphasized that we present single-point cross-sectional measurements of cytokines, and this has its limitations. These data do not show the dynamics and the variability of cytokine production in an individual patient, which one would expect to take place in vivo. The only repeated measures that we have are in VL patients before and after treatment (group 1 versus group 2), and clearly treatment had a significant effect on levels of cytokines. It is noteworthy that in treated VL patients, even though the plasma levels of IP-10 were significantly lower than before treatment (2 = 9.8, P < 0.01), they were still significantly higher (Figure 1e) than the levels in subjects with asymptomatic infections (2 = 35.4, P < 0.01). This observation was also true for Mig.
We performed a regression analysis between levels of cytokines and clinical parameters such as duration of illness, spleen size, liver size, body mass index, hematocrit levels, and total WBC count. We used regression analysis and refrained from grouping data of active VL patients into further subgroups based on these clinical data. Correlation coefficients (r) within 95% confidence intervals (CIs) were not high enough to draw strong associations; for example, r = 0.29 (IL-18 versus duration of illness, F-statistic = 5.39, 95% CI = –0.17–0.33), r = 0.37 (IL-15 versus duration of illness, F-statistic = 9.3, 95% CI = –0.12–0.38), and r = 0.33 (Mig versus hematocrit levels, F-statistic = 4.78, 95% CI = –0.21–0.41). Thus, our data did not show clear relationships between levels of cytokines and severity or duration of illness, and could not highlight their protective role in any way.
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DISCUSSION |
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and IL-12p40, but not of IL-12p70, have been reported.22,23 These reports concur with our present data in patients with symptomatic VL, where we also found elevated increased concentrations of IFN- and IL-12p40, but not of IL-12p70. Both IFN- and IL-12 are implied to exert a protective function in melioidosis, tuberculosis, and VL. Relative overproduction of IL-12p40 has also been found in other in vivo and in vitro conditions.24 It remains to be established whether bioactive IL-12p70 is produced during active VL, e.g., at the tissue level. In vitro, it has been noted that very low levels of IL-12p70 are sufficient to influence IFN- production.22
Interleukin-18, which shares many biologic activities with IL-12, is not by itself an important stimulus for IFN- production. However, it plays a pivotal synergistic role in optimal IFN- synthesis triggered by IL-12, which at least in part is explained by IL-12-induced up-regulation of the IL-18 receptor.25 Mouse studies on the role of IL-18 in host defense against Leishmania are equivocal. Production of IL-18 by stimulated peripheral blood mononuclear cells of VL patients was lower than its production by similar cells from healthy controls.26 Dogs infected with L. chagasi did not show increased IL-18 mRNA in bone marrow aspirates, although IFN- mRNA was clearly detectable.27 We found increased plasma concentrations of IL-18 in active VL patients, which decreased 3-12 months after therapy. In melioidosis patients, it was found that high levels of IL-18 lasted at least three days after the initiation of antibiotic treatment.22
Infection of monocytes with a number of intracellular pathogens is associated with an up-regulation of IL-15 mRNA.28 Interleukin-15 exerts pro-inflammatory effects on many different cell types, including T cells and natural killer cells; and it is an important co-stimulus for IL-12- and endotoxin-induced IFN- production in vitro.29 Knowledge of IL-15 production during human infection is limited. An increased plasma concentration of IL-15 in melioidosis patients has been reported.22 Our present data in VL patients suggest that the presence of IL-15 in the circulation may be a relatively common phenomenon during systemic intracellular infections. In inflammatory diseases such as rheumatoid arthritis,30 multiple sclerosis,31 and sarcoidosis,32 IL-15 is known to induce expression of CXCR3, the receptor for Mig and IP-10. Conversely, expression of IL-15 in human monocytes may also be up-regulated by IFN-.33 These data show that, together with other cytokines, both IL-15 and IFN- may interplay and amplify inflammatory responses induced by infections.
Monokine induced by IFN- and IP-10 are related chemokines of the CXC chemokine subfamily.34 They are potent chemoattractants for Th1 lymphocytes and natural killer cells, and their common receptor, CXCR3, is preferentially expressed on activated Th1 lymphocytes, which are the predominant sources of IFN-.35 It is currently unknown if IP-10 and Mig play a role in the chemotaxis of cytotoxic T lymphocytes or in macrophage leishmanicidal functions. In BALB/c infections of L. donovani, IP-10 mRNA was expressed at high levels during the period of the infection and involved CD4+ and CD8+ T cells.36 Inducible protein 10 has also been found in delayed-type hypersensitivity reaction.37
The high leishmanial antibody titers and the negative LST results in VL patients suggest that type 1 immune responses are suppressed in these patients, despite the increased plasma concentrations of IFN-, IFN- inducing cytokines (IL-12, IL-18, IL-15), and IFN- inducible CXC chemokines (Mig and IP-10).
Based on this observation, one can speculate that in VL, type 1 cytokine responses are not depressed as commonly believed; instead, unresponsiveness to type 1 cytokine stimuli prevails. This may point to the possibility that a parallel increase of cytokines antagonizing type 1 responses or alternatively an extensive blockade of Th1 cytokine receptors and/or CXCR3 are critical immune consequences of Leishmania infections. Conversely, immune processes leading to macrophage deactivation can be implied. In Mycobacterium avium infection of mouse macrophages in vitro, down-regulation of IFN- receptor, which resulted in reduced phosphorylation of IFN- R 
, JAK1, JAK2, and STAT1, has been discussed as a possible cause of macrophage deactivation.38 In fact, based on our observation that plasma levels of type 1 cytokines are elevated during the active stage of VL, one may hypothesize that Th cytokine profiles are not critical. Instead, other factors might better explain the observed immunologic unresponsiveness that prevails amid the increased plasma concentrations of type 1 cytokines. A number of experimental data highlight the importance of events interfering with cell signaling in macrophages affecting expression of inducible nitric oxide synthase and c-FOS.39 Other experimental evidence has also implicated phosphatases,39,40 endogenous IL-10,41 and IFN--induced adenosine receptors42 as immunomodulatory agents leading to macrophage deactivation. It can be implied that during active human VL, the failure of macrophages to respond to prevailing type 1 cytokines may underlie the immunopathologic processes. Thus, novel approaches to develop immunotherapeutic/prophylactic vaccines are needed, and as such a further understanding of the biochemical events of macrophage activation is necessary.
We have recently shown that antibody isotype profiles and immune responses of drug-cured VL patients resemble that of asymptomatic infections of VL.43 Our present report also shows that the cytokine and monokine profiles of asymptomatic infections were indistinguishable from those of healthy subjects. Conversely, increased plasma concentrations of IFN-, IFN--inducing cytokines and IFN--inducible monokines in patients with acute VL are characteristic and quite distinct from that of asymptomatic infections or drug-cured patients, implying that unresponsiveness to, rather than failure to produce, Th1 cytokines characterizes active VL. These data suggest that in human VL, type 1 cytokine responses are intact, and also raise a question about the role of monokines and proinflammatory cytokines in pathogenesis and protection, and in the cross-regulation of immune responses during active human VL. The immunopathogenic mechanisms leading to macrophage deactivation and immunologic unresponsiveness in VL remain to be elucidated.
Received January 22, 2004. Accepted for publication June 9, 2004.
Acknowledgments: We thank the staff of the Karat Health Centre in Konso for the carefully and diligently rendered patient care. The field study was undertaken in the Konso District in southwestern Ethiopia. Pentostam (sodium antimony gluconate; The Wellcome Foundation Ltd., London, United Kingdom) was provided by Médecins sans Frontières of The Netherlands. We also thank the technical staff of the Institute of Pathobiology of Addis Ababa University for clinical laboratory work and collecting blood samples.
Financial support: Asrat Hailu was supported by the Netherlands Foundation for the Advancement of Tropical Research (WOTRO). This study was supported by WOTRO, the Research and Publications Office, Addis Ababa University; and L’Agence pour l’Investis-sement dans la Recherche a l’Etranger Développement of the French Government.
Authors’ addresses: Asrat Hailu, Faculty of Medicine, Department of Microbiology, Immunology and Parasitology, and Institute of Pathobiology, Addis Ababa University, PO Box 9086, Addis Ababa, Ethiopia and Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center; University of Amsterdam, Amsterdam, The Netherlands, Telephone: 251-1-533-197, Fax: 251-1-513-099, E-mails: a_hailu{at}hotmail.com and hailu_a2004{at}yahoo.com. Tom van der Poll, Department of Internal Medicine, Laboratory of Experimental Internal Medicine, and Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS; Academic Medical Center; University of Amsterdam; Amsterdam, the Netherlands. Nega Berhe, Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia. Piet A. Kager, Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center; University of Amsterdam, Amsterdam, The Netherlands.
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REFERENCES |
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are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1-, or CCR2-deficient mice. J Immunol 163: 5519–5525.-inducible protein-10 and monokine induced by IFN-. J Immunol 165: 1548–1556. (Mig), IFN--inducible T cell chemoattractant (I-TAC), and IFN--inducible protein-10 (IP-10) chemokines by human neutrophils. J Immunol 162: 4928–4937. or interleukin-4 during resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets. J Exp Med 169: 59–72.-independent mechanism. J Exp Med 171: 115–127., interleukin-10 and tumor necrosis factor- in patients with visceral leishmaniasis. Rev Inst Med Trop Sao Paulo 40: 31–34.[Medline]
production. Clin Immunol Immunopathol 57: 242–249.[Web of Science][Medline]
production. J Immunol 161: 3400–3407. and IP-10 are expressed in de-myelinating brain lesions. Proc Natl Acad Sci USA 96: 6873–6878.This article has been cited by other articles:
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  A. Banerjee, M. De, and N. Ali Complete Cure of Experimental Visceral Leishmaniasis with Amphotericin B in Stearylamine-Bearing Cationic Liposomes Involves Down-Regulation of IL-10 and Favorable T Cell Responses J. Immunol., July 15, 2008; 181(2): 1386 - 1398. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and whiskers (lines extending from the box to the highest and lowest values excluding the outliers and extremes). Extremes are cases with more than 3.0 box lengths from the upper or lower edge of the box. Outliers are cases with 1.5-3.0 box lengths from the edges of the box. Based on the Mann-Whitney test or the Wilcoxon two-sample test (Kruskal-Wallis test for two groups), statistically significant differences (P < 0.05) were observed in the following pairs of groups: For IFN-gamma, group 1 versus groups 2, 3, 4, and 5; for IL-12p40, group 1 versus groups 2, 3, 4, and 5 and group 2 versus groups 3, 4, and 5; for IL-15, group 1 versus groups 2, 3, 4, and 5; for IL-18, group 1 versus groups 2, 3, 4, and 5; for IP-10, group 1 versus groups 2, 3, 4, and 5 and group 2 versus group 3; for Mig, group 1 versus groups 2, 3, 4, and 5 and group 2 versus group 3.