Antimonial Therapy Induces Circulating Proinflammatory Cytokines in Patients with Cutaneous Leishmaniasis

Abstract

The objective of this study was to evaluate the association between antimonial therapy and circulating levels of proinflammatory cytokines in patients with cutaneous leishmaniasis (CL). Patients were treated with conventional chemotherapy by using pentavalent antimonium salts (Glucantime) for 3 weeks. Circulating plasma levels of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) were determined for CL patients and healthy subjects before and 3 weeks after the treatment was started. Plasma IL-1β, IL-6, IL-8, and TNF-α levels were significantly higher for pretreatment CL patients than for healthy subjects. Proinflammatory cytokines significantly increased after 21 days postinfection compared to levels for the pretreatment patients. These increments were approximately 3-fold for IL-1β and TNF-α levels, 10-fold for IL-6 levels, and 20-fold for IL-8 levels in patients with CL. Taken together these results indicate that circulating proinflammatory cytokine levels were increased in patients with CL as a consequence of host defense strategies, and antimonial therapy may induce these cytokines by affecting the macrophage or other components of the host defense system.

Protozoa from the genus Leishmania cause cutaneous leishmaniasis (CL), which infects macrophages from many mammals, including humans. Depending on the species of the parasite, the disease is characterized by the occurrence of ulcerative lesions in the skin and/or mucosae that result in permanent disfiguration of patients (24). A variety of inflammatory mediators are produced by monocytes/macrophages during the course of this infection.

The principle effector mechanism mediating parasite elimination is the activation of macrophages by proinflammatory cytokines, and production of cytokines that activate macrophages correlates with healing responses (13, 23). The strongest evidence has come from laboratory models of protozoan infections. In malaria, toxoplasmosis, and leishmaniasis, to name just a few, the preferential production of proinflammatory cytokines results in increased synthesis of nitric oxide (NO) and reactive oxygen species, which are involved in host protection processes by direct toxicity on parasites or by inhibiting parasite growth (6). Pentavalent antimonials (Glucantime) remain the drugs of choice in the treatment of all forms of Leishmania infection in spite of their reported toxicity, difficulty in administration, and high cost. However, it is not known whether the antimonial compound acts directly on the parasite or by activating the macrophage or other components of host defense systems which subsequently exert an effect on the Leishmania parasite (14). Several studies have demonstrated that there is an important immunological component in response to antimonial therapy (5, 7). Murray et al. (18) reported that cytokine production is involved in the healing process following antimonial treatment. Recently it has also been demonstrated that antimonium salts potentiate oxidant production in murine visceral leishmaniasis and in human blood (19). However, the association (if any) between proinflammatory cytokines and antimonial therapy in CL patients is not known.

Our main objective in this study was to understand the effects of antimonial compounds on the circulating levels of some proinflammatory cytokines, namely, interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α), before and during therapy for patients with CL.

MATERIALS AND METHODS

The study was conducted at the Harrankapi Leishmania Treatment Centre in Sanliurfa, Turkey. This province is located in the southeastern Anatolia region and is an area where leishmaniasis is hyperendemic. Subjects included 28 patients and 24 healthy individuals aging between 10 to 34 years from the same area. All participants gave informed consent for the present study. Admission criteria for the patient group were no pregnancy and no prior intralesional or systemic antimonial therapy or infection. The criteria did not involve restriction on the basis of age or sex. Patients with lesions of 6 months or greater duration were excluded from the study because of the possibility of spontaneous healing and immunity. The patients who had a single lesion smaller than 2 cm were also excluded from the study.

The clinical diagnosis was confirmed by laboratory demonstration of the parasite in the lesions by direct smears. Lesions were cleaned with ethanol and were punctured at the margins with a sterile lancet. Biopsy material was smeared, dried in air, and fixed by methanol. Parasitologic diagnosis was based on visualization of parasites in Giemsa-stained smears, prepared with material aspirated from borders of skin lesions and from tissue imprints from biopsy. In order to confirm the diagnosis, the materials were also cultured in Novy-MacNeal-Nicolle medium for up to 3 weeks to detect leishmanial promastigotes. The parasites were classified by isoenzyme analyses by using starch gel electrophoresis techniques (9). In all cases the strains corresponded to the Leishmania tropica.

The blood samples were collected after the diagnostic procedure. Prior to blood sample collection all patients and control subjects fasted for 12 h in order to exclude dietary differences.

Heparinized venous blood was drawn from the patients and from the control subjects before and after the antimonial therapy. Plasma was separated and stored at −80°C until assayed.

After the first blood sample collection, the CL patients were cured with Glucantime at daily intramuscular doses of 10 mg/kg for 3 weeks. Glucantime treatment was given only for lesions larger than 2 cm in diameter, for ulcerating lesions, or for multiple lesions. The CL patients were checked monthly for healing or recurrence. Efficacy of antimonial therapy was defined as complete clinical healing of lesion with disappearance of edema, induration, or other signs of inflammation and with a negative culture smear finding. Final evaluation was performed at the end of 3 months after therapy.

Plasma IL-1β, IL-6, IL-8, and TNF-α levels were determined by a chemiluminescence method with a commercial kit (Immulite/DPC) and with an automatic chemiluminassay analyzer (Immulite).

The mean values obtained from the control subjects and the patient group before therapy were compared by Student's t test. For the patient group, circulating cytokine levels from before and during therapy were compared by using a paired t test. All statistical analyses were performed with the program Statistical Package for the Social Sciences for Windows, version 7.5. All results are expressed as means ± standard deviations; statistical significance was defined as P < 0.05.

RESULTS

A total of 52 subjects were enrolled in this study. The CL patients and unmatched controls were in similar ranges in terms of age, height, body weight, and body mass index. All of the patients with CL healed clinically and parasitologically, and no recurrences were observed at the end of the third month after Glucantime treatment.

As seen in the Table 1, pretreatment plasma IL-1β, IL-6, IL-8, and TNF-α levels were significantly higher for the CL group than for the healthy subjects (P < 0.001, P < 0.01, P < 0.001, and P < 0.01, respectively).

TABLE 1.

Proinflammatory cytokine levels for Controls and for patients with CL before and after antimonial therapy

Cytokine	Cytokine levels (pg/ml) fora:
	Control group (n = 24)	Patient group before therapy (n = 28)	Patient group after 3rd week of treatment (n = 28)
IL-1β	2.015 ± 0.89	5.02 ± 2.05***	16.54 ± 9.20***
IL-6	4.78 ± 1.70	6.48 ± 2.85**	66.71 ± 45.91***
IL-8	14.31 ± 13.00	28.45 ± 21.10***	698.05 ± 235.85***
TNF-α	15.77 ± 6.23	21.83 ± 4.92**	77.21 ± 33.40***

^aAll results were expressed as means ± standard deviation. Asterisks at the right of before-therapy and 3rd-week-of-treatment group values describe differences between control and before-therapy groups and before-therapy and 3rd-week-of-treatment groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

The CL patients treated with Glucantime produced significantly higher plasma proinflammatory cytokine levels than they did before treatment. These increments were approximately 3-fold for IL-1β and TNF-α, 10-fold for IL-6, and 20-fold for IL-8 (Table 1 and Fig. 1).

FIG. 1.

Plasma proinflammatory cytokine levels in control and CL patients before (BT∗) and 3 weeks after (AT∗∗) Glucantime therapy.

DISCUSSION

The protozoan parasite Leishmania major infects mononuclear phagocytes, and control of the infection depends on adequate activation of the infected macrophages to kill parasites and inhibit their replication (2). The microbicidal activity of macrophages in an inflammatory milieu has been related to production of a large number of cytokines and intermediary metabolites of oxygen and nitrogen (3, 8, 11). Among them, NO from activated macrophages especially has been demonstrated to be the principle effector molecule mediating intracellular killing of Leishmania parasites (1). Several suggestions have been made about the effects of Glucantime, and the generally accepted argument is that antimonial agents use proinflammatory cytokines to adequately stimulate macrophages (20). We have therefore investigated any possible association between antimonial therapy and proinflammatory cytokines in the present study.

In this study, TNF-α and IL-1β levels were found to be significantly higher for CL patients than for control subjects, and the increments of TNF-α levels were triggered approximately threefold by antimonial therapy. In vitro studies with murine macrophages revealed that soluble factors secreted by activated T cells mediate activation of macrophages to produce NO, resulting in killing or control of L. major parasites (4). TNF-α has been shown to be a major NF-κB-activating signal for macrophage activation (10). Thus, secretion of TNF-α by macrophages is sufficient to mediate production of NO and killing of L. major parasites (2). It has also been demonstrated that TNF-α levels were higher in the sera of pretreatment CL patients (16). However, to date there is no report available on the relationship between antimonial therapy and TNF-α. IL-1 is primarily produced by cells of the mononuclear phagocytic lineage but is also produced by endothelial cells, keratinocytes, synovial cells, astrocytes, osteoblasts, neutrophils, glial cells, and numerous other cells. IL-1 production may be stimulated by a variety of agents, including endotoxins and other cytokines, microorganisms, and antigens. IL-1 is also cytotoxic to cancerous and virus-infected cells. Sodhi et al. (21) demonstrated that IL-1 levels were significantly increased when Leishmania donovani-infected animals were treated with antimonium salts 14 days postinfection. Although they have studied L. donovani-infected animals, their findings appear to support our study. It is thought that TNF-α and IL-1β levels increase as a part of host defense strategies, and induction of the cytokines by antimonial therapy might be dependent on macrophage activation.

Circulating IL-6 levels were significantly elevated in CL patients compared to values seen for the controls. This cytokine was increased about 10-fold during antimonial therapy in the patient group. Mononuclear phagocytic cells are the most important source of IL-6. However, IL-6 is also produced by T and B lymphocytes, fibroblasts, endothelial cells, keratinocytes, hepatocytes, glial cells, and bone marrow stromal cells. Its synthesis is induced by IL-1β and IL-2. Several in vitro studies demonstrated that some herb powders, such as echinacea, activate macrophage to produce TNF-α, IL-1β, and IL-6 as well as oxidative burst and killing of Leishmania parasites (17, 22). It is not inconceivable that Glucantime may act as an echinacea to activate macrophages to produce these cytokines and killing of Leishmania parasites.

In this study, plasma IL-8 levels were significantly higher in CL patients than in the control subjects, and this increment was triggered approximately 20-fold during antimonial therapy. IL-8 is a potent chemotactic cytokine for polymorphonuclear neutrophils, stimulating their chemotaxis and generating reactive oxygen metabolites (26). This chemokine is synthesized by a variety of cells, including monocytes/macrophages, chondrocytes, and fibroblasts (15, 25). TNF-α can also stimulate release of IL-8, which may in turn play an important role in the inflammatory reaction. In synovial culture TNF-α has been shown to stimulate the production of IL-8 in a time- and dose-dependent manner (12). IL-8 can enhance the release of inflammatory cytokines in human mononuclear cells, including that of IL-1β, IL-6, and TNF-α, which may further modulate the inflammatory reaction (12). Although there is no report yet on the relationship between CL and IL-8, we suggest that either antimonium salts or the other proinflammatory cytokines may induce production of IL-8 to generate a reactive oxygen species. These are known to have microbicidal activity and, therefore, may be the direct cause of intracellular killing of Leishmania parasites.

Our results suggest that proinflammatory cytokines may play a crucial role in the resolution of CL infection. Pentavalent antimonial compounds may have immunostimulating effects which may be responsible for its antimicrobial activity. To our knowledge, this is the first report indicating the association between proinflammatory cytokines and antimonial therapy for CL patients. However, the molecular mechanism(s) is yet to be demonstrated. We believe that our findings will help to explore the mechanism of action of pentavalent antimonial compounds.