Cytokine profile and pathology in human leishmaniasis

The clinical spectrum of leishmaniasis and control of the infection are influenced by the parasite-host relationship. The role of cellular immune responses of the Th1 type in the protection against disease in experimental and human leishmaniasis is well established. In humans, production of IFN-γ is associated with the control of infection in children infected by Leishmania chagasi. In visceral leishmaniasis, an impairment in IFN-γ production and high IL-4 and IL-10 levels (Th2 cytokines) are observed in antigen-stimulated peripheral blood mononuclear cells (PBMC). Moreover, IL-12 restores IFN-γ production and enhances the cytotoxic response. IL-10 is the cytokine involved in down-regulation of IFN-γ production, since anti-IL-10 monoclonal antibody (mAb) restores in vitro IFN-γ production and lymphoproliferative responses, and IL-10 abrogates the effect of IL-12. In cutaneous and mucosal leishmaniasis, high levels of IFN-γ are found in L. amazonensis-stimulated PBMC. However, low or absent IFN-γ levels were observed in antigen-stimulated PBMC from 50% of subjects with less than 60 days of disease (24 ± 26 pg/ml). This response was restored by IL-12 (308 ± 342 pg/ml) and anti-IL-10 mAb (380 ± 245 pg/ml) (P<0.05). Later during the disease, high levels of IFN-γ and TNF-α are produced both in cutaneous and mucosal leishmaniasis. After treatment there is a decrease in TNF-α levels (366 ± 224 pg/ml before treatment vs 142 ± 107 pg/ml after treatment, P = 0.02). Although production of IFN-γ and TNF-α might be involved in the control of parasite multiplication in the early phases of Leishmania infection, these cytokines might also be involved in the tissue damage seen in tegumentary leishmaniasis. Correspondence


• Leishmaniasis
• Human leishmaniasis Leishmaniasis is a parasitic infection caused by a protozoan from the Trypanosomatidae family, genus Leishmania.Four clinical forms of the disease have been described: visceral, cutaneous, mucosal and diffuse cutaneous leishmaniasis (1,2).This clinical spectrum of the disease is mostly related to different Leishmania species (1)(2)(3)(4)(5), but the immunological responses to the parasite are certainly an important determinant of the clinical outcome of Leishmania infection (6).For instance, it has been demonstrated that the prevalence of serological conversion in an endemic area of visceral leishmaniasis is higher than the prevalence of disease (7).This finding suggests that there are subjects that are infected and can control the disease.It is known from studies on experimental models of leishmaniasis that the control of Leishmania infection is dependent on T cell responses.The role of Th1 or Th2 cell activation in determining resistance and susceptibility to leishmaniasis has been well demonstrated in two experimental hosts: the susceptible BALB/c mouse and the resistant C3H/HeN mouse infected with L. major (8)(9)(10).While BALB/c mice develop a Th2 response after infection with L. major and have severe and progressive disease, C3H/ HeN mice develop a Th1 type of response, with interferon-gamma (IFN-γ) production, are able to control Leishmania multiplication and have a self-limited disease (10).In these animals, treatment with anti-IL-4 monoclonal antibody (mAb) at the time of infection makes BALB/c mice able to control leishmaniasis (11) while treatment of C3H/ HeN animals with anti-IFN-γ mAb is associated with progression of the infection and severe disease (12).In humans, the strongest evidence that production of IFN-γ is involved in the control of Leishmania infection comes from a longitudinal study evaluating lymphoproliferative response and IFN-γ production by peripheral blood mononuclear cells (PBMC) from serum-converting children from an endemic area of visceral leishmaniasis.In that study, it was demonstrated that, upon Leishmania chagasi infection, children whose Leishmania antigen-stimulated PBMC produce IFN-γ will control the infection, while children who have a poor IFN-γ production will progress to visceral leishmaniasis (13).These observations clearly indicate that absence or poor IFN-γ production is a marker of susceptibility to visceral leishmaniasis.Although it is likely that several mechanisms may participate in the inability of lymphocytes from visceral leishmaniasis patients to produce this cytokine, one important point is related to the imbalance of cytokines produced in response to Leishmania antigens, i.e., high production of IL-4 and IL-10 and low IL-2 and IFN-γ production (14)(15)(16)(17)(18).Although lymphocytes from patients with visceral leishmaniasis have a strong expression of mRNA for IL-4 (16,18) and sera from visceral leishmaniasis patients have high IL-4 levels (14), there is no evidence that IL-4 is involved in the downregulation of the Th1 type of response in human leishmaniasis.For instance, in vitro addition of mAb against IL-4 did not restore the lymphocyte proliferative response or IFNγ production in L. chagasi-stimulated PBMC from visceral leishmaniasis patients (18).Interleukin-4 also did not suppress lymphocyte proliferative response or IFN-γ production in subjects cured of leishmaniasis.Patients with visceral leishmaniasis have increased expression of mRNA for IL-10 in bone marrow and lymph node cells and high levels of IL-10 in L. chagasi-stimulated PBMC supernatants (15,16,18).Moreover, the addition of mAb to anti-IL-10 restores lymphocyte proliferative response and IFNγ production in PBMC from visceral leishmaniasis patients (15,18).In visceral leishmaniasis, there is also an inability of PBMC to produce IL-12 (19) and restoration of IFN-γ production and lymphocyte proliferative response can be achieved by IL-12 addition (19,20).IL-12 also enhances the cytotoxic response, a function that is suppressed in visceral leishmaniasis patients (20).The demonstration that IL-10 abrogates the effect of IL-12 in inducing IFN-γ production in L. chagasi-stimulated PBMC from visceral leishmaniasis patients strongly suggests that IL-10 is the major cytokine involved in the progression of Leishmania infection to visceral disease (20).Interleukin-10 blocks Th1 activation and consequently a cytotoxic response by down-regulating IL-12 and IFN-γ production.Additionally, since IL-10 also inhibits macrophage activation (21) it decreases the ability of these cells to kill Leishmania.
In contrast to visceral leishmaniasis, patients with cutaneous and mucosal leishmaniasis due to L. braziliensis infection have a strong T cell response, characterized by a high lymphocyte proliferative response to Leishmania antigens and IFN-γ production (22)(23)(24)(25).Delayed-type hypersensitivity to Leishmania antigen is also present in these patients and the intradermal skin test is usually employed for the diagnosis of cutaneous and mucosal leishmaniasis.Since IFN-γ, a major cytokine associated with Leishmania killing, is produced during L. braziliensis infection, the reasons why such patients develop disease are not clear.The possibility that immunological responses may be altered in such patients at the site of the lesion is usually considered.However, immunological studies in cells from tissues of leishmaniasis patients have shown an immune response pattern similar to that seen in PBMC, with evidence of a Th0 or Th1/Th2 mixed cytokine pattern (26,27).Some differences were found in the cytokine profile when patients lesions of short duration were compared with those with old lesions.Patients with chronic lesions show a strong expression of pro-inflammatory cytokines such as TNF-α (28).Since the cytokines secreted in the early phases of infection in the experimental models of leishmaniasis are important to determine the progression or control of the infection, we hypothesize that alterations in the immunological responses early in human infection may account for parasite multiplication and for clinical outcome of the disease.In fact, we have previously shown that early in the infection (less than 60 days of disease) patients with cutaneous leishmaniasis may present a decrease in IFN-γ production and an increase in IL-10 production when compared with patients with more than two months of disease (29).More recently, we have observed that absent or low IFN-γ production may occur in up to 50% of the patients with early cutaneous leishmaniasis.To determine the role of cytokines in modulating the immune response in such patients, PBMC were stimulated with L. amazonensis antigen in the presence of IL-12, anti-IL-4 mAb, and anti-IL-10 mAb.IFN-γ levels were determined by a sandwich ELISA technique in the cul-ture supernatants.In patients with early cutaneous leishmaniasis who did not have a T cell response to Leishmania antigen, the mean IFN-γ level in L. amazonensis-stimulated PBMC supernatants was 24 ± 26 pg/ ml.While the addition of anti-IL-4 mAb did not enhance IFN-γ production (48 ± 79 pg/ ml), the addition of anti-IL-10 mAb or of IL-12 enhanced IFN-γ production to 380 ± 245 and 308 ± 342 pg/ml, respectively (P<0.05,Mann-Whitney test).
The down-regulation of the immune response in cutaneous leishmaniasis is a transitory phenomenon since virtually all patients with more than 60 days of history of cutaneous ulcers present strong IFN-γ production.Although this down-regulation of IFN-γ production in the early phases of cutaneous leishmaniasis may account for parasite multiplication, we have no evidence that patients who had low IFN-γ production will have a more severe disease measured by an increase in ulcer size or in the number of lesions, or dissemination of the infection.In contrast, preliminary data from a retrospective study show that the response to pentavalent antimonial therapy was better in patients with low IFN-γ production than in patients with high IFN-γ production.A more careful analysis of this observation is being performed in a prospective study with a larger number of patients.However, these preliminary data suggest that, although IFN-γ is important for the control of parasite multiplication, it can also be involved in the pathogenesis of the disease.One possibility is that IFN-γ, by up-regulating TNF-α production, may lead this pro-inflammatory cytokine to participate in the tissue damage observed in tegumentary leishmaniasis.In fact, TNF-α levels are elevated in tegumentary leishmaniasis, mainly in the severe cutaneous and mucosal diseases (30,31).In addition, serum TNF-α levels drop after therapy of mucosal leishmaniasis (31).
To evaluate the role of TNF-α in the pathology of tegumentary leishmaniasis, determined TNF-α levels in PBMC supernatants from patients with classical cutaneous and mucosal leishmaniasis, before and after therapy.The TNF-α and IFN-γ levels of patients with classical cutaneous leishmaniasis and mucosal disease are shown in Figure 1.Both IFN-γ and TNF-α were elevated in L. amazonensis-stimulated PBMC from patients with cutaneous and mucosal leishmaniasis.After successful therapy there was a reduction in both IFN-γ and TNF-α levels in 42% and 72% of the patients, respectively.A significant reduction in TNF-α levels was observed after successful therapy (366 ± 224 pg/ml before therapy vs 142 ± 107 pg/ml after therapy, P = 0.02, Mann-Whitney test) (Figure 2).
Finally, to determine if by decreasing TNF-α levels it is possible improve the healing of mucosal leishmaniasis lesions, we are performing an open study evaluating the efficacy of pentoxifylline associated with antimonial therapy in patients with mucosal lesions refractory to antimonial therapy.Pentoxifylline is an aminoxanthine that has the ability to decrease mRNA for TNF-α and consequently decreases TNF-α production (32,33).A pilot study with 6 patients with active mucosal disease who had received at least two courses of antimonial therapy (20 mg kg -1 day -1 for 20 days) before entering the study was performed.The mean age of the patients (5 men, 1 woman) was 24 ± 12 years.These patients received a new course of pentavalent antimony (Glucantime Rhodia, 20 mg/kg body weight) associated with pentoxifylline (1.2 g/day for 20 days).The patients were evaluated every 30 days up to 3 months and 6 and 12 months after the end of therapy.The lesions of all patients who received the antimonial therapy associated with pentoxifylline healed after 30 to 60 days of therapy.
Several lines of evidence show that IFNγ and TNF-α are important for the control of leishmaniasis.TNF-α plays an important protective role in experimental murine cutaneous leishmaniasis (34)(35)(36) and synergizes with IFN-γ in mediating killing of Leishmania major through the induction of nitric oxide (34).In human visceral leishmaniasis and diffuse cutaneous leishmaniasis there is evidence that the absence of IFN-γ allows parasite multiplication and progression from infection to disease (13,29).It is also expected that T cell responses and monocyte function are important in the control of L. braziliensis infection.In fact, there is evidence of a Th1 type of response in subjects with subclinical L. braziliensis infection and in subjects with self-healing cutaneous leishmaniasis (37).The observation presented  here that, early in the infection, T cell response is depressed in cutaneous leishmaniasis may indicate that, after infection, there is an absence of IFN-γ production that allows parasite multiplication.Later on, there is production of IFN-γ that may control the infection.However, an exacerbated production of IFN-γ and TNF-α can be deleterious for the tissue.Therefore, the present data indicate that the same cytokines involved in

Figure 2 -
Figure 2 -TNF-α and IFN-γ levels in Leishmania antigen-stimulated PBMC before and after therapy in patients with tegumentary leishmaniasis.P = 0.02 for TNF-α levels before and after treatment (Mann-Whitney test).Different symbols represent the patients.