Tumor Necrosis Factor (Cachectin) in Human Visceral Leishmaniasis M. Barral-Netto, R. Badar6, A. Barral, R. P. Almeida, S. B. Santos, F. Badar6, D. Pedral-Sampaio, E. M. Carvalho, E. Falcoff, and R. Falcoff

Service de Immunologia, Hospital Universidade Professor Edgard Santos, Universidade Federal de Bahia, Salvador-Bahia, Brazil; Unite INSERM 196, Institute Curie, Paris, France

Visceral leishmaniasis (VL), a disease caused by protozoan parasites of the genus Leishmania, is characterized by fever, hepatosplenomegaly, anemia, leukopenia, and hyperglobulinemia. Although various laboratory abnormalities are seen in patients with full-blown VL, or kala-azar, immunologic dysfunction is a hallmark of the disease. The absence of antigeninduced lymphocyte proliferative responses, negative delayed hypersensitivity reactions, and poor generation oflymphokines are well documented [1, 2]. Malnutrition [3] and elevated serum triglyceride levels [4, 5] have been reported in affected individuals. Tumor necrosis factor-a, or cachectin (TNFa), is a cytokine reported to be involved in the mediation of normal host functions, such as tissue remodeling and mobilization of energy [6], as well as in inflammation [7]. TNFa has also been implicated in host reaction against bacteria or parasites, either playing a role in the protection of the host [8-12] or worsening the course of the infection [13-15]. Here we evaluate the levels of TN Fa in serum of patients with VL and the possibility of using these levels as a marker of disease activity.

Received 24 July 1990; revised 29 October 1990. Presented in part: VI International Congress of Parasitology, Paris, August 1990. Informedconsentwasobtainedfrom all patientsor guardians,andthe study wasapprovedby the committee on human rights of the Universityof Bahia. Grantsupport: UnitedNationsDevelopment ProgrammeIWorld Bank/World HealthOrganizationSpecial Program for Researchand Training in Tropical Diseases; INSERM. Reprintsor correspondence: Dr. Manoel Barral-Netto, Hosp. Univ. Prof. Edgard Santos-UFBA,R. Joaodas Botas,sin; 40.140 Salvador-Bahia, Brazil. The Journal of Infectious Diseases 1991;163:853-857 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6304-0029$01.00

Patients, Materials, and Methods Patients. We evaluated 58 patients with leishmanial infection. Group I comprised 28 patients with typical acute VL according to the following criteria: a clinical history of weight loss, fever, hepatosplenomegaly,anemia, and leukopenia, with elevatedantileishmanial IgG antibody and Leishmania organisms demonstrated in Giemsastained splenic aspiration material. Sixteen patients were male. The splenic parasitism index was determined before and after treatment following the criteria described elsewhere [16]. Briefly, Leishmania amastigotes were counted by scanning 1000 oil immersion fields (lOX eyepiece, 100x oil objective) and graded logarithmically as follows: 0 (no parasites/WOO fields), 1+ (1-10parasites/WOO fields), 2+ (1-10parasites/lOOfields), 3+ (1-10parasites/If fields), 4+ (1-10parasites/field), 5+ (10-100 parasites/field), and 6+ (>100 parasites/field) . Group II was composed of 30 patients with cryptic Leishmania infection: 16 asymptomatic, as defined by absence of clinical signs and symptoms, normal laboratory profiles, and high levels of specific antileishmanial antibodies as previously described [17]; 4 with selfhealing subclinical VL as previously described [17]; and 10 posttreatment VL cases with a documented history of acute VL 1-10 years before the present study. At the time of these evaluations, these subjects were asymptomatic and had a positive leishmanin skin test. Controls included 15 normal volunteers (with negative serology and negative skin test to leishmanial antigen), with no history or symptoms ofleishmaniasis and a negative serologic test, and 10 patients with cutaneous leishmaniasis. Treatment. Eight patients received the classic antimonial therapy (Glucantime; Rhodia do Brasil, Sao Paulo, Brazil) 20 mglkglday for 20 days. Twenty refractory cases (full-blown VL unresponsive to conventional antimonial therapy) received a combined therapy of Glucantime plus recombinant human interferon-s (rHuIFN-')'; Roussel Uclaf, Romainville, France) following a research protocol recently reported [18]. Macrophage cultures. Monocyte-derived human macrophages were obtained from normal donors as previously described [19]. After

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High tumor necrosis factor-a (TNFa) levels were present in the serum of24 of28 active visceral leishmaniasis (VL) patients (142.9 ± 113.9pg/ml, mean ± SD), whereas levels were not elevated in 26 of 30 patients with cryptic leishmanial infection (16 asymptomatic, 4 with self-healing subclinical infection, and 10 posttreatment VL cases). Serum TNFa levels were also not elevated in 15 normal volunteers (11.3 ± 15.6 pg/ml) and in 10 patients with tegumentary leishmaniasis (19.1 ± 10.8 pg/ml). Leishmanial infection of human monocyte-derived macrophages enhanced the basal TNFa production by these cells, and this effect was further potentiated by treatment with recombinant interferon-v, After effectivetreatment ofVL patients, serum TNFa levels dropped rapidly (129 ± 112 vs. 9 ± 13 pg/ml in 10 days), even before clinical parameters such as spleen size or parasitism, white blood cell count, or levels of hemoglobin returned to normal values. On the other hand, patients unresponsive to treatment remained with elevated levels (276 ± 69 vs. 155 ± 71 pg/ml in 10 days). Thus, serum TNFa levels in VL patients are a good parameter to monitor in determining host response to therapy.


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Barr al-Netto et al.

6 daysin culture, the cells were infected withstationary-phase Leishmania amazonensis promastigotes (MHOMIBR/76/Josefa) at a proportion of 10:1 for 2 h at 35°C in an atmospherecontaining 5%

Results The levels of TNFa found in 15 normal human sera, obta ined from volunteers from the same area as the patients and collected , stored , and handled in the same way as patients' sera , were 0-50 pglrnl (11.33 ± 15.58 pg/ml , mean ± SD ). Of 28 patients with active VL , 24 (85.7 %) had levels of serum TNFa >50 pglrnl (2.5 SD above levels from normal controls) in samples obtained before treatment. TNFa levels for VL patients ranged from 0 to 450 pg/rnl of serum (142.9 ± 113.96). All 10 tested sera from cutaneous leishmaniasis patients were not elevated, ranging from 10 to 35 pg/ml (mean ± SD, 19.1 ± 10.84; figure 1). In the group with cryptic infections, 26 of 30 patients had normal serum TN Fa levels, three (post-kala azar patients) were in the "gray zone" (30 and 50 pg/ml) , and one (an asymptomatic child) had elevated TN Fa (117 pglrnl). Of 4 sera from patients with subclinical cryptic infection, none had elevated TNFa (group mean shown in figure 1). Considering the values obtained in the two groups of patients, a high serum TNFa value had a predictive value of 86 % for disease activity in VL. In our in vitro studies, supernatants obtained in two different experiments from untreated cultures of monocyte-derived human macrophages contained 93 ± 33 pglrnl TNFa. When cultures were infected by L. amazonensis, production increased to 248.5 ± 3.5 pglrnl. Pretreatment of macrophages with rHuIFN--y (10 IU/ml) did not change significantly the ba sal level of TNFa production (76.5 ± 33.5 pglml) but increased to 359.5 ± 52.5 pglrnl TNFa production after Leishmania infection. A similar effect was obtained when macrophages were treated with interferon: Interferon-treated

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Normal Controls

Tegumen tary

Cr y ptic VL

Acu te VL

Figure 1. Serum tumor necrosis factor-a (TNF) levels in patients with full-blown visceral leishmaniasis (n = 28), cryptic leishmanial infections (asymptomatic [n = 16]. subclinical [n = 4], posttreatment [n = laD. and cutaneous (tegumentary) leishmaniasis (n = 10) compared with those in normal controls (n = 15) from the same area. Data are mean ± SO.

macrophages produced 13:4 f -20.l pg/ml , whereas infected and then interferon-treated macrophages produced 498 ± 57.5 pglrnl. The 28 VL patients were divided into two categosies on the basis of their response to treatment: patients who remained severely ill despite therapy (refractory) and those who responded completely to treatment (re sponsive). Table 1 compares 21 cured and 5 refractory pat ients before, during (5-10 days) , and after therapy. Before treatment, there was no significant difference between groups in sever ity of disease, considering spleen size , white blood cell count, and levels of hemoglobin and spleen parasitism. After treatment , however, responsive patients showed marked improvement in all parameters and had normal temperatures, in contrast to refractory cases who showed slight or no clinical improvement and remained febrile. No difference was observed between groups in weight gain during this short time. Although pretreatment TNFa levels were higher in refractory than in responsive patients, TNFa levels for a single patient could not be used as a predictor for therapeutic prognos is. TNFa levels in the responsive group ranged from 0 to 450 pglrnl and in the refractory group from 60 to 380 pglrnl. However, TNFa levels during and after treatment correlated with the general response. Significant differences were obtained comparing TNFa values for respons ive and refractory group s during (35 ± 40 vs. 150 ± 70 pglml) and after (9 ± 13 vs. 155 ± 71 pglrnl ) treatment. Mean spleen size, white blood cell count, hemoglobin level , and parasite load in cured patients were significantly different from those in refractory cases (table 1). In summary, there was a significant difference between pre compared with posttreatment serum TNFa levels (129 ± 112 vs. 9 ± 13 pg/ml) in patients responsive to therapy, whereas

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C~ . Next, nonphagocytosed parasites were washed away, and cultures were overlaid with fresh medium and cultured at 35°C in a humidified atmosphere containing 5% C02. After 24 h, supernatants were collected and stored at -20°C until tested for TNFa. In some cultures rHuIFN--y (providedby Dr. M. Brandely, RousselUclaf ) at 10 IU/mI, a dose reported effective for activating human macrophages [20], was used before or after infection. TNFa determination; Sera werecollectedand stored frozen. All determinations weremadebya commerciallyavailable immunoradiometric assay kit (TNFa-IRMA; Medgenix Diagnostics, Brussels). Several monoclonal antibodies directed againstdistinct epitopes of TNFa were attached to the inner surface of-the tubes. Standard amounts of TNFa and serum samples (200 ILl/tube) were added in duplicate, immediately followed by 50 ILl of the I25I-1abeled antiTNFa monoclonal antibody. The tubes were aspirated,washed, and counted for 60 s in a gammacounter. The standard curves were prepared with values rangingfrom a to 5000 pg/ml TNFa and sample values interpolated, Sensitivity of the test is 5 pg/ml, and it does not cross-react with TNF-I3. IL-I or -2, or interferon -rz, -13. or -"IStatistical treatment. Comparisons betweengroups were made by the Mann-Whitney test.


TNF in Visceral Leishmaniasis

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Table 1. Comparison of serum tumor necrosis factor-a (TNFa) levels with clinical and laboratory data in acute visceral leishmaniasis patients according to therapeutic response. Responsive (n Before

Parameter Spleen size (em) White blood cell count (X 10 3/rnm3 ) Hemoglobin (g/dl) Parasite index (+) TNFa (pg/ml) NOTE.

11 3.5 8.4 5 129

± ± ± ±

4 1.6 1.3 1 ± 40

During 5.7 5.2 9.7 1 35

± ± ± ±

3.6 1.7 1.5 1 ± 40

= 21)

Refractory (n After 2.8 7.6 10.9 0 9

± 3.3

± 2.2 ± 1

± 13

Before 16 2.8 7.5 5.6 276

±6 ± 1.2 ± 2 ± 1 ± 69

= 5)

During 13 2.7 7.5 3.7 150

±4 ± 1.7 ± 1.3 ± 0.5 ± 70

After 12 2.8 7.2 4 155

±6 ± 1.5 ± 2.5 ± 1 ±71

Data are mean ± SD. Parasite index is explained in Methods.

Discussion We found a marked elevation of serum TNFa levels in patients with VL and an intense in vitro production of TNFa by human macrophages after leishmanial infection. In addition' a correlation between the presence of elevated serum TNFa levels during acute disease and a sharp and rapid fall in such levels after effective therapy in VL was documented. We conclude that serum TNFa levels can be used as an indication of disease activity and their fall as a marker of effective therapeutic response. The clinical picture of VL is similar to effects attributed to TNF in the literature. VL patients have fever, anorexia, anemia and weakness, hypertriglyceridemia [5], and polyclonal B cell activation [21, 22], and TNF can induce all these signs and symptoms [23-28]. Cutaneous leishmaniasis patients have no such clinical signs, and serum TNF levels are in the normal range. It is possible that because fewer macrophages are infected in the localized skin ulcer, serum TNF levels do not become elevated. Alternatively, skin macrophages, which are infected in cutaneous leishmaniasis, produce less TNF than do spleen macrophages, the target cells in visceral leishmaniasis. Elevated serum levels of TNFa have been reported in human malaria [29, 30] and have been correlated with severity of disease [31, 32]. In experimental cutaneous leishmaniasis, TNF has been reported to act as a host protective element, both by exogenous administration and by its neutralization by monoclonal antibodies [11, 12]. We provided evidence that Leishmania organisms themselves are able to induce enhanced TNFa production by hu-

man monocyte-derived macrophages. Although the parasite may not be the only stimulus for TNFa induction, its penetration into macrophages helps to explain elevated TNFa levels in leishmaniasis. Malarial parasites [33, 34] or antigens [34, 35] and Mycoplasma species [36] or bacille Calmette-Guerin antigen [37] also can induce TNF production by macrophages. This suggests a direct participation of external agents in the induction of TNF by host cells. Pretreatment of macrophage cultures with interferon-v led to enhanced TNFa production after in vitro infection by leishmania. The enhancing effect of interferon on TNFa production is well known [38], and this phenomenon was also observed in cultures of macrophages stimulated by malarial parasite-infected erythrocytes [33]. However, some of our patients were treated with rHuIFN-)' and this treatment led to a decrease in their serum TNFa levels. This is only an apparent paradox. The combined treatment of interferon-y and pentavalent antimony, as used, is effectivein reducing the parasite load [18, 20] and, thus, decreasing the stimulus for TNF production. Furthermore, TNF and interferon-y present a potent synergism in cancer regression [39] and induce cytotoxic effector mechanisms in macrophages [40]; such effects may lead to the destruction of parasitized macrophages, also reducing parasitism in VL. High levels of serum TNF in untreated VL patients were previously reported in Africa [29], but no clinical correlation was demonstrated. We documented the association of high serum TNFa levels with acute VL and found normal serum levels in patients with cutaneous leishmaniasis, in children with Leishmania infection but without clinical disease, and in subjects cured of VL. These findings indicate that an elevated TNFa level is a marker of disease activity in VL. In addition, we saw a rapid fall in TNFa levels after effective therapy and a persistence of high TNFa levels in subjects refractory to therapy. Although the immunologic dysfunctions observed in VL (such as poor cell-mediated immune responses and high levels of antileishmanial antibodies) disappear after therapy, none can be used as a marker of therapeutic response because restoration may take months or years [41]. Because of the persistence of leishmanial organisms in treated VL patients [16],

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the decrease was not significant in the refractory patients (276 ± 60 vs. 155 ± 71 pg/ml). Two patients presented an atypical evolution. An apparent response was observed after the first course of treatment, and interruption of therapy was based on clinical parameters. A few weeks later the patients relapsed. TNFa levels decreased at time of clinical improvement (from 160 to 70 and from 75 to 0 pg/ml) and increased during relapses (to 260 pg/rnl 3 months later and to 150 pg/ml4 months later, respectively).


Barral-Netto et al.

search for presence of organisms may be a poor strategy for evaluation of clinical response. The observation of the decrease in TNFa levels in responsive cases ofVL, even when clinical parameters are still abnormal, indicates that monitoring serum TNFa levels is useful in the clinical management of VL patients, being a good parameter in determining host response; to therapy. Acknowledgment We thank Warren Johnson, Jr., and John David for helpful suggestions and Jackson Lemos for manuscript preparation.

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TNF in Visceral Leishmaniasis

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Tumor necrosis factor (cachectin) in human visceral leishmaniasis.

High tumor necrosis factor-alpha (TNF alpha) levels were present in the serum of 24 of 28 active visceral leishmaniasis (VL) patients (142.9 +/- 113.9...
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