A link between chemokine levels and disease severity in human African trypanosomiasis

International Journal for Parasitology 36 (2006) 1057–1065 www.elsevier.com/locate/ijpara

A link between chemokine levels and disease severity in human African trypanosomiasis Bertrand Courtioux a,b,*,1, Caroline Boda a,e,1, Ge´de´on Vatunga c, Lynda Pervieux The´ophile Josenando c, Paulette Mengue M’Eyi d, Bernard Bouteille a, Marie-Odile Jauberteau-Marchan b, Sylvie Bisser a,e

a,e

,

a

b

Equipe Accueil 3174 Neuroparasitologie et Neuroe´pide´miologie Tropicale, Institut d’Epide´miologie Neurologique et de Neurologie Tropicale, Faculte´ de Me´decine, 2 rue du Dr Raymond Marcland, 87025 Limoges Cedex, France Equipe Accueil 3842 Home´ostasie cellulaire et pathologies, Faculte´ de Me´decine, 2 rue du Dr Raymond Marcland, 87025 Limoges Cedex, France c Instituto de Combate e Controlo das Tripanossomiases, Luanda, Angola d Programme National de Lutte contre la THA, Hoˆpital de N’Kembo, Libreville, Gabon e Centre International de Recherches Me´dicales de Franceville, BP 769, Franceville, Gabon Received 24 January 2006; received in revised form 10 April 2006; accepted 26 April 2006

Abstract Trypanosoma brucei gambiense infection is an important public health challenge in sub-Saharan Africa. This parasitic disease is difficult to diagnose due to insidious clinical signs and transient parasitaemias. The clinical course is marked by two stages of increasing disease severity. An early systemic parasitic invasion is followed by the development of a progressive meningo-encephalitis. During this latter stage, a broad spectrum of neurological signs appears, which finally lead to a demyelinating and fatal stage if untreated. Treatment is toxic and difficult to administer when the CNS is invaded. Therefore, accurate diagnostic methods for stage determination are needed. The classically used criteria are not sufficiently specific and mechanisms of parasite invasion through the blood–brain barrier remain poorly understood. As cytokines/chemokines are involved in the early recruitment of leukocytes into the CNS, this study has focused on their potential value to define the onset of CNS involvement. Levels of monocyte chemoattractant protein-1/CCL-2, macrophage inflammatory protein1a/CCL-3, IL-8/CXCL-8, regulated upon activation T cell expressed and secreted (RANTES)/CCL-5 and IL-1b were measured in paired sera and CSF from 57 patients and four controls. Patients were classified into three groups (stage 1, intermediate and stage 2) according to current field criteria for stage determination (CSF cell count, presence of trypanosomes in CSF and neurological signs). In sera, cytokine/ chemokine levels were poorly related to disease stage. Only CXCL-8 was higher in stage 1 patients when compared with stage 2 and CCL-5 was higher in controls when compared with patients. In contrast, in CSF the expression of the selected cytokines, except CCL-5, was associated with the presence of neurological signs, demonstrating their diagnostic value. We observed a relationship between the presence of trypanosomes or trypanosome-related compounds in CSF and levels of IL-1b, CXCL-8, CCL-2 and CCL-3. These cytokines and chemokines may be triggered by the parasite and hence are potential markers of CNS invasion. Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Trypanosoma brucei gambiense; Chemokines; Cytokines; Neurological stage; Stage determination

1. Introduction * Corresponding author. Address: Equipe Accueil 3174 Neuroparasitologie et neuroe´pide´miologie tropicale, Faculte´ de Me´decine, 2 rue du Dr Marcland, 87025 Limoges Cedex, France. Tel.: +33 555 435 820; fax: +33 555 435 821. E-mail address: [email protected] (B. Courtioux). 1 These two authors contributed equally to this paper.

Sleeping sickness or human African trypanosomiasis (HAT) still claims thousands of victims in African countries. Trypanosoma brucei gambiense in West Africa and Trypanosoma brucei rhodesiense in East African countries are the protozoa responsible for the disease. Both are transmitted by the

0020-7519/$30.00 Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.04.011

1058

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065

bite of a tsetse fly belonging to the genus Glossina. Trypanosoma brucei gambiense infection is a chronic disease with transient parasitemia and poor clinical expression during the early stage (S-1) when trypanosomes multiply in lymph and blood. Fever, headaches, pruritus and swelled lymph nodes are common signs at this stage and are followed after weeks or years by a late stage (S-2) corresponding to central nervous system (CNS) invasion. Neurological signs appear but they are diverse and their expression differs widely among patients. Signs can include sleep, endocrine, behaviour or motor disturbances in a single or combined picture. CNS involvement leads to irreversible neurological damage, resulting in coma and death in the absence of treatment (Bouteille and Dumas, 2003). The diagnosis of CNS involvement is based on arbitrary biological criteria and cut-offs, as clinical signs are not specific at the onset of this chronic meningo-encephalitis (Dumas and Bisser, 1999; Kennedy, 2004). However, the diagnosis of CNS involvement is critical, as the treatment for the haemo-lymphatic stage (mainly pentamidine) and neurological stage (mainly melarsoprol or alpha-di-fluoromethylornithine, DFMO) are different and moreover highly toxic and difficult to administer in the second stage (Bouteille et al., 2003). In the field, cerebrospinal fluid (CSF) cell counts and parasite detection remain the main diagnostic tools. Much controversy surrounds this method of diagnosis. The significance of finding trypanosomes in an otherwise normal CSF and the appropriate threshold for CSF cell count to define late-stage patients are unclear (Bisser et al., 2002). It is likely that there is an intermediate stage where the host immune response could, at the blood– brain barrier (BBB), interfere with further development of the nervous disease. The study of the intermediate stage (Sint) and especially the immune mechanisms involved, could capture critical points in the development of the neurological stage and hence its diagnosis (Lundkvist et al., 2004). The first steps of host immune activation by trypanosomes are initiated through variable surface antigens and presumably the glycosylphosphatidylinositol membrane anchors of these molecules, which readily bind to macrophages. This interaction results in an up-regulation of macrophage gene expression, which in turn leads to the potentiation of a Th1 response, development of macrophages with suppressive activity and inflammation (Paulnock and Coller, 2001). Trypanosomes promote their survival through the secretion of a microtubule-binding protein, trypanin (Hill et al., 2000; Hutchings et al., 2002), which stimulates CD8+ cells to produce IFN-c, which in turn stimulates parasite growth (Bakhiet et al., 1993, 1996). We are particularly interested in the relationship between trypanosome factors and host immune activation during the first steps of the disease, particularly those leading to BBB invasion. At this crucial stage, the immune changes are dominated by macrophage and T cell (mainly CD8+) activation (Evens et al., 1970; Schultzberg et al., 1989; Bakhiet et al., 1990). The role of cytokine production induced by trypanosomes is a major event in the disease course in experimental

African trypanosomiasis with chronic meningo-encephalitis. In addition, cytokines are of major importance in the control of parasitic infections (Rhind and Shek, 1999). TNF-a, IFNc, IL-4, IL-6 and IL-1 are induced and their role has been confirmed in experimental models. Cytokines can control levels of parasitaemia by direct trypanocidal effect as observed with TNF-a (Lucas et al., 1994) or IL-4, which promotes antibody production necessary to eliminate the infection (Bakhiet et al., 1996; Sharafeldin et al., 1999). In sera, high levels of TNF-a have been related to disease severity (Okomo-Assoumou et al., 1995) and IL-10 may be a marker of cure in CSF (Lejon et al., 2002; MacLean et al., 2006). Recently, the importance of cytokines/chemokines has been highlighted in protozoan infections (Brenier-Pinchart et al., 2001). Some of them (cysteine–cysteine (CC)–chemokine) exert trypanocidal activity via the nitric oxide pathway (Villalta et al., 1998; Aliberti et al., 1999). Furthermore, astrocytes, the major glial cell-type in the CNS, are a source of cytokine/chemokine production within diseased brain and trypanin can induce cytokine/ chemokine expression in these cells, implicating them in cerebral pathology (Bakhiet et al., 2002). Cytokines/chemokines are some of the major soluble factors that induce recruitment of cells to the brain parenchyma. They belong to a family of low molecular weight proteins that function in leukocyte recruitment and cellular activation. Some chemokines (CXC or a subfamily) attract mainly neutrophils, whereas others (CC or b subfamily) attract monocytes/macrophages, eosinophils, basophils and T cells (Rollins, 1997). Their secretion is induced by pro-inflammatory cytokines such as IL-1b, TNF-a, IFN-c or by factors such as lipopolysaccharide. The b-chemokine or CCL family is likely to be involved in HAT (Rollins, 1997). This family, especially CCL-2 (CCL-2 or monocyte chemo-attractant protein), CCL-3 (CCL-3-a or macrophage inflammatory protein) and CCL-5 (CCL-5 or RANTES: regulated upon activation T cell expressed and secreted), are chemo-attractants for monocytes/macrophages, natural killer (NK) cells, adhesion and proliferation of T cells. Each chemokine has its particularities. For instance, CCL-2 is a potent attractant for activated CD4 and CD8 memory T-lymphocytes, CCL-3 activates monocytes but less effectively than CCL-2 and CCL-5 is the most potent chemokine for CD8+T cell chemo-attraction. Using a sandwich ELISA assay, we quantified blood and CSF levels of CXCL-8, CCL-2, CCL-3, CCL-5 and the pro-inflammatory cytokine IL-1b, in patients infected with T. b. gambiense. We report modulation of cytokine/ chemokine responses during different stages of the disease and their usefulness for diagnosis. 2. Materials and methods 2.1. Patients Patients were recruited in two central African countries. They were detected either actively during field surveys in

Angola (Bengo district, November, 2002) and Gabon (December, 2002) or passively at Viana (Angola, 2003) or N’Kembo hospitals (Gabon, 2003). Patients were included in the study after informed consent was given and the study protocol was approved by the Ministry of Health in Gabon and Angola. Inclusion criteria were: parasitologically confirmed cases in blood/or lymph nodes in patients over 10 years of age and never previously treated for sleeping sickness. Exclusion criteria were co-infections (malaria, filariasis, positive serology for syphilis or human immunodeficiency virus). Co-infections were treated according to national guidelines or directed to the institutes in charge of these diseases. Clinical and neurological signs and symptoms were carefully reported on case report forms. Daytime somnolence or nocturnal insomnia, sensory and gait disturbances, presence of primitive reflexes (palmo-mental reflex, sucking reflex), modified deep tendon reflexes, psychiatric disorders (confusion, mood swings, agitation, aggressive behaviour, euphoria, absent gaze, mutism and indifference), abnormal movements such as tremor (fine and diffuse) or myoclonic jerks were notified. Patients were classified with neurological signs only if at least two major signs or symptoms were present. For example, patients with headaches alone were not considered as having neurological signs or symptoms as this symptom is common and can have many origins other than HAT meningo-encephalitis. Blood and CSF samples were obtained during normal diagnostic procedures. Six mL CSF samples were collected in conic tubes, 4 mL were centrifuged, aliquoted and stored in liquid nitrogen. The remaining 2 mL of CSF were used for stage determination. Hemorrhagic lumbar punctures (>500 red blood cells/mL) were discarded and those patients excluded from this study. Ten millilitre samples of blood were collected by venipuncture and separated into two tubes. One was mixed with anticoagulant for serological diagnosis in plasma. The second one, without anticoagulant or ethylene diamine tetra acetate (EDTA), was used for cytokine studies on serum (according to Thavasu et al., 1992). Samples were aliquoted and stored in liquid nitrogen (Thavasu et al., 1992; Kenis et al., 2002). Serum and CSF samples remained in liquid nitrogen until further cytokine analysis at the Centre International de Recherches Me´dicales de Franceville (CIRMF). As controls, CSF and serum specimens were obtained from patients with non-infectious diseases consulting at Viana (Angola) or N’Kembo hospitals (Gabon). 2.2. Stage determination procedure All patients were screened for antibodies on whole blood by a card agglutination trypanosomiasis test (CATT) (Magnus et al., 1978). A positive CATT test was confirmed on diluted plasma. Parasites were searched in lymph nodes after puncture or in blood using the hematocrit centrifuge technique (Woo, 1970). Disease stage was diagnosed on the basis of cell count and/or presence of trypanosome in

Frequency (Number of patients)

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065

1059

8

Controls

S-int patients

7

S-1 patients

S-2 patients

6 5 4 3 2 1 0 0

0

1

2

3

4

5

6

7

8

9

10 12 18

30- 101-201- > 500 100 200 500

cells/µL

Fig. 1. Histogram of patients’ CSF cell count and representation of the corresponding defined groups of patients. S-1: CSF patients with cell count 5 cells/lL and no trypanosome. S-2: CSF patients with cell count >20 cells/lL and/or presence of trypanosomes. S-int: CSF patients with a different pattern from those defined in S-1 or S-2.

CSF. CSF cells were counted on Kova slides (Hycor Biomedical Inc., Gardengrove, CA). Parasite detection after modified simple centrifugation (Mie´zan et al., 2000) and cell count were done immediately after lumbar puncture. The following classification was used (Fig. 1): S-1 when CSF showed less than or equal to 5 cells/lL and no trypanosomes; S-2 when CSF showed more than 20 cells/lL (with or without presence of trypanosomes); S-int when CSF showed a pattern other than S-1 or S-2, i.e. between 6–20 cells/lL or presence of trypanosomes without cellular increase (Bisser et al., 2002). 2.3. Quantification of cytokine/chemokine levels in serum and CSF of African trypanosomiasis patients The levels of IL-1b, CXCL-8, CCL-2, CCL-3 and CCL-5 in serum and CSF were quantified by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). The mean OD for each sample was calculated and compared with a standard curve of recombinant human cytokine/chemokine and negative controls. The thresholds of detection given by the manufacturer were as follows: IL-1b: 0.1 pg/mL, CXCL-8: 8 pg/mL, CCL-2: 10 pg/mL, CCL-3: 10 pg/mL and CCL-5: 5 pg/mL. 2.4. Statistical analysis Patient data were expressed as median values within stages. Statistical significance between the patients groups was determined with non-parametric tests: the Kruskal Wallis U test (three-tailed) and the Mann–Whitney U test (twotailed). Data were considered statistically significant if P<0.05. 3. Results 3.1. Patients and samples Sixty-one subjects (57 patients and four controls) from Gabon (n=10) and Angola (n=51) were included. Patient ages ranged from 12 to 61 years with a median of 32,

1060

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065

Table 1 Cerebrospinal fluid and neurological characteristics of patients and controls. S-1: CSF patients with cell count 5 cells/lL and no trypanosome. S-2: CSF patients with cell count >20 cells/lL and/or presence of trypanosomes. S-int: CSF patients with a different pattern from those defined in S-1 or S-2 n

Controls 4

S-1 19

S-int 19

S-2 19

Mean CSF cell count/lL (standard deviation) Trypanosomes in CSF Neurological signs

1.75 (±0.5) 0 0

2.60 (±1.59) 0 1

10.07 (±3.77) 3 2

400.57 (±375.18) 9 15

control ages ranged from 20 to 61 with a median of 38. Nineteen patients were classified as S-1, 19 as S-int and 19 as S-2 (Table 1 and Fig. 1). Neurological signs and symptoms were searched for in 57 subjects and were present in 18 patients (31%; one S-1, two S-int, 15 S-2) (Table 2). Among these 18 patients, sleep disturbances were present in 15 (83%), gait disturbances in eight (44%), sensory disturbances in three (17%), abnormal primitive or deep tendon reflexes in 10 (56%), abnormal movements in eight (44%) and psychiatric disturbances in 13 (72%). More than two major signs were associated in 12 cases (67%) (Table 2). Cytokines and chemokines were measured in paired sera and CSF. Some data are missing for technical reasons (Table 3). 3.2. Levels of IL-1b, CXCL-8, CCL-2, CCL-3 and CCL-5 in sera Levels of IL-1b, CXCL-8, CCL-2, CCL-3 and CCL-5 in sera were compared with disease stage (S-1, S-int and S-2).

Median serum concentration, range and the number of samples examined in each group (S-1, S-int, S-2 and controls) are presented in Table 3. When comparing cytokine levels between patients and controls, only CCL-5 differed significantly and was lower in the patients (P=0.02). With regard to disease stage, few data were significantly different. CCL-5 levels in S-int patients and in S-2 patients were significantly lower than in controls (P=0.01 and 0.03, respectively). CXCL-8 levels in S-1 patients were significantly higher than in S-2 patients (P=0.04). 3.3. Levels of IL-1b, CXCL-8, CCL-2, CCL-3 and CCL-5 in CSF: relationship to disease severity and parasitemia and neurological signs Median CSF concentration, range, and the number of samples examined in each group (S-1, S-int, S-2 and controls) are presented in Table 3. Levels of IL-1b, CXCL-8, CCL-3 and CCL-5 were below the detection limit in five (10.2%), 22 (31.8%), 35 (58.3%) and 55 (91.6%) samples, respectively, and CCL-2 was always detected. When comparing results from patients with those from controls, only CXCL-8 levels in patients were significantly higher than in controls (P=0.04). With regard to disease stage (Table 3 and Fig. 2(A)), CXCL-8, CCL-3 and IL-1b levels were significantly higher in S-2 patients compared with controls (P=0.003, 0.03 and 0.02, respectively). CCL-2, CCL-3, CXCL-8 and IL-1b levels were significantly higher in S-2 patients compared with S-1 patients (P=0.01, 0.005, <0.001 and 0.04, respectively). CCL-2, CCL-3 and CXCL-8 levels were significantly higher in S-2 patients compared with S-int patients (P=0.01,

Table 2 Description of the 18 patients (one stage 1 (S-1), two intermediate stage (S-int) and 15 stage 2 (S-2) patients) with neurological signs and symptoms Stage

Neurological disturbances Sleep

Gait

S-1 S-int S-int S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 S-2 Total

+

+

Sensory

Reflexes

Movements

+ +

+

+

+

+ + + +

+ + + + + + + + + + + + +

+ + +

+ + + +

+ +

+ +

+ +

+

+ + + + + + +

+ +

+ 15 (83%)

8 (44%)

+ 3 (17%)

10 (56%)

Ppsychiatry

+ + 8 (44%)

+ + + + + + 13 (72%)

Signs and symptoms evaluated were: sleep—daytime somnolence, nocturnal insomnia or permanent sleep; gait—walking disturbances, positive Romberg sign or cerebellar syndrome; sensory—hyperpathia (Kerandel sign); reflexes—exaggerated deep tendon reflexes, presence of a Babinski sign, presence of primitive reflexes; movements—tremor, myoclonic jerks, chorea or athetosis; psychiatry—confusion, mood swing, agitation, aggressive behaviour, euphoria, absent gaze, mutism, indifference. +: presence of neurological sign, : absence of neurological sign.

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065

1061

Table 3 Pathogen-dependent levels of Il-1b, IL-8/CXCL-8, monocyte chemotactic protein-1 (CCL-2), macrophage inflammatory protein-1a (CCL-3), regulated upon activation of normal T cell, expressed and presumably secreted (RANTES/CCL-5) in sera and CSF of human Aftrican trypanosomiasisHAT patients divided into three stages according to disease severity (stage 1 (S-1), intermediate (S-int), stage 2 (S-2)) and controls IL-1b (pg/mL)

S-1

S-int

S-2

Controls

CXCL-8 (pg/mL)

CCL-2 (pg/mL)

CCL-3 (pg/mL)

CCL-5 (pg/mL)

Sera

CSF

Sera

CSF

Sera

CSF

Sera

CSF

Sera

CSF

Median Range n Median Range n Median Range n Median Range

2.82 0.41–8.20 10 1.11 0.31–2.40 7 1.2 0–9.5 8 1.03 0.42–1.43

0.32 0.01–0.78 15 0.32 0–1.07 14 1.28 0–9.52 16 0.23 0.21–0.25

592 0–2012 13 420 0–1660 15 176 0–993 19 37 0–118

13 0–173 19 24 0–198 19 170 0–351 19 0

485 0–5059 11 72 0–371 8 26 0–149 9 28 0–115

1820 1139–2513 15 2131 1519–29491 11 3187 701–4565 11 1974 701–4565

268 0–1132 14 259 0–1704 15 206 0–1226 19 62 7–178

0.11 0–2.12 18 0.12 0–2.41 19 34.08 0–187.41 19 0

0

n

4

4

4

4

4

4

4

4

108 511 57–211 550 14 65 391 74–185 300 15 74 198 0–209050 19 166 675 128 300–188 425 4

0.0004 and <0.0001, respectively). Relevant significant differences are shown in Fig. 2(A). CCL-5 was observed in one control and one S-2 patient only. The parasite was detected in 12 patients’ CSF. Three patients were classified as S-int and nine patients as S-2 (Table 1). IL-1b, CXCL-8 and CCL-2 levels were correlated to the presence of trypanosomes (P=0.02, 0.001 and 0.004, respectively) in contrast with CCL-3 and CCL-5. However, CCL-3 (19.3±31.3 pg/mL) was detected concomitantly with the presence of trypanosomes in five patients in S-2 and levels were high. CCL-5 was detected concomitantly with trypanosomes in one patient in S-2 (Fig. 2(B)). Neurological signs were present in 18 patients (31%), one in S-1, two in S-int and 15 in S-2. As represented in Fig. 2(C), IL-1b, CXCL-8, CCL-2 and CCL-3 levels were correlated with the presence of neurological signs (P=0.01, <0.001, 0.03, 0.002, respectively). IL-1b and CXCL-8 were present with high levels in the S-1 patient with neurological signs and also in one S-int patient with neurological signs (Fig. 2(C)). 4. Discussion Chemokines are produced upon activation by a wide spectrum of cells such as astrocytes, microglia, macrophages, T cell and endothelial cells. They play key roles in the early events of inflammation and are candidate mediators of leukocyte migration from the circulation into the CSF along a chemotactic gradient at the site of inflammation (Luster, 1998). In the present study, cytokine/chemokine secretion was evaluated for the first time, in paired sera and CSF from sleeping sickness patients. Interestingly, the major and significant differences in cytokine/chemokine levels were observed in CSF in relation to the disease stage. Indeed, a significant increase in IL-1b, CXCL-8, CCL-2 and CCL-3 levels were found in CSF of S-2 HAT patients whereas CCL-5 levels were dramatically decreased during

18 0 19 5.04 0–95.82 19 75 0–300 4

all the stages of HAT, including the neurological stage of the disease. High values of IL-1b, CXCL-8, CCL-2 and CCL-3 were associated with the presence of neurological signs and correlated with disease severity, suggesting they may reflect HAT-induced brain damage as already seen in brain inflammation and neurodegenerative diseases (Cartier et al., 2005). Such data are supported by results obtained in an experimental model (T. b. brucei-infected rat) of African trypanosomiasis, in which an early increase in CCL-5, CCL-3 and CCL-2 production occurred in the brain. Cell types expressing these chemokines were identified as astrocytes and microglial cells, which may be involved in attracting macrophages and T-lymphocytes to the brain, leading to lesions (Sharafeldin et al., 2000). In contrast with this report, CCL-5 was not increased in patients’ CSF, maybe due to the progressive immune suppression associated with parasite development and linked to the switch to a type II cytokine environment (De Baetselier et al., 2001). It seems that this chemokine is of minor importance for the recruitment of leukocytes into the subarachnoidal spaces in HAT, as in meningococcal diseases (Moller et al., 2005). The differences in cytokine/chemokine secretion were not super-imposable regarding patients’ sera and a relation to disease stage could only be assessed for CXCL-8 and CCL-5. Interestingly, CXCL-8 levels were significantly higher at the onset of the disease (S-1 patients) whereas CCL-5 was low in all stages and lower than in controls. A high serum concentration of cytokines/chemokines has been previously observed in infectious diseases of various origins, with a tendency to much higher levels in severe and generalized processes in comparison with milder and localized ones (Halstensen et al., 1993). In HAT, cytokine/chemokine secretions are not correlated with the presence of parasites in blood but with their presence in CSF, hence correlating with disease severity. In CSF, some cytokine/chemokine levels were elevated in the latter stages of the disease and were not present

1062

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065

during the initiating mechanisms of parasite invasion through the BBB. Therefore, they seem to play more of a role in the immune-increasing process and further recruitment of cells in the brain. We demonstrated a relationship between IL-1b, CCL-2 and CXCL-8 levels and the presence of parasites in CSF. It could be hypothesized that parasite

A pg/mL

pg/mL a, b, c

100

a, c

3 2

60

1 20 pg/mL

CSF CCL-3

CSF IL-1ß pg/mL

b, c

a, b, c 300

4000 3000

200

2000 100

1000 CSF CCL-2 Control

CSF CXCL-8 S-1

S-int

S-2

B pg/mL

pg/mL 3

100

2

60

1 20 CSF CCL-3 pg/mL

CSF IL-1ß pg/mL 300

4000 3000

200

2000 100

1000 CSF CCL-2 Control

C

pg/mL

CSF CXCL-8 Absence of trypanosome in CSF

Presence of trypanosome in CSF

presence in CSF induces, directly or indirectly, release of these cytokines/chemokines, depending on some parasitic factor or immune cell activation triggered by the presence of trypanosomes. The pro-inflammatory cytokine IL-1b is known to induce cytokine/chemokine secretion and is synthesized in a wide range of inflammatory conditions, including HAT (Sileghem et al., 1989). Ching et al. (2005) have shown that trypanosome infection induces chronic IL-1 expression in the brain in mice. The IL-1 receptor appears to be important for the recruitment of leukocytes across the BBB. Intra-ventricular injection of interleukin IL-1b induced infiltration of leukocytes. In our study, we confirmed that high levels of IL-1b were detected only in the CSF of S-2 patients, in contrast with base line levels measured in controls and S-1 patients. Parasite detection in CSF was found to be associated with IL-1b secretion. This could suggest the parasite directly or indirectly activates IL-1b production by parasite-derived factors (Tachado and Schofield, 1994). Activation induced by trypanosomes could be argued by the study performed by Quan et al. (1998). They have injected i.p. bacterial LPS. The IL-1b RNA synthesis, detected by in situ hybridization, was present early after injection (in the first 2 h) in circumventricular organs where the BBB is leaky (circumventricular organs—organum vasculosum of the lamina terminalis, subfornical organ, median eminence and area postrema—and in choroid plexus, meninges and blood vessels). Interestingly, these areas are invaded early by trypanosomes (Schultzberg et al., 1988). Therefore, the IL-1b levels in CSF could assess the presence of an early CNS invasion of trypanosomes in patients. In addition, IL-1b is known to activate chemokine production, especially CCL-2 by CNS endothelial cells leading to the leukocyte recruitment across the BBB (Harkness et al., 2003). Indeed, in our study, CSF levels of CCL-2 were highly correlated with cell number, parasite presence and disease severity. In contrast, CCL-2 levels were diminished in sera. The major function of CCL-2 in CNS inflammatory diseases is also linked to the temporal and spatial recruitment of mononuclear cells into the CNS (Banisor et al., 2005). CCL-2 is involved in Th2 polarization

pg/mL 3

100

b

2

60

1 20 CSF CCL-3 pg/mL

CSF IL-1ß pg/mL 300

4000 3000

200

2000 100

1000 CSF CCL-2 Control

CSF CXCL-8 Absence of neurological signs

Presence of neurological signs

Fig. 2. (A) CSF chemokines/cytokines levels (CCL-2, CCL-3, CXCL-8, IL1b) compared with disease stage, respectively, in controls, patients in stage 1 (S-1), intermediate stage (S-int) and in stage 2 (S-2). The minimal and maximal values are represented by the limit of the box and a line symbolizes the mean. (a) significant difference between controls and S-2 patients; (b) significant difference between S-1 and S-2 patients; (c) significant difference between S-int and S-2 patients. (B) CSF chemokine/cytokine levels (CCL-2, CCL-3, CXCL-8, IL-1b) compared with the presence/absence of trypanosomes, respectively, in controls, patients with and without trypanosomes in CSF. The minimal and maximal values are represented by the limit of the box and the mean is symbolized by a line. (C) CSF chemokine/cytokine levels (CCL-2, CCL-3, CXCL-8, IL-1b) compared with the presence of neurological signs, respectively, in controls, patients with and without neurological signs. The minimal and maximal values are represented by the limit of the box and the mean is symbolized by a line.

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065

(Gu et al., 2000), suggesting an effective suppression of the pro-inflammatory Th1 response during the course of the disease. Recently, it has been shown that CCL-2 is strongly implicated in the opening of the BBB by altering tight junction proteins in endothelial cells (Stamatovic et al., 2005). Therefore, CCL-2 plays a key role for parasite entry into the brain. The CXC family, represented by CXCL-8, showed surprisingly interesting results with regards to disease stage in sera and CSF. The high values in sera for S-1 patients and CSF for S-2 patients could suggest a strong activity consistent with findings showing that, in addition to its strong action on neutrophils, CXCL-8 is a chemo-attractant for distinct lymphocyte subpopulations (Larsen et al., 1989) and homing in CSF (Sozzani et al., 1997). CXCL-8 was associated with parasite detection in CSF and the presence of neurological signs. Interestingly, high levels of this cytokine/chemokine reinforced the diagnosis of one patient exhibiting neurological signs but without increased CSF cells and without CSF trypanosomes. Therefore, the CXCL-8 production in CSF could be an early marker of the presence of parasite in CNS. Indeed, it was demonstrated that an acute up-regulation of CXCL-8 in the brain endothelium was induced by a bacterial pathogen (B Streptococcus) (Doran et al., 2003). Such a mechanism could occur in HAT, through a direct activation of the BBB endothelial cells. A recent study demonstrated that T. b. gambiense blood stream-form parasites have the capacity to enter human brain microvascular endothelial cells (Nikolskaia et al., in press). Strikingly, this extra-cellular parasite could cross the BBB by a trans-endothelial pathway. This additional pathway of CNS invasion by trypanosomes is an early phenomenon, which is a potential trigger for endothelial cell activation to produce CXCL-8. This hypothesis could explain why one patient in the present study had neurological symptoms without cell or detectable parasites in CSF. CCL-3 in CSF was only expressed in S-2 patients with high values (>100-fold of stage 1 patients) correlated with cell count but not with the presence of trypanosomes. Its early production in the CNS during inflammation processes was described in an experimental model of African trypanosomiasis (Sharafeldin et al., 2000). Increased concentrations of CCL-3 were also detected in CSF of patients with meningitis of purulent, tuberculous or viral aetiology and CCL-3 is believed to contribute to monocyte migration to the CSF (Spanaus et al., 1997; Inaba et al., 1997; Lahrtz et al., 1997). We demonstrated here that CCL-3 chemotactic and pro-inflammatory effects in recruiting cells to the CSF is of major importance in HAT and confirms S-2 patients. The absence of correlation with the presence of parasites can be linked to a different triggering mechanism than the other chemokines reported here but must still somehow be linked to parasite presence. Some missing links could hamper the strength of our results. Firstly, in sera there were no differences in chemokine secretions between controls and patients except

1063

CCL-5. CSF controls showed surprisingly high values for CCL-2 and IL-1b. But the low number of controls (4 controls compared with 57 patients) could explain the lack of statistically proven differences in some cases. On the other hand, controls were chosen in rural settings with high prevalence of infectious diseases and difficult access to medication. These four controls may be exposed to high immune pressures, even if major co-infections were excluded from the study. These data are consistent with higher CSF levels of chemokines in these controls compared with lower levels in Caucasians (Moller et al., 2005). However, S-1 and S-int patients could not be used as controls for S-2 patients with regard to the neurological symptoms present in some of them, attesting that parasites could be present early in CNS. This hypothesis is measured by the occurrence of high levels of chemokines in some S-1 or S-int patients in CSF, similar to those of S-2 patients. Second, whether chemokine levels in CSF reflect levels present at the sites of brain inflammation is a matter for discussion. Chemokine receptors in the brain are expressed constitutively and upregulated in different pathways during different pathological conditions (Cartier et al., 2005). Their characterization in HAT could give more value to our results. However, the presence of neurological signs, which correlated with CSF results already, confirms the importance of these results. In conclusion, we have shown herein that cytokine/chemokine patterns, especially IL-1b, CXCL-8, CCL-2 and CCL-3 can define neurological involvement in HAT. High levels are concordant with CNS disturbances and help to detect patients who need to be treated with second stage drugs. Indeed, cytokine/chemokine patterns could be used as additional tests in doubtful cases to assess disease stage. The concentrations of some cytokines (IL-6, IL-8 and IL-10) have already been studied in the CSF of sleeping sickness patients. In T.b. gambiense and T.b. rhodesiense infections, IL-10 concentration in CSF of S-2 patients is elevated and could be an interesting marker (MacLean et al., 2001; Lejon et al., 2002). Studies of cytokines/chemokines open the way to identifying patterns of immune responses that could define early CNS invasion, especially in patients without parasites or sufficient cell counts in CSF. Multicentric studies with standard stage determination procedures and including patients’ follow-up, could help to identify patterns of immune response linked to host susceptibility and finally specifically assess HAT CNS invasion. Acknowledgements The authors are indebted to the staff of the Instituto de Combate e Controlo das Tripanossomiases (Angola) for their help and their assistance in the field. This work was supported by grants from Conseil Re´gional du Limousin (France), Universite´ de Limoges (France), Ministe`re Franc¸ais des Affaires Etrange`res (FSP 1997 000 100) and Centre International de Recherches Me´dicales de Franceville. Funds issued from the French Embassy in Angola

1064

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065

at Luanda insured the functioning of the Viana Trypanosomiasis Clinic, travel and accommodation expenses of the investigators. We also are very grateful to Dr J. Cook-Moreau, who kindly agreed to spend time editing this manuscript. References Aliberti, J.C., Machado, F.S., Souto, J.T., Campanelli, A.P., Teixeira, M.M., Gazzinelli, R.T., Silva, J.S., 1999. Beta-chemokines enhance parasite uptake and promote nitric oxide-dependent microbiostatic activity in murine inflammatory macrophages infected with Trypanosoma cruzi. Infect Immun. 67, 4819–4826. Bakhiet, M., Olsson, T., van der Meide, P., Kristensson, K., 1990. Depletion of CD8+T cells suppresses growth of Trypanosoma brucei brucei and interferon-gamma production in infected rats. Clin. Exp. Immunol. 81, 195–199. Bakhiet, M., Mix, E., Kristensson, K., Wigzell, H., Olsson, T., 1993. T cell activation by a Trypanosoma brucei brucei-derived lymphocyte triggering factor is dependent on tyrosine protein kinases but not on protein kinase C and A. Eur. J. Immunol. 23, 535–539. Bakhiet, M., Bu¨scher, P., Harris, R.A., Kristensson, K., Wigzell, H., Olsson, T., 1996. Different trypanozoan species possess CD8 dependent lymphocyte triggering factor-like activity. Immunol. Lett. 50, 71–80. Bakhiet, M., Mousa, A., Seiger, A., Andersson, J., 2002. Constitutive and inflammatory induction of alpha and beta chemokines in human first trimester forebrain astrocytes and neurons. Mol. Immunol. 38, 921–929. Banisor, I., Leist, T.P., Kalman, B., 2005. Involvement of beta-chemokines in the development of inflammatory demyelination. J. Neuroinflamm. 2, 1–7. Bisser, S., Lejon, V., Preux, P.M., Bouteille, B., Stanghellini, A., Jauberteau, M.O., Buscher, P., Dumas, M., 2002. Blood–cerebrospinal fluid barrier and intrathecal immunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness. J. Neurol. Sci. 193, 127–135. Bouteille, B., Dumas, M., 2003. Human African trypanosomiasis. In: Aminoff, M., Daroff, R. (Eds.), Encyclopedia of the Neurological Sciences, vol. 2. Academic Press, San Diego, CA, pp. 587–594. Bouteille, B., Oukem, O., Bisser, S., Dumas, M., 2003. Treatment perspectives for human African trypanosomiasis. Fundam. Clin. Pharmacol. 17, 171–181. Brenier-Pinchart, M.P., Pelloux, H., Derouich-Guergour, D., AmbroiseThomas, P., 2001. Chemokines in host–protozoan-parasite interactions. Trends Parasitol. 17, 292–296. Cartier, L., Hartley, O., Dubois-Dauphin, M., Krause, K.H., 2005. Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases. Brain Res. Rev. 48, 16–42. Ching, S., He, L., Lai, W., Quan, N., 2005. IL-1 type I receptor plays a key role in mediating the recruitment of leukocytes into the central nervous system. Brain Behav. Immun. 19, 127–137. De Baetselier, P.D., Namangala, B., Noel, W., Brys, L., Pays, E., Beschin, A., 2001. Alternative versus classical macrophage activation during experimental African trypanosomosis. Int. J. Parasitol. 31, 575–587. Doran, K.S., Liu, G.Y., Nizet, V., 2003. Group B streptococcal betahemolysin/cytolysin activates neutrophil signalling pathways in brain endothelium and contributes to development of meningitis. J. Clin. Invest. 112, 736–744. Dumas, M., Bisser, S., 1999. Clinical aspect of human African trypanosomiasis (Chapter 13). In: Dumas, M., Bouteille, B., Buguet, A. (Eds.), Progress in human African trypanosomiasis, Sleeping Sickness, vol. 344. Springer, Paris, France, pp. 215–249. Evens, F., Marsboom, R., Mortelmans, J., 1970. Experimental infection with Trypanosoma brucei, Trypanosoma rhodesiense, and Trypanosoma gambiense in beagle-dogs. Trans R. Soc. Trop. Med. Hyg. 64, 161–162.

Gu, L., Tseng, S., Horner, R.M., Tam, C., Loda, M., Rollins, B.J., 2000. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404, 407–411. Halstensen, A., Ceska, M., Brandtzaeg, P., Redl, H., Naess, A., Waage, A., 1993. Interleukin-8 in serum and cerebrospinal fluid from patients with meningococcal disease. J. Infect. Dis. 167, 471–475. Harkness, K.A., Sussman, J.D., Davies-Jones, G.A., Greenwood, J., Woodroofe, M.N., 2003. Cytokine regulation of MCP-1 expression in brain and retinal microvascular endothelial cells. J. Neuroimmunol. 142, 1–9. Hill, K.L., Hutchings, N.R., Grandgenett, P.M., Donelson, J.E., 2000. T lymphocyte-triggering factor of African trypanosomes is associated with the flagellar fraction of the cytoskeleton and represents a new family of proteins that are present in several divergent eukaryotes. J. Biol. Chem. 275, 39369–39378. Hutchings, N.R., Donelson, J.E., Hill, K.L., 2002. Trypanin is a cytoskeletal linker protein and is required for cell motility in African trypanosomes. J. Cell Biol. 156, 867–877. Inaba, Y., Ishiguro, A., Shimbo, T., 1997. The production of macrophage inflammatory protein-1a in the cerebrospinal fluid at the initial stage of meningitis in children. Pediatr. Res. 42, 788–793. Kenis, G., Teunissen, C., De Jongh, R., Bosmans, E., Steinbusch, H., Maes, M., 2002. Stability of interleukin 6, soluble interleukin 6 receptor, interleukin 10 and CC16 in human serum. Cytokine 19, 228–235. Kennedy, P.G., 2004. Human African trypanosomiasis of the CNS: current issues and challenges. J. Clin. Invest. 113, 496–504. Lahrtz, F., Piali, L., Nadal, D., Pfister, H.W., Spanaus, K.S., Baggiolini, M., Fontana, A., 1997. Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-c inducible protein-10 and monocyte chemotactic protein-1. Eur. J. Immunol. 27, 2484–2489. Larsen, C.G., Anderson, A.O., Oppenheim, J., Matsushima, K., 1989. Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-1 or tumor necrosis factor. Immunology 68, 31–36. Lejon, V., Lardon, J., Kenis, G., Pinoges, L., Legros, D., Bisser, S., N’Siesi, X., Bosmans, E., Bu¨scher, P., 2002. Interleukin (IL)-6, IL-8 and IL-10 in serum and CSF of Trypanosoma brucei gambiense sleeping sickness patients before and after treatment. Trans. R. Soc. Trop. Med. Hyg. 96, 329–333. Lucas, R., Magez, S., De Leys, R., Fransen, L., Scheerlinck, J.P., Rampelberg, M., Sablon, E., De Baetselier, P., 1994. Mapping the lectin-like activity of tumor necrosis factor. Science 263, 814–817. Lundkvist, G.B., Kristensson, K., Bentivoglio, M., 2004. Why trypanosomes cause sleeping sickness. Physiology 19, 198–206. Luster, A.D., 1998. Chemokines-chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338, 436–445. MacLean, L., Odiit, M., Sternberg, J.M., 2001. Nitric oxide and cytokine synthesis in human African trypanosomiasis. J. Infect. Dis. 184, 1086–1090. MacLean, L., Odiit, M., Sternberg, J.M., 2006. Intrathecal cytokine responses in Trypanosoma brucei rhodesiense sleeping sickness patients. Trans. R. Soc. Trop. Med. Hyg. 100, 270–275. Magnus, E., Vervoort, T., Van Meirvenne, N., 1978. A card-agglutination test with stained trypanosomes (CATT) for the serological diagnosis of T.b. gambiense trypanosomiasis. Ann. Soc. Belg. Med. Trop. 58, 169–176. Mie´zan, T.W., Meda, H.A., Doua, F., Dje, N.N., Lejon, V., Buscher, P., 2000. Single centrifugation of cerebrospinal fluid in a sealed pasteur pipette for simple, rapid and sensitive detection of trypanosomes. Trans. R. Soc. Trop. Med. Hyg. 94, 293. Moller, A.S., Bjerre, A., Brusletto, B., Joo, G.B., Brandtzaeg, P., Kierulf, P., 2005. Chemokine patterns in meningococcal disease. J. Infect. Dis. 191, 768–775. Nikolskaia, O.V., Kim, Y.V., Kovbasnjuk, O., Kim, K.J., Grab, D.J., 2006. Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood–brain barrier. Int. J. Parasitol. 36, 513–519.

B. Courtioux et al. / International Journal for Parasitology 36 (2006) 1057–1065 Okomo-Assoumou, M.C., Daulouede, S., Lemesre, J.L., N’Zila-Mouanda, A., Vincendeau, P., 1995. Correlation of high serum levels of tumor necrosis factor-alpha with disease severity in human African trypanosomiasis. Am. J. Trop. Med. Hyg. 53, 539–543. Paulnock, D.M., Coller, S.P., 2001. Analysis of macrophage activation in African trypanosomiasis. J. Leukoc. Biol. 69, 685–690. Quan, N., Whiteside, M., Herkenham, M., 1998. Time course and localization patterns of interleukin-1beta messenger RNA expression in brain and pituitary after peripheral administration of lipopolysaccharide. Neuroscience 83, 281–293. Rhind, S.G., Shek, P.N., 1999. Cytokines in the pathogenesis of human African trypanosomiasis: antagonistic roles of TNF-a and IL-10 (Chapter 7). In: Dumas, M., Bouteille, B., Buguet, A. (Eds.), Progress in Human African Trypanosomiasis, Sleeping Sickness, vol. 344. Springer, Paris, France, pp. 119–135. Rollins, B.J., 1997. Chemokines. Blood 90, 909–928. Schultzberg, M., Ambatsis, M., Samuelsson, E.B., Kristensson, K., van Meirvenne, N., 1988. Spread of Trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia. J. Neurosci. Res. 21, 56–61. Schultzberg, M., Olsson, T., Samuelsson, E.B., Maehlen, J., Kristensson, K., 1989. Early major histocompatibility complex (MHC) class I antigen induction in hypothalamic supraoptic and paraventricular nuclei in trypanosome-infected rats. J. Neuroimmunol. 24, 105–112. Sharafeldin, A., Hamadien, M., Diab, A., Li, H., Shi, F., Bakhiet, M., 1999. Cytokine profiles in the central nervous system and the spleen during the early course of experimental African trypanosomiasis. Scand. J. Immunol. 50, 256–261. Sharafeldin, A., Eltayeb, R., Pashenkov, M., Bakhiet, M., 2000. Chemokines are produced in the brain early during the course of experimental African trypanosomiasis. J Neuroimmunol. 103, 165–170.

1065

Sileghem, M., Darji, A., Hamers, R., De Baetselier, P., 1989. Modulation of IL-1 production and IL-1 release during experimental trypanosome infections. Immunology 68, 137–139. Sozzani, S., Luini, W., Borsatti, A., Polentarutti, N., Zhou, D., Piemonti, L., D’Amico, G., Power, C.A., Wells, T.N., Gobbi, M., Allavena, P., Mantovani, A., 1997. Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines. J. Immunol. 159, 1993–2000. Spanaus, K.S., Nadal, D., Pfister, H.W., Seebach, J., Widmer, U., Frei, K., Gloor, S., Fontana, A., 1997. C-X-C and C-C chemokines are expressed in the cerebrospinal fluid in bacterial meningitis and mediate chemotactic activity on peripheral blood-derived polymorphonuclear and mononuclear cells in vitro. J. Immunol. 158, 1956–1964. Stamatovic, S.M., Shakui, P., Keep, R.F., Moore, B.B., Kunkel, S.L., Van Rooijen, N., Andjelkovic, A.V., 2005. Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability. J. Cereb. Blood Flow. Metab. 25, 593–606. Tachado, S.D., Schofield, L., 1994. Glycosylphosphatidylinositol toxin of Trypanosoma brucei regulates IL-1 alpha and TNF-alpha expression in macrophages by protein tyrosine kinase mediated signal transduction. Biochem. Biophys. Res. Commun. 205, 984–991. Thavasu, P.W., Longhurst, S., Joel, S.P., Slevin, M.L., Balkwill, F.R., 1992. Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions. J. Immunol. Methods 153, 115–124. Villalta, F., Zhang, Y., Bibb, K.E., Kappes, J.C., Lima, M.F., 1998. The cysteine-cysteine family of chemokines RANTES, MIP-1alpha, and MIP-1beta induce trypanocidal activity in human macrophages via nitric oxide. Infect. Immun. 66, 4690–4695. Woo, P.T.K., 1970. The hematocrit centrifuge technique for the diagnosis of African trypanosomiasis. Acta Trop. 74, 676–684.