Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by Th3/Tr1-type cytokines IL-10 and transforming growth factor-ß but not by a Th1 to Th2 shift
Andrea Doetze3,
Judith Satoguina,
Gerd Burchard,
Thomas Rau1,
Cornelius Löliger2,
Bernhard Fleischer and
Achim Hoerauf
Bernhard Nocht Institute of Tropical Medicine, 20359 Hamburg, Germany
1 Institute of Clinical Pharmacology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany
2 University Clinic Eppendorf, University of Hamburg, 20246 Hamburg, Germany
Correspondence to:
A. Hoerauf
 |
Abstract
|
---|
Exposure to infective larvae of the filarial nematode Onchocerca volvulus (Ov) either results in patent infection (microfilaridermia) or it leads to a status called putative immunity, characterized by resistance to infection. Similar to other chronic helminth infections, there is a T cell proliferative hyporesponsiveness to Ov antigen (OvAg) by peripheral blood mononuclear cells (PBMC) from individuals with patent infection, i.e. generalized onchocerciasis (GEO), compared to PBMC from putatively immune (PI) individuals. In this study, mechanisms mediating this cellular hyporesponsiveness in GEO were investigated: the low proliferative response in PBMC from GEO individuals was associated with a lack of IL-4 production and significantly lower production of IL-5 compared to those from PI individuals, arguing against a general shift towards a Th2 response being the cause of hyporesponsiveness. In contrast, IL-10 and transforming growth factor (TGF)-ß, two cytokines associated with a Th3 response, seemed to mediate hyporesponsiveness: PBMC from individuals with GEO produced significantly more IL-10, and T cell proliferative hyporesponsiveness in this group could be reversed by the addition of anti-IL-10 and anti-TGF-ß antibodies. Hyporesponsiveness was specific for OvAg and not observed upon stimulation with related nematode antigens, arguing for a T cell-mediated, Ov-specific down-regulation. Ov-specific T cells could be cloned from GEO PBMC which have a unique cytokine profile (no IL-2 but high IL-10 and/or TGF-ß production), similar to the T cell subsets known to suppress ongoing inflammation (Th3 and Tr1), indicating that this cell type which has not been found so far in infectious diseases may be involved in maintaining Ov-specific hyporesponsiveness.
Keywords: cytokines, helminth parasites, human, suppression, Th1/Th2, Th3/Tr1, tolerance
 |
Introduction
|
---|
Infection with the filaria Onchocerca volvulus (Ov) affects ~20 million people in the tropical region of the world and constitutes a major cause of blindness in endemic regions. Immunoepidemiological studies suggest that man's immune system is able to acquire resistance to onchocerciasis (13). Thus, some individuals living in areas hyperendemic for Ov remain free of infection despite heavy exposure to transmitting vectors and infective L3 larvae. These individuals have been termed putatively immune (PI). Compared to individuals with patent infection, PI display a cellular immune response to Ov antigen (OvAg) characterized by strong proliferation of peripheral blood mononuclear cells (PBMC); in addition, production of both IFN-
(47) and IL-5 (710) have been described. In contrast, individuals with generalized onchocerciasis (GEO) show little or no parasite-specific proliferation and cytokine production by PBMC (13,8,11). Given that in GEO there is a calculated daily turnover of >50,000 offspring worms, i.e. microfilariae (mf) (12), down-regulation of T cell reactivity to OvAg seems to be a necessary compromise between host and parasite to avoid too extensive an inflammatory reaction in the organ where mf reside, i.e. the skin.
T cell proliferative hyporesponsiveness in GEO appears to be antigen specific (13), but the molecular mechanisms by which it is induced and maintained are unclear. It is assumed that antigen-specific hyporesponsiveness is not due to clonal deletion since after therapy with the microfilariacidal drug ivermectin, proliferation is restored (11). In this study we show that in onchocerciasis, hyporesponsiveness is mediated by the effect of Th3-type cytokines IL-10 and transforming growth factor (TGF)-ß, while the Th2 cytokine IL-5 was found associated with high cellular Ov-specific proliferation. Ov-specific down-regulation is inferred from the fact that GEO PBMC do not show hyporesponsiveness to antigens from the related intestinal nematode Ascaris suum (As) and Ascaris lumbricoides (Al). We also show that that Ov-specific down-regulation may be mediated by T cells clonable from GEO PBMC which bear a cytokine profile similar to recently described T cell subsets (Th3 and Tr1) able to actively suppress inflammatory responses (14,15).
 |
Methods
|
---|
Study subjects
PBMC were obtained from 58 individuals living in the forest zone of the Republic of Guinea (West-Africa), an area hyperendemic for onchocerciasis and not included in WHO onchocerciasis eradication programs. None of the study subjects had received previous chemotherapy for onchocerciasis. Participants were assigned to either one of the groups PI (16) (n = 18) or GEO (n = 40), based upon clinical histories, parasitological examinations and physical findings as described (17). Briefly, the criteria for PI were as follows: persons having lived in hyperendemic villages for more than 12 years, without palpable nodules, signs of dermatitis, lacking mf in six skin snips taken at different sites and time points (>6 months apart); further criteria were no prior diethylcarbamazine (DEC) or ivermectin treatment and a negative Mazzotti reaction (failure to develop pruritus within 24 h after 50 mg DEC administered orally), as well as a positive IgG1 serum antibody response to OvAg indicating exposure to the parasite (18). Onchocerciasis was defined by a mf+ skin snip or by a palpable nodule containing adult worms detected later during nodulectomy.
All procedures were conducted according to the Declaration of Helsinki principles.
Collection of lymphocytes
PBMC were isolated from heparinized venous blood by centrifugation on Ficoll-Paque. PBMC were washed in RPMI 1640 medium (Gibco, Eggenstein, Germany) supplemented with 2 mM L-glutamine and 50 µM gentamicin, then resuspended in the same medium complemented with 5% heat-inactivated human AB serum (Sigma, Munich, Germany).
Parasite antigen preparations
Saline-soluble extracts from adult female worms of Ov, As and Al were prepared as described (7). Briefly, worms frozen in nitrogen were minced at about 70°C, and the resulting powder was extracted overnight at 4°C in PBS containing PMSF (0.5 mM), E64 (0.01 mM) and Benzamidine (6.4 mM; all from Sigma). Debris was removed by ultracentrifugation at 10,000 g for 1 h. The supernatant was dialyzed against PBS, sterile filtered and stored at 20°C until use. All antigen preparations were found to be negative for bacterial endotoxin effects at the concentrations used (LAL assay < 0.06 ng/ml; negativity in the test for pyrogen activity in rabbits for OvAg).
PBMC culture procedures
PBMC (105) were cultured in a total volume of 200 µl/ml RPMI/5% human AB serum in 96-well round-bottom microtiter plates. Cells were either left unstimulated or they were stimulated (i) with phytohemagglutinin (PHA) (1 µg/ml); (ii) with OvAg (50 µg/ml) with or without the following neutralizing mAb (all from PharMingen, Hamburg, Germany): anti-human IL-10 (10 µg/ml), anti-human TGF-ß (5 µg/ml) or rat IgG1 (clone R3-34; PharMingen) as isotype control; or (iii) with AsAg or AlAg (50 µg/ml). After 4 days of culture, 100 µl supernatant was removed from culture wells for cytokine detection and cultures were pulsed with BrdU (Boehringer, Mannheim, Germany) according to the manufacturer's instructions. Proliferation was quantified using an ELISA to measure the BrdU incorporation. The optical density was read at 450 nm.
T cell clones
Aliquots of PBMC which had shown OvAg-specific proliferation were subject to secondary stimulation using irradiated (4000 rad) MHC II haplotype-matched PBMC (1x105/well) from healthy European donors as antigen-presenting cells. T cells from those lines were cloned at a density of 10, 3 and 0.3 cells/well in Terasaki plates (Nunc, Wiesbaden, Germany) together with irradiated feeder cells (1.2x104), PHA (2 µg/ml) and IL-2 (100 U/ml). Growing T cell clones (TCC) were re-stimulated in 96-well round-bottom microtiter plates at 1014 day intervals. The specificity of TCC was determined by testing their proliferation in response to OvAg, using 3x104 T cells as responders and 1x105 MHC II haplotype-matched PBMC as antigen-presenting cells. After 3 days, cultures were pulsed with 0.2 µCi of [3H]thymidine for the last 18 h and [3H]thymidine incorporation was measured by liquid scintillation spectrometry (7). For the analysis of TCC cytokine production, 5x104 T cells were stimulated with anti-CD3 (OKT3, affinity purified, 1 µg/ml) and anti-CD28 (2.5 µg/ml; clone CD28.2; PharMingen) antibodies in the presence of 105 feeder cells; supernatants were removed after 3 days (2 days in the case of IL-2) of culture. For TGF-ß measurements, cultures supplemented with 1% (instead of 5%) human AB serum were used to minimize the background.
Cytokine assays
Cytokines were quantified in supernatants by ELISA using the following standard cytokines as well as mAb pairs for capture and detection (all from PharMingen): IL-2: 5344.111, biotinylated B33-2; IL-4: IL-41, biotinylated MP4-25D2; IL-5: TRFK5, biotinylated JES1-5A10; IL-10: JES3-9D7, biotinylated JES3-12G8; IFN-
: NIB42, biotinylated 4S.B3. Recombinant human cytokines IL-5, IL-10 and IFN-
(PharMingen) were used as reference standards. Immuno-Plate F96 Maxisorp (Nunc, Wiesbaden, Germany) plates were coated with 50 µl capture antibody (1 µg/ml) in 0.1 M NaHCO2/Na2CO3 buffer (pH 9.6) overnight at 4°C. After blocking with 1% BSA plates were washed with PBS/0.05% Tween and incubated overnight at 4°C with 50 µl culture supernatant or cytokine standard dilution. The biotinylated detection antibodies were used at 0.5 µg/ml in PBS/Tween/0.1% BSA. Plates were developed after incubation with streptavidinperoxidase complex (1:10,000; Boehringer) using 100 µl/well tetramethylbenzidine (Roth, Karlsruhe, Germany; dissolved 6 mg/ml in DMSO) as substrate. Substrate reactions were stopped with 25 µl/well 4 N H2SO4 and measured at 450 nm. The working sensitivity of all ELISA assays was 32 pg/ml. TGF-ß was measured using the following reagents from R & D systems (Wiesbaden, Germany): anti-human TGF-ß1 mAb (clone 9016.2) for coating, and biotinylated polyclonal chicken anti-TGF-ß antibodies (cat. no. BAF240) for detection as well as rTGF-ß1 (cat. no. 240-B-002) as standard. For this ELISA the manufacturer's protocol was strictly followed, i.e. sample `activation' by incubation with 1 N HCl for 10 min and subsequent neutralization with 1.2 N NaOH/0.5 M HEPES before addition of the detection antibody and further processing as with the other ELISA above.
Statistical analysis
Computerized statistical analyses using Statview version 5 were performed by means of non-parametric testing, due to a non-Gaussian distribution of the data. In a first step the Wilcoxon signed-rank test was used to analyze separately for each group of individuals (PI and GEO) if the enhancement of proliferation or cytokine production induced by the addition of antigen (OvAg and AsAg) in comparison to RPMI alone was significant (e.g. `is there a significant enhancement of IL-10 production by PBMC in the group of GEO after addition of OvAg compared to RPMI alone?', e.g. Fig. 2B
). In the same manner, differences in proliferation or cytokine production induced by the addition of anti-IL-10 or anti-TGF-ß antibodies were analyzed by the Wilcoxon signed-rank test. In a second step, those parameters that had been found to be significant in the Wilcoxon test (i.e. within a group) were further analyzed by the MannWhitney U-test for differences between the groups [e.g. `is the extent of OvAg-induced IL-5 production (pre-tested for each group by Wilcoxon to be significant over background) different between PI and GEO?', e.g. Fig. 3B
]. P < 0.05 was considered significant.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2. Higher spontaneous and OvAg-driven IL-10 production in GEO individuals. IL-10 secretion by PBMC from GEO and PI individuals. IL-10 was measured in supernatants of PBMC (1x105/well) after 4 days of culture in the absence of a stimulus (A), or after stimulation with PHA (1 µg/ml) (B) or with OvAg (50 µg/ml) (C). The horizontal line inside the box plots depicts the median; the lower and upper end show the 25 and 75% percentile respectively. Error bars show the range. (A) The P value denotes a significant difference in spontaneous IL-10 secretion between the GEO and PI groups (analyzed by MannWhitney U-test). (B) There is equivalent IL-10 production in response to mitogen (MannWhitney U-test). (C) In the GEO group there is a significant increase in antigen-driven versus spontaneous IL-10 production (analyzed by Wilcoxon test).
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3. PBMC from PI individuals produce more IL-5 than GEO individuals upon OvAg but not AsAg. OvAg-driven (50 µg/ml) production of IFN- (A) and IL-5 (B), as well as AsAg-driven (50 µg/ml) IL-5 production (C) by PBMC from GEO and PI individuals. Cytokine production was measured after 4 days. Spontaneous cytokine secretion was subtracted to calculate antigen-specific values. (B) P values denote a significantly higher IL-5 production in PBMC from PI compared to GEO individuals (analyzed by MannWhitney U-test).
|
|
 |
Results
|
---|
Study population
Persons with GEO had a median age of 38 years (range 1275 years) and PI had a median age of 23 years (range 1250 years). There were no significant correlations between age and any of the parameters analyzed below, as studied by multivariate analysis (not shown).
PBMC from patients with GEO are characterized by low proliferation upon OvAg as well as by high IL-10 production
PBMC from both groups proliferated in response to OvAg, although to a different extent: only a small increase was observed in the GEO group, whereas a significantly higher increase was seen in PI (P < 0.01 for the difference in OvAg-specific proliferation between GEO and PI individuals, MannWhitney U-Test; Fig. 1
). Those differences between the groups were not seen upon stimulation with PHA (not shown). PBMC from patients with GEO, compared to PI, showed a significantly higher spontaneous IL-10 production in medium control (P < 0.01 for the difference between GEO and PI individuals, MannWhitney U-Test, Fig. 2A
). In response to mitogen, equivalent production of IL-10 was observed (Fig. 2B
). However, OvAg stimulation resulted in a significant increase of IL-10 production in the GEO group only (P < 0.03, Wilcoxon signed-rank test, Fig. 2C
).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1. Higher proliferation of PBMC from PI compared to GEO individuals in response to Ov but not to AsAg extract. PBMC (1x105/well) from 40 individuals with the generalized disease (GEO) and 18 PI individuals were stimulated (A) with OvAg (50 µg/ml) or (B) with AsAg (50 µg/ml). Proliferation was measured after 4 days by BrdU incorporation. The stimulation index was calculated as the quotient between OvAg-driven and spontaneous proliferation (measured in wells with PBMC without a stimulus). The horizontal line inside the box plots depicts the median; the lower and upper end show the 25 and 75% percentile respectively. Error bars show the range. The P value denotes a significant difference in proliferation between the GEO and PI groups in response to OvAg but not to AsAg (analyzed by MannWhitney U-test).
|
|
High Ov-specific proliferation in the PI group is associated with elevated IL-5 production but not with a Th1-type response
After exposure to OvAg, PBMC from some PI individuals did produce measurable amounts of IFN-
compared to RPMI (Fig. 3A
), but the increase in the whole group was not significant (P < 0.2, Wilcoxon signed-rank test). Patients with GEO produced very little IFN-
(Fig. 3A
). When IL-5 production was looked at, PBMC from both groups displayed a significant increase after OvAg exposure (GEO P < 0.0001, PI P = 0.01, Wilcoxon signed-rank test, not shown). However, the extent of IL-5 production was significantly lower in the GEO compared to the PI group; the strongest response was seen in the PI group (GEO versus PI P < 0.001, analyzed by MannWhitney U-test; Fig. 3B
). OvAg-induced IL-4 was undetectable in either of the groups (not shown). It was also attempted to measure TGF-ß but all serum charges used for cell culture gave to high a background to detect antigen-specific production of this cytokine.
The immunosuppression is OvAg specific and not detectable upon Ascaris antigen
Individuals living in areas hyperendemic for onchocerciasis are usually co-infected with other parasites, in particular with intestinal nematodes. Proteins of these nematodes display a high degree of homology to Ov (19,20). In order to detect if the hyporeactivity of PBMC from GEO patients to OvAg is specific for Ov, PBMC were also stimulated with Ascaris antigen. Contrary to the differential response to OvAg by PBMC from GEO and PI groups (see Fig. 1A
), there was equivalent proliferation between GEO and PI PBMC in response to AsAg (Fig. 1B
) and AlAg (data not shown). Consistent with the proliferation data, AsAg-induced IL-5 production was equally enhanced in both GEO and PI groups (Fig. 3C
), in contrast to IL-5 production upon OvAg which was significantly lower in the GEO group (see above, Fig. 3B
).
IL-10 and TGF-ß in combination are involved in the hyporesponsiveness to OvAg in the GEO group
The results obtained in Figs 1 and 2
suggest that the hyporeactivity in the GEO group is mediated by the activity of down-regulatory cytokines. It has been reported that both IL-10 and TGF-ß can act to inhibit proliferation of immune cells (2123).
To examine the possibility that these cytokines inhibit T cell proliferation, PBMC were cultured with OvAg in the presence or absence of neutralizing antibodies to IL-10 or/and to TGF-ß. A slight but not significant elevation of proliferation was observed in PBMC from only a few GEO individuals after addition of either neutralizing antibody alone. However, the addition of both antibodies led to a significant increase in OvAg-specific T cell proliferation in the whole GEO patient group (P < 0.025, Wilcoxon signed-rank test), whereas no significant enhancing effect could be observed in the PI group (Fig. 4
). Addition of antibodies to IL-10 or/and to TGF-ß without OvAg did not induce proliferation (not shown), indicating that the enhancement of OvAg-driven proliferation in GEO patients is not an unspecific effect. These data suggest that IL-10 and TGF-ß, in patent onchocerciasis but not after exposure without infection (PI), act in concert to down-modulate proliferative cellular responses to this parasite.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4. Augmentation of OvAg-induced proliferation PBMC from GEO individuals in the presence of neutralizing antibodies to IL-10 and TGF-ß. PBMC (1x105) were stimulated for 4 days with OvAg (50 µg/ml) in the presence of control Ig (left GEO or PI plots) or with OvAg in the presence of neutralizing antibodies anti-IL-10 (10 µg/ml) and anti-TGF-ß (5 µg/ml). Proliferation was measured by BrdU incorporation and quantified using an ELISA. Optical density was read at 450 nm. After addition of both antibodies a significant increase in the Ov-specific T cell response was observed in the GEO group (P < 0.025, analyzed by Wilcoxon test).
|
|
Cloning of T cells with a regulatory cell (Th3/Tr1)-like phenotype from PBMC of a GEO donor
In search of the cellular source of IL-10 and TGF-ß production, 27 OvAg-specific TCC were obtained from a T cell line from a GEO individual. While all TCC proliferated strongly in response to OvAg, they could be subdivided into two groups on the basis of IL-2 production (one TCC of each group is shown in Fig. 5
). The first, `normal' type (nine of the 27 TCC for which 29H shown in Fig. 5
is representative) was characterized by strong production of IL-2 (0.53.5 ng/ml) but lacked IL-10 and TGF-ß. In contrast, the other type (18 of the 27 TCC) was characterized by high amounts of either IL-10 (three TCC, 1.23.5 ng/ml) or TGF-ß (11 TCC, 280930 pg/ml) or both (four TCC) and a lack of IL-2 production (42 B; Fig. 5B
); even in the presence of IL-2 receptor antibodies blocking endogenous consumption, this type of TCC did not secrete detectable amounts of IL-2 (not shown). Both types of TCC produced IL-5 (34 ng/ml) but little IFN-
(100200 pg/ml) and IL-4 (0200 pg/ml). While the first type of TCC would be most appropriately named Th0, this latter type of TCC has a cytokine profile very similar to TCC of the Th3 (14) or Tr1 (15). Cytokine production from two Tr1 and three Th0 clones was also measured upon Ov-specific stimulation; the TCC displayed the same cytokine pattern as with stimulation by anti-CD3/anti-CD28. Notably, TCC of the Th3/Tr1 type could not be cloned using PBMC from PI individuals, as also shown in an earlier study (7).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5. Existence of T cells with a Th3/Tr1-like cytokine profile in GEO. (A) Proliferation of OvAg-specific TCC 29H and 42B in response to OvAg (50 µg/ml). T cells (5x104) were cultured for 4 days together with and 1x105 -irradiated PBMC from an MHC-matched healthy European donor which were used as antigen-presenting cells. Proliferation was measured by thymidine incorporation and the stimulation index was calculated. (B) Production of cytokines IL-2 (white bars), IL-10 (black bars) and TGF-ß (hatched bars) by TCC in response to stimulation with anti-CD3/anti-CD28 antibodies. TCC 29H predominantly produced IL-2 and neither IL-10 not TGF-ß, whereas TCC 42B produced remarkable amounts of IL-10 and TGF-ß but no IL-2.
|
|
Collectively, these data suggest that in GEO individuals, T cells exist that are characterized by a preferential production of deactivating cytokines and by consumption of IL-2, indicating that increased numbers of the latter type at sites of infection would consume IL-2 produced by other T cells, at the same time providing the environment with deactivating cytokines IL-10 and TGF-ß.
 |
Discussion
|
---|
GEO is characterized by high mf loads with a calculated daily turnover rate of >50,000 (12). The host therefore must limit the extent of immune reaction in order not to impose serious damage to itself, i.e. it has to render itself tolerant to the many antigens displayed by dying mf. It was suggested that T cell tolerance, as part of the hyporesponsiveness seen in GEO, is not due to clonal deletion since after ivermectin treatment and reduction of mf loads, there was a recovery of the cellular response (10,11).
In the present study we analyzed mechanisms mediating this type of hyporesponsiveness: we show that the hyporesponsiveness in GEO is not related to an elevated but to a lowered Ov-specific production of the Th2 cytokine IL-5 (Fig. 3B
) when compared to PI individuals, i.e. those with high proliferative responses. In contrast, IL-10 and TGF-ß, two cytokines characteristic for a Th3 response, seem to be associated with hyporesponsiveness: in the GEO group the spontaneous IL-10 secretion was significantly elevated compared to PI and there was OvAg-induced IL-10 production (Fig. 2
); in addition, T cell proliferative hyporesponsiveness could be reversed by the simultaneous addition of anti-IL-10 and anti-TGF-ß antibodies in PBMC from patients with GEO (Fig. 4A
). Furthermore, the fact that hyporesponsiveness in GEO was observed to OvAg but not to the extract from the related nematodes As and Al (cf. Fig. 3B and C
), argues for an antigen-specific hyporesponsiveness regulated by T cells. In this regard, it was of importance that Ov-specific TCC could be generated from GEO PBMC (Fig. 5
) which have a cytokine profile similar to regulatory cells of the Th3 (14) or Tr1 (15) phenotypes, indicating that those cells are involved in the regulation of T cell proliferative hyporesponsiveness. To the best of our knowledge, this is the first time that human TCC of this regulatory type have been generated which are specific for an infectious agent.
In lymphatic filariasis, data from several reports suggest that T cell proliferative hyporesponsiveness is associated with a Th1 tolerance (for review, see 24). Individuals exposed to Brugia malayi and Wuchereria bancrofti in endemic areas but without patent disease (`endemic normals') were shown to display a high frequency of IFN-
-producing cells, but few IL-4 producers compared to individuals with disease (23,24). In a more recent study, in an area in which brugian filariasis is endemic, it was found that both IFN-
and IL-5 were suppressed in mf carriers compared to endemic normals, but that IL-4 was unabated (25).
The situation in onchocerciasis seems to be different with regard to IFN-
, given that several studies reveal that PI (the group comparable to endemic normals in lymphatic filariasis) in general do not show a Th1 pattern (79) and that PBMC from only a minority of PI do respond to OvAg with IFN-
production (5,7 and this study, Fig. 3A
). It is feasible that this difference between lymphatic filariasis and onchocerciasis reflects the need for the host to react differentially to filarial nematodes residing in different compartments of the body. On the other hand, in both lymphatic filariasis (25) and onchocerciasis (810 and this study), IL-5 apparently correlates with immunity. Thus, while immunity in lymphatic filariasis is apparently associated with parts of both a Th1 and a Th2 response, in onchocerciasis there is a correlation rather between IL-5 (a marker of a Th2 response) and PI.
Recently, it has become clear that Th2 responses are not as uniform as originally thought (26). It was shown, using IL-4 knockout mice, that IL-5 can be regulated independently from IL-4 (27,28). In human T cells, distinct stimulatory requirements were observed of the induction of IL-5 compared with IL-4 (29). Reminiscent to this, in the present study, PBMC from PI produced large amounts of IL-5 (Fig. 3B
) but did not show elevated IL-4 [a lack of measurable IL-4 was also described earlier (5)]. Also in chronic human schistosomiasis, IL-4 and IL-5 responses do not seem to be regulated in parallel (30). In addition, IL-10 production, in humans, is not a marker for Th2 cells but is also found in Th1 cells (31). OvAg-induced IL-5 and IL-10 production may thus easily diverge, as seen in Figs 2 and 3(B)
.
It has been attempted to overcome hyporesponsiveness in PBMC from individuals with several chronic helminth infections such as lymphatic filariasis (23,32) and schistosomiasis (33) by adding anti-IL-10 antibodies to the culture assays. Our data are in part consistent with these studies, since it was possible only with some (but not all) PBMC samples from GEO patients to raise proliferative responses by addition of anti-IL-10 alone (not shown). However, we show for the first time that hyporesponsiveness in the whole group of individuals with GEO is subject to reversal only if anti-TGF-ß antibodies are used simultaneously to anti-IL-10 antibodies (Fig. 4A
). The fact that it was necessary to block both down-regulatory cytokines at the same time may reflect the reported synergism between these two molecules (21,34) or it may be due to particular high amounts of IL-10 and TGF-ß produced by PBMC from GEO individuals, such that the blockade of only one cytokine would not have been sufficient.
T cell proliferation was still reduced after anti-IL-10 and anti-TGF-ß mAb treatment in PBMC of GEO patients, as compared to the PBMC of PI. This suggests that additional factors may be produced by PBMC of GEO patients which contribute to immunosuppression. We can, at present, not exclude an immunosuppressive activity of IL-13 or of IL-4; the latter, however, would have to be consumed since it was not measurable in the assays. Anti-IL-10- and anti-TGF-ß-mediated reversal in hyporesponsiveness (Fig. 4A
) was antigen specific in our study since there was no proliferation enhancement with addition of these two antibodies in the absence of OvAg. Interestingly, antigen-specific hyporesponsiveness is also suggested by immunoepidemiological data showing that Ov infection does not alter the Th response towards mycobacterial antigen (13). Similar data were obtained with B. malayi infection (35). From an evolutionary point of view, it is reasonable to assume that a long-lasting parasitic infection could not have been survived by the host species if it led to a more general immunosuppression.
Thus the question arises how a hyporesponsiveness specific for Ov antigens can be maintained. Over the years, basic immunology has made several attempts to define the phenomenon of T cell-mediated down-regulation of cellular responses (3638). Until recently one might have argued that also in onchocerciasis, this phenomenon is best explained by an immune deviation from Th1 to Th2. However, this is not corroborated by recent data from other reports (710) as well as from this study, showing that PI have increased IL-5 production.
Importantly, new CD4+ T cell subsets (Th3 and Tr1) different from Th0, Th1 or Th2 have been discovered recently, characterized at the level of TCC and shown to confer suppression of ongoing inflammation upon cellular transfer in mice (14,15). Clones with similar profiles have also been generated from human PBMC (15,39). A common feature of these subsets is that they produce TGF-ß and/or IL-10 but low to undetectable levels of IL-2 (14,15). Those cells have also been implicated in the development of infectious tolerance and their lack of IL-2 production has been interpreted as a property in common with anergic T cells (40). In the present study, T cells showing a similar cytokine profile (Fig. 5
) were cloned for the first time in an infectious disease. Although we could not formally prove that these cells are able to suppress immune responses, their production of IL-10 and TGF-ß, combined with a lack of IL-2 secretion, is suggestive of their inhibition of T cell proliferation, both by IL-2 consumption and IL-10 and TGF-ß delivery. Tr1 cells have been suggested to drive antigen-presenting cells to IL-10 production and thus to inhibit immune responses to third-party antigens (40). Theory would predict that for this to occur a large number of Tr1 cells must be present and, thus, that only in situations with high specific unresponsiveness, down-regulation of immune responses to third-party antigens will occur. It is possible that those mechanisms also operate in chronic helminth infection (41) in addition to a specific down-regulation.
Taken together, this study showed that hyporesponsiveness in onchocerciasis is mediated not by Th2 in general but by processes involving the action of IL-10 and TGF-ß. T cells similar to the recently discovered suppressive phenotypes may be involved in maintaining the antigen specificity of the hyporesponsiveness. It will be interesting in the future to identify the filarial antigens that induce those down-regulatory T cells. The chronicity of the course of infection in onchocerciasis makes this disease very well suited to study further the mechanisms of T cell tolerance in infection.
 |
Acknowledgments
|
---|
We thank all individuals from the Macenta region (Republic of Guinea) who declared their willingness to take part in this study, as well as Dr T. Kruppa and Dr E. Diekmann, then Macenta, Guinea, for help with logistics and patient examinations. We also thank M. Badusche for expert technical assistance. The work formed part of a doctoral study of J. S. at the Faculty of Biology, University of Hamburg, Germany. This study received financial support from the Deutsche Forschungsgemeinschaft (grant Ho 2009/1-1 to A. H.), the Edna McConnell Clark Foundation (to A. H.) and the German Academic Exchange Service (DAAD) (to J. S.).
 |
Abbreviations
|
---|
Al Ascaris lumbricoides |
AlAg A. lumbricoides antigen |
As Ascaris suum |
AsAg A. suum antigen |
DEC diethylcarbamazine |
GEO generalized onchocerciasis |
mf microfilariae |
Ov Onchocerca volvulus |
OvAg O. volvulus antigen |
PBMC peripheral blood mononuclear cells |
PHA phytohemagglutinin |
PI putatively immune (individuals) |
TCC T cell clone |
TGF transforming growth factor |
 |
Notes
|
---|
3 Present address: Max Planck Institut für Immunbiologie, 79108 Freiburg, Germany 
Transmitting editor: S. H. E. Kaufmann
Received 27 September 1999,
accepted 13 January 2000.
 |
References
|
---|
-
Gallin, M., Edmonds, K., Ellner, J. J., Erttmann, K. D., White, A. T., Newland, H. S., Taylor, H. R. and Greene, B. M. 1988. Cell-mediated immune response in human infection with Onchocerca volvulus. J. Immunol. 140:1999.[Abstract/Free Full Text]
-
Ward, D. J., Nutman, T. B., Zea-Flores, G., Portocarrero, C., Luhan, A. and Ottesen, E. A. 1988. Onchocerciasis and immunity in humans: enhanced T-cell responsiveness to parasite antigen in putatively immune individuals. J. Infect. Dis. 157:536.[ISI][Medline]
-
Elson, L. H., Guderian, R. H., Araujo N., E., Bradley, J. E., Days, A. and Nutman, T. B. 1994. Immunity to onchocerciasis: identification of a putative immune population in a hyperendemic area of Ecuador. J. Infect. Dis. 169:588.[ISI][Medline]
-
Soboslay, P. T., Lüder, C. G., Hoffmann, W. H., Michaelis, I., Helling, G., Heuschkel, C., Dreweck, C. M., Blanke, C. H., Pritze, S., Banla, M. and Schulz-Key, H. 1994. Ivermectin-facilitated immunity in onchocerciasis; activation of parasite-specific Th1-type responses with subclinical Onchocerca volvulus infection. Clin. Exp. Immunol. 96:238.[ISI][Medline]
-
Elson, L. H., Calvopina H., M., Paredes Y., W., Araujo N., E., Bradley, J. E., Guderian, R. H. and Nutman, T. B. 1995. Immunity to onchocerciasis: putative immune persons produce a Th1-like response to Onchocerca volvulus. J. Infect. Dis. 171:652.[ISI][Medline]
-
Lüder, C. G., Schulz-Key, H., Banla, M., Pritze, S. and Soboslay, P. T. 1996. Immunoregulation in onchocerciasis: predominance of Th1-type responsiveness to low molecular weight antigens of Onchocerca volvulus in exposed individuals without microfilaridermia and clinical disease. Clin. Exp. Immunol. 105:245.[ISI][Medline]
-
Doetze, A., Erttmann, K. D., Gallin, M. Y., Fleischer, B. and Hoerauf, A. 1997. Production of both IFN-
and IL-5 by Onchocerca volvulus S1 antigen specific CD4+ T cells from putatively immune individuals. Int. Immunol. 9:721.[Abstract]
-
Steel, C. and Nutman, T. B. 1993. Regulation of IL-5 in onchocerciasis. A critical role for IL-2. J. Immunol. 150:5511.[Abstract/Free Full Text]
-
Brattig, N., Nietz, C., Hounkpatin, S., Lucius, R., Seeber, F., Pichlmeier, U. and Pogonka, T. 1997. Differences in cytokine responses to Onchocerca volvulus extract and recombinant Ov33 and OvL3-1 proteins in exposed subjects with various parasitologic and clinical states. J. Infect. Dis. 176:838.[ISI][Medline]
-
Soboslay, P. T., Geiger, S. M., Weiss, N., Banla, M., Lüder, C. G., Dreweck, C. M., Batchassi, E., Boatin, B. A., Stadler, A. and Schulz-Key, H. 1997. The diverse expression of immunity in humans at distinct states of Onchocerca volvulus infection. Immunology 90:592.[ISI][Medline]
-
Soboslay, P. T., Dreweck, C. M., Hoffmann, W. H., Lüder, C. G., Heuschkel, C., Gorgen, H., Banla, M. and Schulz-Key, H. 1992. Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment. Clin. Exp. Immunol. 89:407.[ISI][Medline]
-
Duke, B. O. L. 1993. The population dynamics of Onchocerca volvulus in the human host. Trop. Med. Parasitol. 44:61.[ISI][Medline]
-
Cooper, P. J., Guderian, R. H., Nutman, T. B. and Taylor, D. W. 1997. Human infection with Onchocerca volvulus does not affect the T helper cell phenotype of the cellular immune response to mycobacterial antigen. Trans. R. Soc. Trop. Med. Hyg. 91:350.[ISI][Medline]
-
Weiner, H. L. 1997. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18:335.[ISI][Medline]
-
Groux, H., O'Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E. and Roncarolo, M. G. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[ISI][Medline]
-
Ottesen, E. A. 1995. Immune responsiveness and the pathogenesis of human onchocerciasis. J. Infect. Dis. 171:659.[ISI][Medline]
-
Meyer, C. G., Gallin, M., Erttmann, K. D., Brattig, N., Schnittger, L., Gelhaus, A., Tannich, E., Begovich, A. B., Erlich, H. A. and Horstmann, R. D. 1994. HLA-D alleles associated with generalized disease, localized disease, and putative immunity in Onchocerca volvulus infection. Proc. Natl Acad. Sci. USA 91:7515.[Abstract]
-
Brattig, N. W., Krawietz, I., Abakar, A. Z., Erttmann, K. D., Kruppa, T. F. and Massougbodji, A. 1994. Strong IgG isotypic antibody response in sowdah type onchocerciasis. J. Infect. Dis. 170:955.[ISI][Medline]
-
Chandrashekar, R., Masood, K., Alvarez, R. M., Ogunrinade, A. F., Lujan, R., Richards, F. O., Jr and Weil, G. J. 1991. Molecular cloning and characterization of recombinant parasite antigens for immunodiagnosis of onchocerciasis. J. Clin. Invest. 88:1460.[ISI][Medline]
-
Liebau, E., Walter, R. D. and Henkle-Dührsen, K. 1994. Onchocerca volvulus: isolation and sequence of a second glutathione S-transferase cDNA. Exp. Parasitol. 79:68.[ISI][Medline]
-
Bogdan, C. and Nathan, C. 1993. Modulation of macrophage function by transforming growth factor beta, interleukin-4, and interleukin-10. Ann. NY Acad. Sci. 685:713.[Abstract]
-
Letterio, J. J. and Roberts, A. B. 1998. Regulation of immune responses by TGF-beta. Annu. Rev. Immunol. 16:137.[ISI][Medline]
-
King, C. L., Mahanty, S., Kumaraswami, V., Abrams, J. S., Regunathan, J., Jayaraman, K., Ottesen, E. A. and Nutman, T. B. 1993. Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset. J. Clin. Invest. 92:1667.[ISI][Medline]
-
Maizels, R. M., Sartono, E., Kurniawan, A., Partono, F., Selkirk, M. E. and Yazdanbakhsh, M. 1995. T-cell activation and the balance of antibody isotypes in human lymphatic filariasis. Parasitol. Today 11:50.[ISI]
-
Sartono, E., Kruize, Y. C., Kurniawan, A., Maizels, R. M. and Yazdanbakhsh, M. 1997. Depression of antigen-specific interleukin-5 and interferon-gamma responses in human lymphatic filariasis as a function of clinical status and age. J. Infect. Dis. 175:1276.[ISI][Medline]
-
Mahanty, S., Abrams, J. S., King, C. L., Limaye, A. P. and Nutman, T. B. 1992. Parallel regulation of IL-4 and IL-5 in human helminth infections. J. Immunol. 148:3567.[Abstract/Free Full Text]
-
Johnson, E. H., Schynder-Candrian, S., Rajan, T. V., Nelson, F. K., Lustigman, S. and Abraham, D. 1998. Immune responses to third stage larvae of Onchocerca volvulus in interferon-gamma and interleukin-4 knockout mice. Parasite Immunol. 20:319.[ISI][Medline]
-
Hogarth, P. J., Taylor, M. J. and Bianco, A. E. 1998. IL-5-dependent immunity to microfilariae is independent of IL-4 in a mouse model of onchocerciasis. J. Immunol. 160:5436.[Abstract/Free Full Text]
-
Palmer, E. M. and van Seventer, G. A. 1997. Human T helper cell differentiation is regulated by the combined action of cytokines and accessory cell-dependent costimulatory signals. J. Immunol. 158:2654.[Abstract]
-
Grogan, J. L., Kremsner, P. G., Deelder, A. M. and Yazdanbakhsh, M. 1998. Antigen-specific proliferation and interferon-gamma and interleukin-5 production are down-regulated during Schistosoma haematobium infection. J. Infect. Dis. 177:1433.[ISI][Medline]
-
Mosmann, T. R. and Sad, S. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17:138.[ISI][Medline]
-
Mahanty, S., Ravichandran, M., Raman, U., Jayaraman, K., Kumaraswami, V. and Nutman, T. B. 1997. Regulation of parasite antigen-driven immune responses by interleukin-10 (IL-10) and IL-12 in lymphatic filariasis. Infect. Immun. 65:1742.[Abstract]
-
Sartono, E., Kruize, Y. C., Partono, F., Kurniawan, A., Maizels, R. M. and Yazdanbakhsh, M. 1995. Specific T cell unresponsiveness in human filariasis: diversity in underlying mechanisms. Parasite Immunol. 17:587.[ISI][Medline]
-
Oswald, I. P., Gazzinelli, R. T., Sher, A. and James, S. L. 1992. IL-10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity. J. Immunol. 148:3578.[Abstract/Free Full Text]
-
Sartono, E., Kruize, Y. C., Kurniawan, A., Maizels, R. M. and Yazdanbakhsh, M. 1996. In Th2-biased lymphatic filarial patients, responses to purified protein derivative of Mycobacterium tuberculosis remain Th1. Eur. J. Immunol. 26:501.[ISI][Medline]
-
Bloom, B. R., Salgame, P. and Diamond, B. 1992. Revisiting and revising suppressor T cells. Immunol. Today 13:131.[ISI][Medline]
-
Modlin, R. L. 1994. Th1Th2 paradigm: insights from leprosy. J. Invest. Dermatol. 102:828.[Abstract]
-
Röcken, M. and Shevach, E. M. 1996. Immune deviationthe third dimension of nondeletional T cell tolerance. Immunol. Rev. 149:175.[ISI][Medline]
-
Fukaura, H., Kent, S. C., Pietrusewicz, M. J., Khoury, S. J., Weiner, H. L. and Hafler, D. A. 1996. Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-beta1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J. Clin. Invest. 98:70.[Abstract/Free Full Text]
-
Cobbold, S. and Waldmann, H. 1998. Infectious tolerance. Curr. Opin. Immunol. 10:518.[ISI][Medline]
-
Cooper, P. J., Espinel, I., Paredes, W., Guderian, R. H. and Nutman, T. B. 1998. Impaired tetanus-specific cellular and humoral responses following tetanus vaccination in human onchocerciasis: a possible role for interleukin-10. J. Infect. Dis. 178:1133.[ISI][Medline]