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*
Department of Molecular Parasitology and
Institute of Medical Immunology, Charite Medical School, Humboldt University, Berlin, Germany;
Division of Clinical Chemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany;
Institute of Biochemistry, University of Jena, Jena, Germany; and
¶ Department of Biological Sciences, University of Salford, Manchester, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. At the same time, rOv17 reduced the expression of HLA-DR
proteins and of the costimulatory molecule CD86 on human monocytes.
Neutralization of IL-10 by specific Abs restored the expression of
HLA-DR and CD86, whereas the proliferative block remained unaffected.
Depletion of monocytes from the PBMC reversed the rOv17-induced
cellular hyporeactivity, indicating monocytes to be the target cells of
immunomodulation. Therefore, onchocystatin has the potential to
contribute to a state of cellular hyporesponsiveness and is a possible
pathogenicity factor essential for the persistence of O.
volvulus within its human host. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20 million
persons in tropical regions of Africa and Latin America. The parasites
persist in their hosts for 10 years or longer (1, 2), and
in the majority of infected persons onchocerciasis is associated with
markedly reduced cellular immune reactions and polarization of T cell
responses toward a Th2 and Th3 type (3, 4, 5). It is believed
that these alterations are a key element in the persistence of the
worms within their hosts. The down-regulation of inflammatory host
immune reactions is suggested to be induced by secreted products of the
nematodes (6, 7). Despite the probable importance of this immunomodulation for the balance of the host-parasite relationships, there is little information on pathogenicity factors accounting for it. Several studies demonstrated that phosphorylcholine, a lipid component bound to proteins of filariae, inhibits murine B and T cell proliferative responses by interference with signaling cascades leading to cellular activation (8). Phosphorylcholine was also shown to increase the IL-10 production of B1 cells (9) and could thus contribute to the polarization of Th cell responses. Homologues of various cytokines, such as macrophage migration-inhibitory factor (10) or tgh-2 (11, 12) released by the filarial nematode Brugia malayi, appears to mimic host cytokines and to interfere with the host’s cytokine network. Furthermore, a secreted protein, the cysteine protease inhibitor (cystatin) of the rodent filaria Acanthocheilonema viteae,was shown to down-regulate proliferative T cell responses and to up-regulate the production of IL-10 by splenocytes in vitro by hitherto unknown mechanisms (13). In addition, B. malayi cystatin inhibits the Ag presentation of human B lymphocyte lines (14). These examples show that nematode components have the potential to interfere with regulatory host immune responses very much like certain pathogenicity factors described from bacterial and protozoan infections (15, 16).
Filarial cystatin of O. volvulus (onchocystatin) was described by Lustigman et al. (17, 18), who suggested a role in the regulation of parasite cysteine proteases during molting or hatching of the worms. Such an activity is compatible with the described function of cystatins, which are tight-binding natural protease inhibitors. They represent important regulators of proteolytic processes catalyzed by cysteine proteases (19). Among others, the cystatins have important functions in the regulation of proteases relevant in immune responses. Thus, the endogenous inhibitor cystatin C influences the fate of newly synthesized peptide-MHC class II complexes by regulating the activity of cathepsin S, a cysteine protease that is essential in Ii-chain degradation in B cells and dendritic cells (20, 21, 22). In addition, human cystatin C has been described to inhibit the phagocytic function of monocytes and granulocytes (23, 24), and it has been shown that various members of the cystatin superfamily up-regulate the inducible NO production of murine macrophages (25).
These functions of cystatins imply that the role of filarial cystatin is not necessarily restricted to the molting process of the nematodes. This is also underlined by the fact that A. viteae cystatin is secreted by nematode stages that do not molt, i.e., the male and female worms and blood microfilariae (13). Indeed, our initial data, obtained in a murine spleen cell model system, show A. viteae cystatin to have several immunomodulatory activities, e.g., up-regulation of IL-10 and of NO production by murine spleen cells (13).3 These data encouraged us to study the immunomodulatory role of filarial cystatin in a homologous system. Using a combination of human peripheral blood cells and recombinant onchocystatin, we could establish that filarial cystatin is a potential pathogenicity factor of O. volvulus in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cDNA of the mature 17-kDa onchocystatin
(Ov17),4 was cloned by
PCR from a 
Uni-ZAP XR cDNA library of infective larvae of O.
volvulus (provided by S. Williams, Smith College, Northampton,
MA). The cDNA was amplified by PCR with degenerate primers derived from
the internal sequence of onchocystatin. Forward primer,
gttcagttgcaaggagcc; reverse primer, tcatacttcttttgttccc. PCR
amplification yielded a 423-bp fragment which was cloned into a
T-overhang vector (pGEM-T Easy Vector Systems; Promega, Madison, WI).
The recombinant protein was termed rOv17. The cDNA was subcloned into
the EcoRI site of an expression vector yielding polypeptides
with a six-histidine tag (pET System; Novagen, Madison, WI). The
recombinant plasmid was transformed into BL21 (DE3) or BL21 (DE3)
pLysS-competent Escherichia coli and screened for expression
by SDS-PAGE. rOv17 was purified under nondenaturing conditions by
affinity chromatography using Ni-NTA resin (Qiagen, Hilden, Germany)
and dialyzed twice against PBS. The control protein, recombinant 33-kDa
onchocystatin (rOv33), an O. volvulus protein with
homologies to aspartic protease inhibitors (26), was
expressed and purified under identical conditions.
Because rOv17 and rOv33 were expressed in E. coli, the endotoxin contamination was evaluated by quantitative, chromogenic Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). The endotoxin concentration of rOv17 was determined to be 0.04–0.13 pg LPS/0.01 µM protein. The control rOv33 showed endotoxin concentrations of 1.0–2.0 pg/0.01 µM protein.
Human cathepsin inhibition assays
The inhibitory activity of rOv17 toward human cathepsins was characterized by Ki value determination. Human cathepsin B was purchased from Sigma (Deisenhofen, Germany), human cathepsin L was purchased from Calbiochem (Schwalbach, Germany), and human cathepsin S was provided by Dr. Wiederanders (Institute of Biochemistry, University of Jena, Jena, Germany). Ki values were determined by measuring the activity of human cathepsin B (1.2 nM), cathepsin L (0.1 nM), or cathepsin S (0.1 nM) in the presence of various concentrations of rOv17 using the fluorogenic substrates Z-Arg-Arg-AMC (40 µM; Bachem, Heidelberg, Germany) for cathepsin B, z-Phe-Arg-AMC (5 µM; Bachem) for cathepsin L, and Z-Val-Val-Arg-AMC (40 µM; Bachem) for cathepsin S (27, 28). The Ki values were calculated using the program GraphPadPrism.
Quantification of secreted Ov17 by ELISA
Five female O. volvulus worms isolated from patients suffering from Onchocerca volvulus infections were cultured individually for 96 h in 1 ml RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. The culture supernatants were harvested, and fresh medium was added every 24 h. ELISA plates were coated with 50 µl of the culture supernatant of a 24-h culture, mixed with 25 µl 0.1 M carbonate buffer (pH 9.5) and in parallel with graded concentrations of rOv17. A 1/5000 dilution of a monospecific rabbit serum against rOv17 was reacted with the proteins. The amount of bound Abs was quantified with a peroxidase-labeled goat anti-rabbit Ab diluted 1/10,000 (Dianova, Hamburg, Germany). Values obtained with the preimmune serum were subtracted.
Ag-driven and polyclonally stimulated proliferation of human PBMC
The peripheral mononuclear cell fraction of healthy donors was
isolated from citrated venous blood by density gradient sedimentation
using Ficoll-Hypaque (Pharmacia Biotech Products, Freiburg, Germany).
Cells were resuspended in very low endotoxin RPMI 1640 (Biochrom,
Berlin, Germany), supplemented with 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM glutamine, and 10% FCS (Biochrom). PBMC (3.5
x 105/well) were cultured in 96-well flat-bottom
plates at 37°C and stimulated with 10 IU/ml purified protein
derivative (PPD; Chiron Behring, Marburg, Germany), 10 µg/ml PHA
(Sigma), or immobilized anti-CD3 Ab (4 µg/ml in a final volume of
50 µl for 2 h; Orthoclone-Okt3; gift from Janssen-Cliag, Neuss,
Germany). Recombinant proteins were added at a concentration of
0.1–0.5 µM. Additionally, human PBMC were cultured with LPS (100
pg/ml; E.coli 0127:B8; Sigma) for 72 h. PPD-stimulated
PBMC were cultured for 96 h, and polyclonally stimulated PBMC were
cultured for 72 h at 37°C. Proliferation was quantified by
[3H]thymidine incorporation during the last
20 h of incubation. For neutralization studies, anti-rOv17
rabbit immune serum (1 µl/well), a rabbit control serum against
sporozoites of Eimeria tenella (1 µl/well), anti-IL-10
Ab (5 µg/ml, clone CB/RS/1; gift from Prof. Dr. H. D. Volk,
Charité Medical School, Berlin, Germany), anti-TNF- Ab (5
µg/ml; clone 1825.121; R&D Systems, Wiesbaden-Norderstadt, Germany),
and a mouse IgG1 isotype control Ab (5 µg/ml; R&D Systems) were used.
All experiments were performed in triplicate. Viability of the cells in
presence of recombinant proteins were controlled by trypan blue
exclusion.
Depletion of monocytes (CD14+ cells) and isolation of purified monocytes from human PBMC
CD14+ cells were depleted from human PBMC
using CD14 MicroBeads and VS+ columns (Miltenyi
Biotec, Bergisch Gladbach, Germany). Depletion of
CD14+ cells was done according to the
manufacturers’ instructions and resulted in the enrichment of
CD14- cells of 98–99% as determined by FACS
analysis using FITC-labeled anti-human CD14 Ab (10 µg/ml; UCHM-1;
Dianova). Furthermore, CD14+ depletion
was estimated by measuring TNF- production.
CD14+-depleted cells were stimulated with LPS (5
ng/ml), and TNF- was quantified after 6 h in the culture
supernatant. CD14+-depleted cells produced on the
average 92 pg/ml TNF- in comparison with 2308 pg/ml TNF- produced
by nondepleted PBMC.
Purified monocytes were isolated from MNC by counterflow centrifugation using a Beckmann JE-6B elutriator (Beckmann Instuments, Munich, Germany). The monocyte fraction collected consisted of <95% monocytes, as determined by modified Randolph staining and by FACS analysis after staining with anti-CD14 Ab (10 µg/ml; Dianova).
Quantification of cytokines in the cell culture supernatant
Cytokine production was determined in the culture supernatants
of unstimulated and stimulated PBMC as well as in purified monocyte
cultures cocultured with 0.5 µM recombinant proteins. Culture
supernatants were collected after 6, 24, and 48 h of incubation.
Cytokines were quantified by ELISA according to the manufacturer’s
instructions (TNF-, IL-2, IL-12p40: OptEIA; BD PharMingen, Hamburg,
Germany; IL-10 and IFN-
: DuoSet, R&D Systems). The assays were
performed in triplicates.
Expression of monocyte surface molecules
Unstimulated human PBMC (8 x 105/well) were cultured for 72 h at 37°C in 48-well flat-bottom plates with different concentrations of rOv17 and rOv33 (10–100 nM). The expression of monocyte surface molecules was determined in a final volume of 200 µl using the following Abs for staining: R-PE -labeled anti-human HLA-DR mAb (0.48 µg/ml; clone L243; Becton Dickinson, Heidelberg, Germany); FITC-labeled anti-human CD40 mAb (1 µg/ml; clone 5C3; PharMingen); FITC-labeled anti-human CD80 mAb (1 µg/ml; clone BB1; PharMingen); and PE-labeled anti-human CD86 mAb (4 µg/ml; clone 2331; PharMingen). FITC-labeled mouse IgG1 mAb (1.5 µg/ml; clone 679.1 Mc7; Coulter Immunotech, Hamburg, Germany) and PE-labeled mouse IgG2a mAb (4 µg/ml; clone X 39; Becton Dickinson) were used as isotype controls.
FACS analyses of 3 x 104 PBMC/measurement were done by using a FACScan instrument (Becton Dickinson). The monocyte population was identified by its light scatter properties and in addition by staining of CD14 with PE-cyanin 5-labeled anti-human CD14 mAb (0.1 µg/ml; clone RMO52; Coulter Immunotech).
Statistical analysis
Statistical analysis of T cell proliferation data and expression of the cell surface molecules were performed with the Student t test. Cytokine data were analyzed with the Wilcoxon test. Data were presented as means ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the immunomodulatory potential of onchocystatin, a full length mature recombinant protein (rOv17) with an N-terminal six-histidine tag was produced in E. coli. Affinity-purified rOv17 was tested for its capacity to inhibit the activity of the human cysteine proteases cathepsins B, L, and S. These proteases are involved in the proteolytic processing of Ag and in Ii chain degradation (29, 30). Ki determinations showed that rOv17 strongly inhibited the activity of human cathepsin L (Ki = 0.038 nM) and cathepsin S (Ki = 0.033 nM), whereas the activity of human cathepsin B was less efficiently inhibited by rOv17 (Ki = 494 nM). Therefore, onchocystatin is an inhibitor of immunologically relevant human cysteine proteases. Consequently, the inhibitor could theoretically interfere with Ag processing and Ii chain degradation of APC. The inhibitory domains of the parasite protein are likely to correspond to the structures of human cystatin; as a sequence alignment shows the typical evolutionary conserved motifs of cysteine protease inhibitors, which bind and inhibit the protease. The N-terminal inhibitory domain reaches from aa 51–53, the central domain from aa 98–102, and the C-terminal domain from aa 153–154. In contrast, most of the other regions vary considerably between onchocystatin and human cystatins, such that the overall amino acid identity of the proteins was comparatively low with values between 18 and 22%. As a particular element, onchocystatin contains a stretch of amino acids (aa 37–48) which is lacking in the human cystatins. This stretch does not show any homologies to known sequences.
Quantification of Ov17 secretion by female O. volvulus worms
The cDNA of onchocystatin encodes for a hydrophobic leader sequence which is typical for secreted proteins. Therefore, we evaluated whether onchocystatin is secreted by O. volvulus worms in vitro. To this end, the quantity of onchocystatin in the culture supernatants of intact individual female worms was determined by a calibrated ELISA. A monospecific rabbit serum raised against recombinant Ov17 detected a release of 10–40 ng onchocystatin per female worm in 24 h, suggesting that onchocystatin has the potential to interfere with the host immune system in vivo.
Inhibition of polyclonally stimulated and Ag-driven proliferation of human PBMC by rOv17
As one model for polyclonally stimulated T cell responses, we
studied the effect of rOv17 on the PHA-induced T cell proliferation.
rOv17 inhibited the cellular proliferation in a dose-dependent manner
(12–42% inhibition with concentrations between 0.1 and 0.5 µM
rOv17), whereas the control protein rOv33 had significantly less
activity. The most effective concentration of 0.5 µM was subsequently
used to analyze the antiproliferative activity of rOv17 toward PBMC of
10 healthy blood donors. In this concentration, rOv17 inhibited the
proliferative response of human PBMC by 39% (p
> 0.001; Fig. 1
a), whereas
the control protein rOv33 induced only a slight inhibition of
proliferation (difference between rOv17 and rOv33, p >
0.001). In a further set of experiments, we used immobilized
anti-CD3 Ab to stimulate PBMC of 10 blood donors. Again, 0.5 µM
rOv17 resulted in a mean inhibition of the anti-CD3 Ab-stimulated
proliferation of human PBMC by 42% (p = 0.002;
Fig. 1b), whereas the control protein rOv33 had a
significantly weaker effect. The specificity of the inhibitory effect
of rOv17 was determined using monospecific rabbit Abs against rOv17.
Addition of rabbit anti-Ov17 serum to the cultures completely
abolished the rOv17-induced inhibition of PHA-stimulated proliferation,
whereas a rabbit control serum had no effect (Fig. 1c).
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d). In contrast, an 0.5 µM concentration of the
control protein rOv33 did not inhibit the PPD-induced Ag-driven T cell
proliferation. The inhibition of both Ag-driven and polyclonally stimulated T cell proliferative responses suggests that rOv17 interferes with an immune mechanism that is involved in both types of cellular proliferation.
Cytokine production of unstimulated and stimulated PBMC in presence of rOv17
To test whether the rOv17-induced suppression of T cell
proliferation is due to cytokines produced by PBMC, we quantified
cytokines that mediate activation and regulation of T cell
proliferation, such as IL-2, IL-4, TNF-, IFN-, IL-10, and
IL-12p40. The amounts of these cytokines in the presence or absence of
rOv17 were determined in the culture supernatants of
anti-CD3-stimulated PBMC of 10 donors after 48 h. The tests
revealed that rOv17 induced a significant increase in IL-10 production
(p = 0.002) and a significant decrease in
IL-12p40 production (p = 0.025) as well as a
diminished IFN- production which was, however, not significant (Fig. 2). The control protein rOv33 had
significantly weaker effects on these cytokines. The production of
IL-2, IL-4, and TNF- by stimulated PBMC was not significantly
affected by rOv17 or rOv33 (data not shown).
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in the
presence or absence of rOv17 were determined in the culture
supernatants of unstimulated PBMC of five individuals after 6, 24, and
48 h. The tests revealed that onchocystatin induced an initial
increase of the proinflammatory cytokine TNF- at 6 h
(p > 0.001). This peak in TNF- production
was followed by a significant increase of the inhibitory cytokine IL-10
(p > 0.001) at 24 h (Fig. 3, a and b) which
was also elevated at 48 h. The effect of the control protein rOv33
on both cytokines was significantly lower (Fig. 3). No significant
production of IL-12p40 was observed by unstimulated PBMC by rOv17 (data
not shown). Thus, the increase of the IL-10 production was the most
prominent effect in the cytokine pattern of stimulated and of
unstimulated human PBMC exposed to rOv17.
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Expression of monocyte surface molecules in presence of rOv17
Both the Ag-driven and the polyclonally stimulated proliferations
of PBMC require costimulatory signals for full T cell activation.
Because important costimulatory molecules are expressed by
monocytes, we examined the influence of various concentrations of
rOv17 on the expression of the costimulatory surface markers of
monocytes CD40, CD80, and CD86. The analysis of CD86 on human monocytes
of PBMC of four blood donors revealed that rOv17 reduced the expression
of CD86 by 37% (p = 0.018) at a concentration
of 10 nM (Fig. 4a). An
increase in the rOv17 concentration did not significantly enhance this
effect. The control protein rOv33 did not significantly affect the CD86
expression (Fig. 4a). The expression of the two other
surface markers, CD40 and CD80, which were poorly expressed on
monocytes, remained unchanged in the presence of rOv17 (data not
shown).
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b). In contrast, the control protein rOv33 did
not affect HLA-DR expression (Fig. 4b). Similar to the
experiments with PBMC, rOv17 reduced the expression of HLA-DR and, less
pronounced, of CD86 on purified monocytes (median reduction, 66% for
HLA-DR and 40% for CD86).
Neutralization studies using anti-IL-10 and TNF- Abs
IL-10 exerts suppressive and deactivating effects on T cells and
macrophages, alters the pattern of cytokine production of T cells, and
induces changes in the expression of monocyte surface molecules
(31, 32, 33).To test the role of IL-10 for the observed
alterations, we exposed PBMC to rOv17 and neutralized IL-10 by
addition of anti-IL-10 Abs. The experiment revealed that
addition of anti-IL-10 Abs did not restore the inhibition of
anti-CD3-induced proliferation of PBMC (n = 4; Fig. 5). This suggests that IL-10 is not a
major component of the rOv17-induced inhibition of T cell
proliferation.
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a). In addition,
neutralization of IL-10 restored the diminished expression of HLA-DR on
monocytes (n = 3) to 72–93% of the control levels
(Fig. 6b). Therefore, IL-10 is responsible for the
down-regulated expression of CD86 and HLA-DR on human monocytes in this
scenario.
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may stimulate T cells and monocytes, triggering among
others the production of IL-10 (34), this proinflammatory
cytokine could contribute to the effects exerted by rOv17.
Neutralization of TNF- by addition of
anti-TNF- Abs did not restore the diminished cellular
proliferation (data not shown). However, addition of anti-TNF-
Abs led to a reduction by 31.2% of the rOv17-induced IL-10 production
of unstimulated human PBMC (data not shown), showing that TNF-
accounts for a part of the increase in IL-10. Therefore, one could
expect that this cytokine indirectly acts on the levels of CD86 and
HLA-DR.
Together, these data suggest that the proliferative block induced by
cystatin acts independently of TNF- and IL-10, whereas the
expression of CD86 and HLA-DR is influenced by IL-10.
Determination of the target cell of onchocystatin within the PBMC
The fact that purified monocytes are IL-10 producers in the
presence of rOv17 as well as the observed changes in monocyte surface
molecules induced by rOv17 suggested that these cells are involved in
the rOv17-induced immunomodulation. To study their role, PBMC depleted
of monocytes and complete PBMC were compared with regard to
the rOv17-induced inhibition of proliferation. Monocytes were depleted
from PBMC by CD14 MicroBeads, resulting in a reduction of
CD14+ cells to 1–2% of the total PBMC
population. Monocyte-depleted PBMC (n = 4) were
normalized for the number of lymphocytes, stimulated with PHA, and
cocultured with rOv17 and the control protein rOv33, respectively. The
PHA-stimulated proliferation of complete PBMC were inhibited by 37% in
the presence of 0.5 µM rOv17, whereas the proliferation of
monocyte-depleted PBMC was not suppressed in presence of rOv17 (Fig. 7). The control protein rOv33 had a
slight, but significantly weaker inhibitory effect on unmanipulated
PBMC, which was completely abolished in monocyte-depleted PBMC
cultures. These data show that the inhibition of proliferation of
PHA-stimulated human PBMC induced by rOv17 is mediated by
monocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Filarial parasites persist within their vertebrate hosts for extended time spans, e. g. up to 14 years as shown for O. volvulus (2). This survival within an immunocompetent host might be due to a modulation of the host’s immune system exerted by excretory/secretory (E/S) products of the parasites. Indeed, E/S products of filariae suppressed proliferative lymphocyte responses (6, 8, 13, 36), but despite the possible importance for the understanding of the pathogenicity mechanisms only few components of E/S products were thus far characterized on the molecular level. One of these components is the cystatin protein of the filaria A. viteae, an E/S Ag that inhibits proliferative responses and induces changes of the cytokine pattern of murine spleen cells, as shown in an earlier publication (13).
To establish the relevance of this possible pathogenicity factor for a human pathogenic filarial infection and to enlarge our knowledge of the underlying mechanisms, we characterized the immunomodulatory potential of onchocystatin using a recombinant, biologically active cystatin (rOv17) which comprised the full sequence of the mature inhibitor. Models of the protease-inhibitor interaction suggest that three conserved domains of the cystatins mediate the inhibition of papain-like cysteine proteases (37). The N-terminal conserved inhibitory domain has been shown to have a predominant role because it blocks the active site cleft of the protease (38, 39, 40), whereas the inhibitory potential of the two other domains is dependent on the respective protease (41, 42). Given that onchocystatin contains all three conserved domains, it is not surprising that rOv17 strongly inhibits human cysteine proteases like cathepsins S and L. These cathepsins have essential functions in the processing of Ags and in Ag presentation. Cathepsin L degrades Ag, and it mediates the final proteolytic steps in Ii chain degradation in thymus epithelial cells (29, 43). Cathepsin S processes Ag and is essential in the completion of Ii chain degradation in dendritic cells and B cells (20, 22) Therefore, it is tempting to speculate that onchocystatin acts through inhibition of immunologically relevant host proteases. Fluorescein-labeled rOv17 is taken up by macrophages (our unpublished data) and could thus reach the cellular compartments where Ag processing and the subsequent steps of Ag presentation by MHC II molecules take place.
The strong inhibition of the Ag-driven T cell proliferation suggests that the target molecule in this situation is a cysteine protease with a role in immune processes. Indeed, synthetic as well as microbial cysteine protease inhibitors have been shown to modulate Ag-specific cellular responses. Katunuma et al. (44) describe a marked suppression of proliferation of primed splenocytes to hepatitis B virus Ag in the presence of two cathepsin B inhibitors, E64 and CA-074. Furthermore, another study by Vidard et al. (45) demonstrates that the cysteine protease inhibitor leupeptin influences the generation of immunogenic OVA peptides, which leads to modulation of immune responses. The target proteases of the above mentioned cysteine protease inhibitors remain to be determined. Moreover, it has been shown that cysteine proteases are responsible for invariant chain degradation in APCs and that the ratio of cystatin C to these cathepsins determines the fate of newly synthesized peptide-MHC class II molecules as well as the differentiation of Th cell subclasses (21, 43). Therefore, cysteine protease inhibitors have the capacity to modulate immune responses, and filarial parasites may use this mechanism to interfere with immune responses of their hosts.
However, our data show that onchocystatin down-regulates not only the Ag-driven cellular proliferation but also the polyclonally induced T cell proliferation. Using PHA and anti-CD3 Ab as stimuli of human PBMC, we showed that onchocystatin also inhibits T cell proliferation independent of Ag processing and presentation. Polyclonally induced and Ag-driven T cell proliferation have in common that they require cytokines and coreceptors for full T cell activation (46). Our results indicate that the cellular hyporeactivity induced by onchocystatin is accompanied by a modulation of the cytokine production and by alterations of the expression of monocyte surface molecules. One main characteristic was the up-regulation of the IL-10 production of PBMC in the presence of rOv17.
IL-10 is a cytokine that shows immunosuppressive effects due to its
ability to suppress the production of IL-12 and IFN-, to
down-regulate MHC class II expression, and to reduce the expression of
the adhesion molecule CD54 (ICAM-1) as well as the costimulatory
molecules CD80 and CD86 on APC (31, 32, 33). As a consequence,
IL-10 causes a reduced T cell proliferation. Our data support some of
the described effects of IL-10, in that neutralization of IL-10 by an
anti-IL-10 Ab reversed the production of IFN- by PBMC and
increased the expression of CD86 and HLA-DR by monocytes. High levels
of IL-10 production by Ag-stimulated or by unstimulated PBMC have also
been described in lymphatic filariasis. Mahanty et al.
(47) demonstrated increased IL-10 values that coincided
with T cell hyporeactivity in lymphatic filariasis patients. Similarly,
experimental studies in mice infected with Brugia
pahangi revealed that parasite-reactive Th1 cells are suppressed
in vivo by a mechanism that involves IL-10 and the resident APC
(48). Another study investigating cellular hyporeactivity
of O. volvulus-infected people has shown that both IL-10 and
TGF-
mediate cellular hyporeactivity (5). An
up-regulation of IL-10 coincident with cellular hyporesponsiveness has
also been described for urinary schistosomiasis (49) as
well as for Toxoplasma gondii infection in mice
(50). Thus, the observed induction of IL-10 by
onchocystatin could be an essential feature in the host-parasite
interaction, the increased levels of IL-10 leading among others to a
polarization of host T cell responses toward Th2/Th3 response, which is
characteristic for filarial infections (5, 51, 52).
However, in our experimental set-up, IL-10 does clearly not account for
the inhibition of proliferative PBMC responses, because neutralization
of IL-10 did not abrogate the inhibition of proliferative responses.
Other, IL-10-independent mechanisms, like the contact-dependent
macrophage-mediated cell cycle arrest exerted by filarial parasites
(53), could account for this effect.
Furthermore, this study determines that the target cells of rOv17-induced down-regulation of proliferation are monocytes. Depletion of monocytes from the PBMC reversed the inhibitory effects on the cellular proliferation induced by onchocystatin. Recently, macrophages were shown to be important regulatory cells by which filariae inhibit cellular proliferation (53). Our study is in line with these findings. In addition, this report characterizes for the first time a single filarial protein that is responsible for the modulation of macrophage functions observed in infections with filariae. Modulation of monocyte functions is not restricted to filarial cystatin but seems to be an intrinsic feature of these proteins. Leung-Tack et al. (24) reports that human cystatin C, a protein constitutively secreted by mononuclear phagocytes (54, 55), interferes with the phagocytic function and the oxidative burst of monocytes via a bioactive peptide that is released during the interaction of the inhibitor with a cysteine protease. Furthermore, it has been shown that members of the three subgroups of the cystatin superfamily up-regulate the inducible NO production of murine macrophages (25). It is interesting that NO is a potent inhibitor of murine T cell proliferation (56). Moreover, it has been shown that cystatin C limits acute inflammatory reactions due to cysteine proteases released during inflammation. This is compatible with the fact that cystatin C is down-regulated in chronic inflammatory reactions that are dominated by mononuclear phagocytes and lead to tissue pathology (54). Therefore, cystatins can be considered regulatory proteins, which can modulate monocyte activities in addition to its protease-inhibitory function. From this perspective, it is conceivable that filariae interfere with regulatory functions of host macrophages by releasing cystatin.
The receptor on the target cell by which rOv17 mediates the
immunomodulations is thus far unclear. The early increase of TNF-
with a subsequent decrease and rising of the IL-10 production by PBMC
after contact with rOv17, as well as the modulation of monocyte surface
molecules, is reminiscent to the reaction of cells to bacterial
endotoxins LPS (57, 58). Our controls showed that the
observed effects are not due to LPS, because the rOv33 control protein,
which contained higher levels of LPS than rOv17, did not induce similar
effects. In addition, 10-fold higher quantities of free LPS than
contained in rOv17 preparations did not induce the changes observed for
rOv17. Because it is not LPS that induces an early TNF- and a
subsequent IL-10 response, we hypothesize that exposure of PBMC to
rOv17 has similar effects. rOv17 possibly acts on receptors or signal
pathways, which are also targeted by LPS and which would represent a
possible parallel between antibacterial immune responses and immune
responses to a helminth parasite. Toll-like receptors (TLR), especially
TLR4, are reported to mediate signal transduction by LPS
(59). Interestingly, the TLR4 complex was recently shown
to mediate signaling of a protein, heat shock protein 60. Heat shock
protein 60 induces murine macrophages to produce TNF- and NO via
TLR4 (60). Therefore, it is tempting to speculate that
proteins like onchocystatin might be ligands of a TLR as well.
Our data demonstrate that onchocystatin is a potent immunomodulator of
human PBMC, which targets monocytes and acts through different
mechanisms. It is conceivable that the inhibition of immunologically
relevant target proteases leads to changes of Ag processing and
presentation. In addition, the increased production of IL-10 (partly
mediated through induction of TNF-) could lead to changes in the
cross-regulation of T cell responses, with a resulting predominance of
Th2/Th3-like responses. Furthermore, the inhibition of T
cell-proliferative responses by rOv17, which is independent of IL-10,
could account for the cellular hyporesponsiveness observed in filarial
infections. It is likely that other mechanisms, e.g., the release of
phosphorylcholine-containing proteins (8) and of cytokine
homologues (10, 11), contribute to immunomodulation
exerted by filarial E/S products. Further studies will have to
determine the in vivo role of these molecules and focus on the
identification of the cell ligand and the molecular mechanisms of
immunomodulation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Susanne Hartmann, Department of Molecular Parasitology, Institute of Biology, Philippstrasse 13, 10115 Berlin, Germany. E-mail address: susanne.hartmann{at}rz.hu-berlin.de
3 S. Hartmann, A. Schönemeyer, B. Sonnenburg, B. Vray, and R. Lucius. 2001. Cystatins of filarial nematodes up-regulate the nitric oxide production of IFN--activated murine macrophages through the production of TNF- and IL-10. Submitted for publication.
4 Abbreviations used in this paper: Ov17, 17-kDa onchocystatin; rOv17, recombinant onchocystatin; Ov33, 33-kDa O. volvulus protein; rOV33, recombinant Ov33; PPD, purified protein derivative; E/S, excretory/secretory; TLR, Toll-like receptors.
Received for publication May 8, 2001. Accepted for publication July 24, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but not by a Th1 to Th2 shift. Int. Immunol. 12:623. (TGF-) superfamily from the filarial nematodes Brugia malayi and B. pahangi. Exp. Parasitol. 88:200.[Medline]
, expressed in microfilarial and adult stages of Brugia malayi. Infect. Immun. 68:6402.-activated mouse peritoneal macrophages via cytokine synthesis. Eur. J. Biochem. 266:1111.[Medline]
-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041. and cAMP elevating drugs. Int. Immunol. 7:517.-trace) by monocytes and macrophages and its downregulation after stimulation. J. Exp. Med. 166:1912. and interleukin-10 expression. Eur. J. Immunol. 26:1580.[Medline]
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