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Modulation of Human T Cell Responses and Macrophage Functions by Onchocystatin, a Secreted Protein of the Filarial Nematode Onchocerca volvulus This information is current as of September 27, 2021. Annett Schönemeyer, Richard Lucius, Bettina Sonnenburg, Norbert Brattig, Robert Sabat, Klaus Schilling, Janette Bradley and Susanne Hartmann J Immunol 2001; 167:3207-3215; ;

doi: 10.4049/jimmunol.167.6.3207 Downloaded from http://www.jimmunol.org/content/167/6/3207

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Modulation of Human T Cell Responses and Macrophage Functions by Onchocystatin, a Secreted Protein of the Filarial Nematode Onchocerca volvulus1

Annett Scho¨nemeyer,* Richard Lucius,* Bettina Sonnenburg,* Norbert Brattig,† Robert Sabat,‡ Klaus Schilling,§ Janette Bradley,¶ and Susanne Hartmann2*

Immune responses of individuals infected with filarial nematodes are characterized by a marked cellular hyporesponsiveness and a shift of the cytokine balance toward a Th2/Th3 response. This modulation of cellular immune responses is considered as an important mechanism to avoid inflammatory immune responses that could eliminate the parasites. We investigated the immunomodulatory potential of a secreted cysteine protease inhibitor (onchocystatin) of the human pathogenic filaria Onchocerca volvulus. Recombinant onchocystatin (rOv17), a biologically active cysteine protease inhibitor that inhibited among others the Downloaded from human cysteine proteases cathepsins L and S, suppressed the polyclonally stimulated and the Ag-driven proliferation of human PBMC. Stimulated as well as unstimulated PBMC in the presence of rOv17 produced significantly more IL-10, which was paralleled in some situations by a decrease of IL-12p40 and preceded by an increase of TNF-␣. 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 http://www.jimmunol.org/ 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. The Journal of Immunology, 2001, 167: 3207–3215.

nchocerca volvulus is the causative agent of river blind- IL-10 production of B1 cells (9) and could thus contribute to the ness, a vector-transmitted nematode infection that affects polarization of Th cell responses. Homologues of various cyto- Oϳ20 million persons in tropical regions of Africa and kines, such as macrophage migration-inhibitory factor (10) or Latin America. The parasites persist in their hosts for 10 years or tgh-2 (11, 12) released by the filarial nematode Brugia malayi, by guest on September 27, 2021 longer (1, 2), and in the majority of infected persons onchocercia- appears to mimic host cytokines and to interfere with the host’s sis is associated with markedly reduced cellular immune reactions cytokine network. Furthermore, a secreted protein, the cysteine and polarization of T cell responses toward a Th2 and Th3 type protease inhibitor (cystatin) of the rodent filaria Acanthochei- (3–5). It is believed that these alterations are a key element in the lonema viteae,was shown to down-regulate proliferative T cell re- persistence of the worms within their hosts. The down-regulation sponses and to up-regulate the production of IL-10 by splenocytes of inflammatory host immune reactions is suggested to be induced in vitro by hitherto unknown mechanisms (13). In addition, B. by secreted products of the nematodes (6, 7). malayi cystatin inhibits the Ag presentation of human B lympho- Despite the probable importance of this immunomodulation for cyte lines (14). These examples show that nematode components the balance of the host-parasite relationships, there is little infor- have the potential to interfere with regulatory host immune re- mation on pathogenicity factors accounting for it. Several studies sponses very much like certain pathogenicity factors described demonstrated that phosphorylcholine, a lipid component bound to from bacterial and protozoan infections (15, 16). proteins of filariae, inhibits murine B and T cell proliferative re- Filarial cystatin of O. volvulus (onchocystatin) was described by sponses by interference with signaling cascades leading to cellular Lustigman et al. (17, 18), who suggested a role in the regulation of activation (8). Phosphorylcholine was also shown to increase the 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 cat- *Department of Molecular Parasitology and †Institute of Medical Immunology, Charite Medical School, Humboldt University, Berlin, Germany; ‡Division of Clin- alyzed by cysteine proteases (19). Among others, the cystatins ical Chemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; have important functions in the regulation of proteases relevant in §Institute of Biochemistry, University of Jena, Jena, Germany; and ¶Department of Biological Sciences, University of Salford, Manchester, United Kingdom immune responses. Thus, the endogenous inhibitor cystatin C in- Received for publication May 8, 2001. Accepted for publication July 24, 2001. fluences the fate of newly synthesized peptide-MHC class II com- The costs of publication of this article were defrayed in part by the payment of page plexes by regulating the activity of cathepsin S, a cysteine protease charges. This article must therefore be hereby marked advertisement in accordance that is essential in Ii-chain degradation in B cells and dendritic with 18 U.S.C. Section 1734 solely to indicate this fact. cells (20–22). In addition, human cystatin C has been described to 1 This study was supported by a grant from Deutsche Forschungsgemeinschaft inhibit the phagocytic function of monocytes and granulocytes (23, (to S.H.). 24), and it has been shown that various members of the cystatin 2 Address correspondence and reprint requests to Dr. Susanne Hartmann, Department of Molecular Parasitology, Institute of Biology, Philippstrasse 13, 10115 Berlin, Ger- superfamily up-regulate the inducible NO production of murine many. E-mail address: [email protected] macrophages (25).

These functions of cystatins imply that the role of filarial cys- quantified with a peroxidase-labeled goat anti-rabbit Ab diluted 1/10,000 tatin is not necessarily restricted to the molting process of the (Dianova, Hamburg, Germany). Values obtained with the preimmune se- nematodes. This is also underlined by the fact that A. viteae cys- rum were subtracted. tatin is secreted by nematode stages that do not molt, i.e., the male Ag-driven and polyclonally stimulated proliferation of and female worms and blood microfilariae (13). Indeed, our initial human PBMC data, obtained in a murine spleen cell model system, show A. The peripheral mononuclear cell fraction of healthy donors was isolated viteae cystatin to have several immunomodulatory activities, e.g., from citrated venous blood by density gradient sedimentation using Ficoll- up-regulation of IL-10 and of NO production by murine spleen Hypaque (Pharmacia Biotech Products, Freiburg, Germany). Cells were cells (13).3 These data encouraged us to study the immunomodu- resuspended in very low endotoxin RPMI 1640 (Biochrom, Berlin, Ger- ␮ latory role of filarial cystatin in a homologous system. Using a many), supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine, and 10% FCS (Biochrom). PBMC (3.5 ϫ 105/well) were combination of human peripheral blood cells and recombinant on- cultured in 96-well flat-bottom plates at 37°C and stimulated with 10 IU/ml chocystatin, we could establish that filarial cystatin is a potential purified protein derivative (PPD; Chiron Behring, Marburg, Germany), 10 pathogenicity factor of O. volvulus in humans. ␮g/ml PHA (Sigma), or immobilized anti-CD3 Ab (4 ␮g/ml in a final volume of 50 ␮l for 2 h; Orthoclone-Okt3; gift from Janssen-Cliag, Neuss, Materials and Methods Germany). Recombinant proteins were added at a concentration of 0.1–0.5 ␮M. Additionally, human PBMC were cultured with LPS (100 pg/ml; Cloning and expression of recombinant proteins E.coli 0127:B8; Sigma) for 72 h. PPD-stimulated PBMC were cultured for The cDNA of the mature 17-kDa onchocystatin (Ov17),4 was cloned by 96 h, and polyclonally stimulated PBMC were cultured for 72 h at 37°C. 3 PCR from a ␭Uni-ZAP XR cDNA library of infective larvae of O. volvulus Proliferation was quantified by [ H]thymidine incorporation during the last

(provided by S. Williams, Smith College, Northampton, MA). The cDNA 20 h of incubation. For neutralization studies, anti-rOv17 rabbit immune Downloaded from was amplified by PCR with degenerate primers derived from the internal serum (1 ␮l/well), a rabbit control serum against sporozoites of Eimeria sequence of onchocystatin. Forward primer, gttcagttgcaaggagcc; reverse tenella (1 ␮l/well), anti-IL-10 Ab (5 ␮g/ml, clone CB/RS/1; gift from Prof. primer, tcatacttcttttgttccc. PCR amplification yielded a 423-bp fragment Dr. H. D. Volk, Charite´ Medical School, Berlin, Germany), anti-TNF-␣ Ab which was cloned into a T-overhang vector (pGEM-T Easy Vector Sys- (5 ␮g/ml; clone 1825.121; R&D Systems, Wiesbaden-Norderstadt, Ger- tems; Promega, Madison, WI). The recombinant protein was termed many), and a mouse IgG1 isotype control Ab (5 ␮g/ml; R&D Systems) rOv17. The cDNA was subcloned into the EcoRI site of an expression were used. All experiments were performed in triplicate. Viability of the vector yielding polypeptides with a six-histidine tag (pET System; Nova- cells in presence of recombinant proteins were controlled by trypan blue http://www.jimmunol.org/ gen, Madison, WI). The recombinant plasmid was transformed into BL21 exclusion. (DE3) or BL21 (DE3) pLysS-competent Escherichia coli and screened for Depletion of monocytes (CD14ϩ cells) and isolation of purified expression by SDS-PAGE. rOv17 was purified under nondenaturing conditions by affinity chromatography using Ni-NTA resin (Qiagen, Hilden, monocytes from human PBMC Germany) and dialyzed twice against PBS. The control protein, recombi- CD14ϩ cells were depleted from human PBMC using CD14 MicroBeads nant 33-kDa onchocystatin (rOv33), an O. volvulus protein with homolo- and VSϩ columns (Miltenyi Biotec, Bergisch Gladbach, Germany). De- gies to aspartic protease inhibitors (26), was expressed and purified under pletion of CD14ϩ cells was done according to the manufacturers’ instruc- identical conditions. tions and resulted in the enrichment of CD14Ϫ cells of 98–99% as deter- Because rOv17 and rOv33 were expressed in E. coli, the endotoxin mined by FACS analysis using FITC-labeled anti-human CD14 Ab (10 contamination was evaluated by quantitative, chromogenic Limulus ame- ␮g/ml; UCHM-1; Dianova). Furthermore, CD14ϩ depletion was estimated bocyte lysate assay (BioWhittaker, Walkersville, MD). The endotoxin con- ␣ ϩ by guest on September 27, 2021 ␮ by measuring TNF- production. CD14 -depleted cells were stimulated with centration of rOv17 was determined to be 0.04–0.13 pg LPS/0.01 M LPS (5 ng/ml), and TNF-␣ was quantified after6hintheculture supernatant. protein. The control rOv33 showed endotoxin concentrations of 1.0–2.0 ϩ ␣ ␮ CD14 -depleted cells produced on the average 92 pg/ml TNF- in compar- pg/0.01 M protein. ison with 2308 pg/ml TNF-␣ produced by nondepleted PBMC. Human cathepsin inhibition assays Purified monocytes were isolated from MNC by counterflow centrifu- gation using a Beckmann JE-6B elutriator (Beckmann Instuments, Munich, The inhibitory activity of rOv17 toward human cathepsins was character- Germany). The monocyte fraction collected consisted of Ͻ95% mono- ized by Ki value determination. Human cathepsin B was purchased from cytes, as determined by modified Randolph staining and by FACS analysis Sigma (Deisenhofen, Germany), human cathepsin L was purchased from after staining with anti-CD14 Ab (10 ␮g/ml; Dianova). Calbiochem (Schwalbach, Germany), and human cathepsin S was provided by Dr. Wiederanders (Institute of Biochemistry, University of Jena, Jena, Quantification of cytokines in the cell culture supernatant Germany). K values were determined by measuring the activity of human i Cytokine production was determined in the culture supernatants of un- cathepsin B (1.2 nM), cathepsin L (0.1 nM), or cathepsin S (0.1 nM) in the stimulated and stimulated PBMC as well as in purified monocyte cultures presence of various concentrations of rOv17 using the fluorogenic sub- cocultured with 0.5 ␮M recombinant proteins. Culture supernatants were strates Z-Arg-Arg-AMC (40 ␮M; Bachem, Heidelberg, Germany) for ca- collected after 6, 24, and 48 h of incubation. Cytokines were quantified by thepsin B, z-Phe-Arg-AMC (5 ␮M; Bachem) for cathepsin L, and Z-Val- ELISA according to the manufacturer’s instructions (TNF-␣, IL-2, IL- Val-Arg-AMC (40 ␮M; Bachem) for cathepsin S (27, 28). The K values i 12p40: OptEIA; BD PharMingen, Hamburg, Germany; IL-10 and IFN-␥: were calculated using the program GraphPadPrism. DuoSet, R&D Systems). The assays were performed in triplicates. Quantification of secreted Ov17 by ELISA Expression of monocyte surface molecules Five female O. volvulus worms isolated from patients suffering from On- Unstimulated human PBMC (8 ϫ 105/well) were cultured for 72 h at 37°C chocerca volvulus infections were cultured individually for 96 h in 1 ml in 48-well flat-bottom plates with different concentrations of rOv17 and RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with rOv33 (10–100 nM). The expression of monocyte surface molecules was 100 U/ml penicillin and 100 ␮g/ml streptomycin. The culture supernatants determined in a final volume of 200 ␮l using the following Abs for stain- were harvested, and fresh medium was added every 24 h. ELISA plates ing: R-PE -labeled anti-human HLA-DR mAb (0.48 ␮g/ml; clone L243; were coated with 50 ␮l of the culture supernatant of a 24-h culture, mixed Becton Dickinson, Heidelberg, Germany); FITC-labeled anti-human CD40 with 25 ␮l 0.1 M carbonate buffer (pH 9.5) and in parallel with graded mAb (1 ␮g/ml; clone 5C3; PharMingen); FITC-labeled anti-human CD80 concentrations of rOv17. A 1/5000 dilution of a monospecific rabbit serum mAb (1 ␮g/ml; clone BB1; PharMingen); and PE-labeled anti-human against rOv17 was reacted with the proteins. The amount of bound Abs was CD86 mAb (4 ␮g/ml; clone 2331; PharMingen). FITC-labeled mouse IgG1 mAb (1.5 ␮g/ml; clone 679.1 Mc7; Coulter Immunotech, Hamburg, Ger- ␮ 3 S. Hartmann, A. Scho¨nemeyer, B. Sonnenburg, B. Vray, and R. Lucius. 2001. Cys- many) and PE-labeled mouse IgG2a mAb (4 g/ml; clone X 39; Becton tatins of filarial nematodes up-regulate the nitric oxide production of IFN-␥-activated Dickinson) were used as isotype controls. murine macrophages through the production of TNF-␣ and IL-10. Submitted for pub- FACS analyses of 3 ϫ 104 PBMC/measurement were done by using a lication. FACScan instrument (Becton Dickinson). The monocyte population was 4 Abbreviations used in this paper: Ov17, 17-kDa onchocystatin; rOv17, recombinant identified by its light scatter properties and in addition by staining of CD14 onchocystatin; Ov33, 33-kDa O. volvulus protein; rOV33, recombinant Ov33; PPD, with PE-cyanin 5-labeled anti-human CD14 mAb (0.1 ␮g/ml; clone purified protein derivative; E/S, excretory/secretory; TLR, Toll-like receptors. RMO52; Coulter Immunotech). The Journal of Immunology 3209

Statistical analysis PBMC by 42% ( p ϭ 0.002; Fig. 1b), whereas the control protein Statistical analysis of T cell proliferation data and expression of the cell rOv33 had a significantly weaker effect. The specificity of the in- surface molecules were performed with the Student t test. Cytokine data hibitory effect of rOv17 was determined using monospecific rabbit were analyzed with the Wilcoxon test. Data were presented as Abs against rOv17. Addition of rabbit anti-Ov17 serum to the cul- means Ϯ SEM. tures completely abolished the rOv17-induced inhibition of PHA- stimulated proliferation, whereas a rabbit control serum had no Results effect (Fig. 1c). Production of rOv17 and inhibition of human cathepsins B, L, The influence of rOv17 on the Ag-driven proliferation of human and S by rOv17 PBMC was assessed by stimulation of PBMC of three well-re- To investigate the immunomodulatory potential of onchocystatin, sponding healthy blood donors with PPD. Addition of 0.5 ␮M a full length mature recombinant protein (rOv17) with an N-ter- rOv17 resulted in a significant reduction of the Ag-driven prolif- minal six-histidine tag was produced in E. coli.Affinity-purified eration of the PBMC by a mean of 52% (Fig. 1d). In contrast, an rOv17 was tested for its capacity to inhibit the activity of the 0.5 ␮M concentration of the control protein rOv33 did not inhibit human cysteine proteases cathepsins B, L, and S. These proteases the PPD-induced Ag-driven T cell proliferation. are involved in the proteolytic processing of Ag and in Ii chain The inhibition of both Ag-driven and polyclonally stimulated T degradation (29, 30). Ki determinations showed that rOv17 cell proliferative responses suggests that rOv17 interferes with an ϭ strongly inhibited the activity of human cathepsin L (Ki 0.038 immune mechanism that is involved in both types of cellular ϭ nM) and cathepsin S (Ki 0.033 nM), whereas the activity of proliferation. ϭ Downloaded from human cathepsin B was less efficiently inhibited by rOv17 (Ki 494 nM). Therefore, onchocystatin is an inhibitor of immunolog- Cytokine production of unstimulated and stimulated PBMC in ically relevant human cysteine proteases. Consequently, the inhib- presence of rOv17 itor could theoretically interfere with Ag processing and Ii chain To test whether the rOv17-induced suppression of T cell prolifer- degradation of APC. The inhibitory domains of the parasite protein ation is due to cytokines produced by PBMC, we quantified cyto- are likely to correspond to the structures of human cystatin; as a kines that mediate activation and regulation of T cell proliferation, sequence alignment shows the typical evolutionary conserved mo- such as IL-2, IL-4, TNF-␣, IFN-␥, IL-10, and IL-12p40. The http://www.jimmunol.org/ tifs of cysteine protease inhibitors, which bind and inhibit the pro- amounts of these cytokines in the presence or absence of rOv17 tease. The N-terminal inhibitory domain reaches from aa 51–53, were determined in the culture supernatants of anti-CD3-stimu- the central domain from aa 98–102, and the C-terminal domain lated PBMC of 10 donors after 48 h. The tests revealed that rOv17 from aa 153–154. In contrast, most of the other regions vary con- induced a significant increase in IL-10 production ( p ϭ 0.002) and siderably between onchocystatin and human cystatins, such that a significant decrease in IL-12p40 production ( p ϭ 0.025) as well the overall amino acid identity of the proteins was comparatively as a diminished IFN-␥ production which was, however, not sig- low with values between 18 and 22%. As a particular element, nificant (Fig. 2). The control protein rOv33 had significantly onchocystatin contains a stretch of amino acids (aa 37–48) which weaker effects on these cytokines. The production of IL-2, IL-4, is lacking in the human cystatins. This stretch does not show any and TNF-␣ by stimulated PBMC was not significantly affected by by guest on September 27, 2021 homologies to known sequences. rOv17 or rOv33 (data not shown). In parallel, the amounts of IL-10, IL-12p40, and TNF-␣ in the Quantification of Ov17 secretion by female O. volvulus worms presence or absence of rOv17 were determined in the culture su- The cDNA of onchocystatin encodes for a hydrophobic leader se- pernatants of unstimulated PBMC of five individuals after 6, 24, quence which is typical for secreted proteins. Therefore, we eval- and 48 h. The tests revealed that onchocystatin induced an initial uated whether onchocystatin is secreted by O. volvulus worms in increase of the proinflammatory cytokine TNF-␣ at6h(p Ͼ vitro. To this end, the quantity of onchocystatin in the culture 0.001). This peak in TNF-␣ production was followed by a signif- supernatants of intact individual female worms was determined by icant increase of the inhibitory cytokine IL-10 ( p Ͼ 0.001) at 24 h a calibrated ELISA. A monospecific rabbit serum raised against (Fig. 3, a and b) which was also elevated at 48 h. The effect of the recombinant Ov17 detected a release of 10–40 ng onchocystatin control protein rOv33 on both cytokines was significantly lower per female worm in 24 h, suggesting that onchocystatin has the (Fig. 3). No significant production of IL-12p40 was observed by potential to interfere with the host immune system in vivo. unstimulated PBMC by rOv17 (data not shown). Thus, the increase of the IL-10 production was the most prominent effect in the Inhibition of polyclonally stimulated and Ag-driven proliferation cytokine pattern of stimulated and of unstimulated human PBMC of human PBMC by rOv17 exposed to rOv17. As one model for polyclonally stimulated T cell responses, we Because IL-10 is mainly produced by monocytes, we exposed studied the effect of rOv17 on the PHA-induced T cell prolifera- purified monocytes to rOv17 and rOv33 and quantified the amount tion. rOv17 inhibited the cellular proliferation in a dose-dependent of IL-10 in the culture supernatant. Again, rOv17 induced a sig- manner (12–42% inhibition with concentrations between 0.1 and nificant production of IL-10 at a concentration of 5 nM (medians 0.5 ␮M rOv17), whereas the control protein rOv33 had signifi- of three experiments: rOv17, 168 pg/ml, p Ͼ 0.05; rOv33, 0 pg/ cantly less activity. The most effective concentration of 0.5 ␮M ml). These data demonstrate a direct effect of rOv17 on monocytes was subsequently used to analyze the antiproliferative activity of and suggest that the measured increase of IL-10 production is due rOv17 toward PBMC of 10 healthy blood donors. In this concen- to monocytes. tration, rOv17 inhibited the proliferative response of human PBMC by 39% ( p Ͼ 0.001; Fig. 1a), whereas the control protein Expression of monocyte surface molecules in presence of rOv17 rOv33 induced only a slight inhibition of proliferation (difference Both the Ag-driven and the polyclonally stimulated proliferations between rOv17 and rOv33, p Ͼ 0.001). In a further set of exper- of PBMC require costimulatory signals for full T cell activation. iments, we used immobilized anti-CD3 Ab to stimulate PBMC of Because important costimulatory molecules are expressed by 10 blood donors. Again, 0.5 ␮M rOv17 resulted in a mean inhi- monocytes, we examined the influence of various concentrations bition of the anti-CD3 Ab-stimulated proliferation of human of rOv17 on the expression of the costimulatory surface markers of 3210 IMMUNOMODULATION BY ONCHOCYSTATIN Downloaded from http://www.jimmunol.org/

FIGURE 1. T cell proliferation in the presence of recombinant onchocystatin. PHA (a) and anti-CD3 (b) Ab-stimulated proliferation of human PBMC of 10 blood donors in the presence of 0.5 ␮M rOv17 or of the control protein rOv33. Proliferation of PBMC in presence of PHA or anti-CD3 Ab corresponds to 100% proliferation. c, T cell proliferation in the presence of recombinant onchocystatin and a rabbit anti-rOv17 serum; representative experiment. PBMC, were stimulated with PHA, and proliferation was determined in the presence of 0.5 ␮M rOv17 and rabbit anti-rOv17 serum or a rabbit control serum. d, Ag-driven proliferation of human PBMC in the presence of recombinant onchocystatin. PPD-stimulated proliferation of PBMC of three blood donors in by guest on September 27, 2021 the presence of 0.5 ␮M rOv17 or of the control protein rOv33. monocytes CD40, CD80, and CD86. The analysis of CD86 on and induces changes in the expression of monocyte surface mol- human monocytes of PBMC of four blood donors revealed that ecules (31–33).To test the role of IL-10 for the observed alter- rOv17 reduced the expression of CD86 by 37% ( p ϭ 0.018) at a ations, we exposed PBMC to rOv17 and neutralized IL-10 by concentration of 10 nM (Fig. 4a). An increase in the rOv17 con- addition of anti-IL-10 Abs. The experiment revealed that addition centration did not significantly enhance this effect. The control of anti-IL-10 Abs did not restore the inhibition of anti-CD3- protein rOv33 did not significantly affect the CD86 expression induced proliferation of PBMC (n ϭ 4; Fig. 5). This suggests that (Fig. 4a). The expression of the two other surface markers, CD40 IL-10 is not a major component of the rOv17-induced inhibition of and CD80, which were poorly expressed on monocytes, remained T cell proliferation. unchanged in the presence of rOv17 (data not shown). Because the expression of CD86 and HLA-DR on monocytes Ag-driven T cell stimulation is also dependent on presentation was described to be negatively regulated by IL-10, we tested of Ags in association with MHC II molecules of APC. To assess whether IL-10 was the cause of the down-regulation in our situa- whether the inhibition of Ag-driven T cell responses by rOv17 acts tion. Neutralization of IL-10 not only restored the diminished ex- by interference with the Ag presentation, we quantified the pression of CD86 on human monocytes (n ϭ 3) by rOv17 but led HLA-DR surface molecules of human monocytes of PBMC of to expression of 3-fold higher CD86 levels than those of the me- four blood donors. In the presence of 10 nM rOv17, the expression dium controls (Fig. 6a). In addition, neutralization of IL-10 re- of HLA-DR was significantly reduced by 57.9% ( p ϭ 0,0014), stored the diminished expression of HLA-DR on monocytes (n ϭ whereas an increase of the concentration to 50 nM rOv17 en- 3) to 72–93% of the control levels (Fig. 6b). Therefore, IL-10 is hanced this effect (72% reduction in comparison with cells without responsible for the down-regulated expression of CD86 and addition of rOv17; p ϭ 0.0015; Fig. 4b). In contrast, the control HLA-DR on human monocytes in this scenario. protein rOv33 did not affect HLA-DR expression (Fig. 4b). Similar Because TNF-␣ may stimulate T cells and monocytes, trigger- to the experiments with PBMC, rOv17 reduced the expression of ing among others the production of IL-10 (34), this proinflamma- HLA-DR and, less pronounced, of CD86 on purified monocytes tory cytokine could contribute to the effects exerted by rOv17. (median reduction, 66% for HLA-DR and 40% for CD86). Neutralization of TNF-␣ by addition of anti-TNF-␣ Abs did not restore the diminished cellular proliferation (data not shown). ␣ Neutralization studies using anti-IL-10 and TNF- Abs However, addition of anti-TNF-␣ Abs led to a reduction by IL-10 exerts suppressive and deactivating effects on T cells and 31.2% of the rOv17-induced IL-10 production of unstimulated macrophages, alters the pattern of cytokine production of T cells, human PBMC (data not shown), showing that TNF-␣ accounts The Journal of Immunology 3211 Downloaded from http://www.jimmunol.org/

FIGURE 3. Production of TNF-␣ and IL-10 of unstimulated human PBMC in presence of recombinant onchocystatin. a, Spontaneous production of TNF-␣ by PBMC of ﬁve blood donors in the presence of 0.5 ␮M by guest on September 27, 2021 rOv17 or of the control protein rOv33 after 6 h. b, Spontaneous production of IL-10 by PBMC of ﬁve blood donors in the presence of 0.5 ␮M rOv17 or the control protein rOv33 after 24 h.

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 CD14ϩ cells to 1–2% of the total PBMC population. Monocyte-depleted PBMC (n ϭ 4) were normalized for the number of lymphocytes, stimulated FIGURE 2. Production of IL-10, IL-12, and IFN-␥ of human PBMC in with PHA, and cocultured with rOv17 and the control protein the presence of recombinant onchocystatin. Anti-CD3 Ab-stimulated pro- rOv33, respectively. The PHA-stimulated proliferation of com- duction of IL-10 (a), IL-12 (b) and IFN-␥ (c) of PBMC of 10 healthy plete PBMC were inhibited by 37% in the presence of 0.5 ␮M donors after 48 h. The concentration of rOv17 and of the control protein rOv17, whereas the proliferation of monocyte-depleted PBMC rOv33 was 0.5 ␮M. 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 abol- for a part of the increase in IL-10. Therefore, one could expect ished in monocyte-depleted PBMC cultures. These data show that this cytokine indirectly acts on the levels of CD86 and that the inhibition of proliferation of PHA-stimulated human HLA-DR. PBMC induced by rOv17 is mediated by monocytes. Together, these data suggest that the proliferative block induced by cystatin acts independently of TNF-␣ and IL-10, whereas the Discussion expression of CD86 and HLA-DR is influenced by IL-10. This report describes immunomodulatory activities of onchocystatin, a secreted protein of the filaria O. volvulus. We show that Determination of the target cell of onchocystatin within recombinant onchocystatin inhibits Ag-driven and polyclonally in- the PBMC duced T cell-proliferative responses of human PBMC, induces The fact that purified monocytes are IL-10 producers in the pres- changes of their cytokine expression, and down-regulates the ex- ence of rOv17 as well as the observed changes in monocyte sur- pression of CD86 as well as HLA-DR. The data suggest that on- face molecules induced by rOv17 suggested that these cells are chocystatin could act as a pathogenicity factor of O. volvulus, with involved in the rOv17-induced immunomodulation. To study their properties similar to certain bacterial modulins (15, 35), which 3212 IMMUNOMODULATION BY ONCHOCYSTATIN

FIGURE 5. T cell proliferation in the presence of recombinant onchocystatin and anti-IL-10 Ab. PBMC of four blood donors were stimulated

with anti-CD3 Ab in the presence of 0.5 ␮M rOv17 or the control protein Downloaded from rOv33 and 5 ␮g/ml anti-IL-10 Ab. Anti-CD3 Ab-induced proliferation corresponds to 100%.

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 http://www.jimmunol.org/ N-terminal conserved inhibitory domain has been shown to have a predominant role because it blocks the active site cleft of the protease (38–40), 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 de- grades Ag, and it mediates the final proteolytic steps in Ii chain by guest on September 27, 2021 degradation in thymus epithelial cells (29, 43). Cathepsin S pro- FIGURE 4. Expression of CD86 and HLA-DR on human monocytes in cesses Ag and is essential in the completion of Ii chain degradation presence of recombinant onchocystatin. PBMC of four blood donors were in dendritic cells and B cells (20, 22) Therefore, it is tempting to exposed to various concentrations of rOv17 and the control protein rOv33. speculate that onchocystatin acts through inhibition of immuno- Expression of CD86 (a) and HLA-DR (b) on monocytes was determined logically relevant host proteases. Fluorescein-labeled rOv17 is by FACScan. Data are presented in percent expression in comparison with taken up by macrophages (our unpublished data) and could thus unstimulated control cells. The monocyte population was identified by its reach the cellular compartments where Ag processing and the sub- light scatter properties and by staining of the surface molecule CD14. sequent 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 modulate host immune responses and enhance the survival and with a role in immune processes. Indeed, synthetic as well as mi- reproduction of the bacteria. crobial cysteine protease inhibitors have been shown to modulate Filarial parasites persist within their vertebrate hosts for ex- Ag-specific cellular responses. Katunuma et al. (44) describe a tended time spans, e. g. up to 14 years as shown for O. volvulus (2). marked suppression of proliferation of primed splenocytes to hep- This survival within an immunocompetent host might be due to a atitis B virus Ag in the presence of two cathepsin B inhibitors, E64 modulation of the host’s immune system exerted by excretory/ and CA-074. Furthermore, another study by Vidard et al. (45) secretory (E/S) products of the parasites. Indeed, E/S products of demonstrates that the cysteine protease inhibitor leupeptin influ- filariae suppressed proliferative lymphocyte responses (6, 8, 13, ences the generation of immunogenic OVA peptides, which leads 36), but despite the possible importance for the understanding of to modulation of immune responses. The target proteases of the the pathogenicity mechanisms only few components of E/S prod- above mentioned cysteine protease inhibitors remain to be deter- ucts were thus far characterized on the molecular level. One of mined. Moreover, it has been shown that cysteine proteases are these components is the cystatin protein of the filaria A. viteae,an responsible for invariant chain degradation in APCs and that the E/S Ag that inhibits proliferative responses and induces changes of ratio of cystatin C to these cathepsins determines the fate of newly the cytokine pattern of murine spleen cells, as shown in an earlier synthesized peptide-MHC class II molecules as well as the differ- publication (13). entiation of Th cell subclasses (21, 43). Therefore, cysteine pro- To establish the relevance of this possible pathogenicity factor tease inhibitors have the capacity to modulate immune responses, for a human pathogenic filarial infection and to enlarge our knowl- and filarial parasites may use this mechanism to interfere with edge of the underlying mechanisms, we characterized the immu- immune responses of their hosts. nomodulatory potential of onchocystatin using a recombinant, bi- However, our data show that onchocystatin down-regulates not ologically active cystatin (rOv17) which comprised the full only the Ag-driven cellular proliferation but also the polyclonally The Journal of Immunology 3213

FIGURE 7. T cell proliferation of monocyte-depleted PBMC in the presence of recombinant onchocystatin. Monocyte-depleted PBMC and complete PBMC of four blood donors were stimulated with PHA in the Downloaded from presence of 0.5 ␮M rOv17 or of the control protein rOv33. Proliferation in presence of PHA corresponds to 100% proliferation.

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 http://www.jimmunol.org/ 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 by guest on September 27, 2021 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 Toxo- plasma gondii infection in mice (50). Thus, the observed induction FIGURE 6. Expression of CD86 and HLA-DR on human monocytes in of IL-10 by onchocystatin could be an essential feature in the host- the presence of recombinant onchocystatin and anti-IL-10 Ab. PBMC of parasite interaction, the increased levels of IL-10 leading among four blood donors were incubated with various concentrations of rOv17 or others to a polarization of host T cell responses toward Th2/Th3 the control protein rOv33 and 5 ␮g/ml anti-IL-10 Ab. Expression of CD86 response, which is characteristic for filarial infections (5, 51, 52). (a) and HLA-DR (b) on monocytes was determined by FACScan. Data are However, in our experimental set-up, IL-10 does clearly not ac- presented in percent expression in comparison with unstimulated control count for the inhibition of proliferative PBMC responses, because cells. The monocyte population was identified by its light scatter properties neutralization of IL-10 did not abrogate the inhibition of prolifer- and by CD14 staining. ative responses. Other, IL-10-independent mechanisms, like the contact-dependent macrophage-mediated cell cycle arrest exerted induced T cell proliferation. Using PHA and anti-CD3 Ab as stim- by filarial parasites (53), could account for this effect. uli of human PBMC, we showed that onchocystatin also inhibits T Furthermore, this study determines that the target cells of cell proliferation independent of Ag processing and presentation. rOv17-induced down-regulation of proliferation are monocytes. Polyclonally induced and Ag-driven T cell proliferation have in Depletion of monocytes from the PBMC reversed the inhibitory common that they require cytokines and coreceptors for full T cell effects on the cellular proliferation induced by onchocystatin. Re- activation (46). Our results indicate that the cellular hyporeactivity cently, macrophages were shown to be important regulatory cells induced by onchocystatin is accompanied by a modulation of the by which filariae inhibit cellular proliferation (53). Our study is in cytokine production and by alterations of the expression of mono- line with these findings. In addition, this report characterizes for cyte surface molecules. One main characteristic was the up-regu- the first time a single filarial protein that is responsible for the lation of the IL-10 production of PBMC in the presence of rOv17. modulation of macrophage functions observed in infections with IL-10 is a cytokine that shows immunosuppressive effects due to filariae. Modulation of monocyte functions is not restricted to fi- its ability to suppress the production of IL-12 and IFN-␥, to down- larial cystatin but seems to be an intrinsic feature of these proteins. regulate MHC class II expression, and to reduce the expression of Leung-Tack et al. (24) reports that human cystatin C, a protein the adhesion molecule CD54 (ICAM-1) as well as the costimula- constitutively secreted by mononuclear phagocytes (54, 55), inter- tory molecules CD80 and CD86 on APC (31–33). As a conse- feres with the phagocytic function and the oxidative burst of quence, IL-10 causes a reduced T cell proliferation. Our data sup- monocytes via a bioactive peptide that is released during the in- port some of the described effects of IL-10, in that neutralization of teraction of the inhibitor with a cysteine protease. Furthermore, it 3214 IMMUNOMODULATION BY ONCHOCYSTATIN has been shown that members of the three subgroups of the cys- 5. Doetze, A., J. Satoguina, G. Buchard, T. Rau, C. Lo¨liger, B. Fleischer, and tatin superfamily up-regulate the inducible NO production of mu- A. Hoerauf. 2000. Antigen-spezific cellular hyporesponsiveness in a chronic human helminth infection is mediated by Th3/Tr1-type cytokines IL-10 and trans- ␤ rine macrophages (25). It is interesting that NO is a potent inhibitor forming growth factor- but not by a Th1toTh2 shift. Int. Immunol. 12:623. of murine T cell proliferation (56). Moreover, it has been shown 6. Allen, J. E., and A. S. McDonalds. 1998. Profound suppression of cellular pro- that cystatin C limits acute inflammatory reactions due to cysteine liferation mediated by the secretions of nematodes. Parasite Immunol. 20:241. 7. Whelan, M., M. M. Harnett, K. M. Houston, V. Patel, W. Harnett, and proteases released during inflammation. This is compatible with K. P. Rigley. 2000. A filarial nematode-secreted product signals dendritic cells to the fact that cystatin C is down-regulated in chronic inflammatory acquire a phenotype that drives development of Th2 cells. J. Immunol. 164:6453. 8. Harnett, W., M. R. Deehan, K. M. Houston, and M. M. Harnett. 1999. Immu- reactions that are dominated by mononuclear phagocytes and lead nomodulatory properties of a phosphorylcholine-containing secreted filarial gly- to tissue pathology (54). Therefore, cystatins can be considered coprotein. Parasite Immunol. 21:601. regulatory proteins, which can modulate monocyte activities in 9. Al Quad, K. M., B. Fleischer, and A. Hoerauf. 1998. The Xid defect imparts susceptibility to experimental murine filariosis-association with a lack of anti- addition to its protease-inhibitory function. From this perspective, body and IL-10 production by B cells in response to phosphorylcholine. Int. it is conceivable that filariae interfere with regulatory functions of Immunol. 10:17. host macrophages by releasing cystatin. 10. Pastrana, D., N. Raghavan, P. Fitzgerald, S. Eisinger, C. Metz, R. Bucala, R. Schleimer, C. Bickel, and A. Scott. 1998. Filarial nematode parasites secrete The receptor on the target cell by which rOv17 mediates the a homologue of the human cytokine macrophage migration inhibitory factor. immunomodulations is thus far unclear. The early increase of Infect. Immun. 66:5955. TNF-␣ with a subsequent decrease and rising of the IL-10 pro- 11. Gomez-Escorbar, N., E. Lewis, and R. M. Maizels. 1998. 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