2Instituto de Ciencias Básicas y Medicina Experimental,Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina; 3Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús CONICET, Universidad Nacional de General San Martín, Pcia. de Buenos Aires, Argentina; 4Laboratory of Molecular Pathology, Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina; 5Laboratory of Clinical Immunology, Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina; and 6Laboratory of Immunogenetics from Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina
Received on May 9, 2001; revised on June 27, 2001; accepted on August 17, 2001.
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Abstract |
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Key words: immunomodulation/mucins/T. cruzi
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Introduction |
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In the present study we have designed a strategy with the objective of assessing the utility of expressing a single T. cruzi mucin gene in higher eukaryotic cells and to evaluate its immunomodulatory activity on a xenogeneic response in vitro. The gene chosen for this purpose (TcMuc-e2) encodes for a T. cruzi mucin that is expressed in the blood-circulating stage of the parasite. The rationality of that strategy was based on the following:
1. The mucins that coat the surface of T. cruzi are potentially responsible for some of the parasites capacity to circumvent the vertebrate host response (Kierszenbaum et al., 1999).
2. Despite the predictable ocurrence of differences in the pattern of glycosylation, the organisation of TcMuc genes resembles those present in mammalian cells (central domains, rich in codons for Thr and/or Ser and Pro residues made up of a variable number of repeat units in tandem; Di Noia et al., 1995).
3. Mucin-type glycoproteins expressed in higher eukaryotic cells can mediate pathological interactions among leukocytes modulating the immune response (Kim et al., 1999).
4. The above-mentioned software prediction (Hansen et al., 1998).
In addition, in previous experiments we showed that when expressed on Vero cells, TcMuc-e2 inhibits lymphocytic proliferation in Balb C mice, both in vivo and in vitro (Argibay et al., 2000). In this work, we found that mammalian cells expressing the TcMuc-e2 mucin induce a nonresponding phenotype in human lymphocytes in vitro. In addition, we elucidated some of the mechanisms involved in this effect, such as downregulation of IL-2 receptor CD25 and the necessity of glycosylation on TcMuc-e2 to acquire immunomodulatory effects.
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Results |
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Discussion |
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We know that the O-glycosylation, both in parasites and in mammalian cells can be different. However, there are interesting functional analogies between these two systems that could be utilized to study the immunomodulatory role of a particular T. cruzi gene encoding a heavily O-glycosylated surface mucin (Pollevick et al., 2000) when expressed in mammalian cells. Mucins are involved in immunomodulatory effects both in mammals and in lower organisms (Rudd et al., 1998
; Kierszenbaum et al., 1999
). In recent years, in vitro studies have revealed not only that T. cruzi mucins are the most abundant glycoproteins on the surface of the parasite but also that they play a relevant role in the mechanisms displayed by the parasite for cell invasion and evasion from the immune system (Schenkman et al., 1992
; Pereira-Chioccola et al., 2000
). On the other hand, it was shown that the inefficient host immune response to cancer antigens is at least in partly modulated by the presence of carcinoma-associated mucins (Gimmi et al., 1996
; Agrawal et al., 1998
; Kim et al., 1999
).
In consistency with those studies, our study shows that the expression of the T. cruzi gene TcMuc-e2 in mammalian cells can inhibit the human T cell proliferative response to a xenogeneic stimulus. The mechanism involved in such anergy presents two sides:
1. On the side of the stimulus, it is known that the TcMuc products are O-glycosylated in T. cruzi (Pollevick et al., 2000) as well as in mammalian cells (Di Noia et al., 1996
). In this regard, our results with the apomucin expressed in bacteria suggest that some posttranslational changes are needed to achieve the immunomodulatory effect. The total abolition of the anergy-inducing effect of E2-Vc by OSGPE treatment and partially by neuraminidase treatment, points to O-glycosylation and sialic acid.
2. On the lymphocytes side, previous studies regarding the induction of unresponsiveness either by cancer mucins or glycoconjugates from T. cruzi have focused on two potential mechanisms: anergy and apoptosis. It is known that tumor-infiltrating lymphocytes express the early activation marker CD69 but express low levels of the IL-2 receptor (Alexander et al., 1995). This anergy phenotype associated with cancer mucins is compatible with our results. We found that human lymphocytes exposed to xenogeneic cells, both TcMuc-e2 positive or controls, express the CD69 antigen despite the fact that this expression presents some delay in the case of T cells exposed to E2-Vc. But, as is the case in anergy associated with cancer mucins, T cells exposed to E2-Vc remain as resting lymphocytes with no expression of the CD25 antigen. This mechanism of anergy coincides with the fact that T. cruzi has a selective suppressive effect on IL-2 gene expression (Soong and Tarleton, 1992
), and with the recent results showing that lymphocytes cocultured with a mucin from T. cruzi show a significant decrease in their capacity to express IL-2 receptor molecules (Kierszenbaum et al., 1999
).
In concordance with other communications regarding anergy (Sad and Mosmann, 1995), we found that E2-Vc-inhibited human lymphocytes retained their capacity to secrete cytokines in response to a xenogeneic challenge. Therefore, partial anergy was achieved, so that cell proliferation and upregulation of CD25 were inhibited, but the T helper 1 cytokine pattern was unaffected.
In the present work, we found only a low degree of early apoptosis in lymphocytes exposed to E2-Vc by day 5, but not before. This process could not explain the early inhibition of proliferation seen by days 3 and 4, and it is probably an associated phenomenon. T cell reversible anergy (Beverly et al., 1992) and lack of stimulation through the IL-2 pathway could be a mechanism to explain the TcMuc-e2 mucin-induced inhibition of T cell proliferation and poor expression of the CD25 antigen. In further support of this hypothesis, the addition of exogenous human IL-2 overcame TcMuc-e2 mucin-induced inhibition of proliferation.
Regardless of the mechanism, the fact that TcMuc-e2 mucin expressed on Vero cells can cause such profound inhibitory effects on human lymphocytes is compatible with experimental studies suggesting an immunosuppressive effect of T. cruziassociated mucins. Furthermore, our results are compatible with clinical studies showing that hosts acutely infected with T. cruzi develop manifestations of immunosuppression, including functional alterations in lymphocytes and other cells relevant to mount an immune response. However, although suggestive, the similarities between the anergic effects of TcMuc-e2 positive Vero cells and those elicited by natural T. cruziderived factors must be taken with caution. The structure of posttranslational modifications, in particular O-glycosylation, of both the Vero expressed TcMuc-e2 product and the natural parasite molecule are still not well defined. So, the immunomodulatory effects reported for the native parasite mucins and those observed herein might be achieved by different pathways or due to different effectors. Further work is needed to clarify if any mucin-type molecule expressed in Vero or other cell types would have the same effect, or if there is some particular feature to the T. cruzi apomucin that facilitates the induction of anergy.
The model here described might find, in addition to any possible application to Chagas disease, a diversity of applications ranging from parasitology to clinical glycoimmunology. In particular, the field of transplant glycoimmunology could be enriched.
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Materials and methods |
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The gene TcMuc-e2 (GeneBank accession number AF036409) encoding a T. cruzi mucin (Di Noia et al., 1998), with its signal peptide replaced by the human IgE signal peptide, was cloned into the eukaryotic vector pCDNA3.1 (Invitrogen, San Diego, CA). The resulting plasmid pCTcMuce2/IgEsp was purified using Qiagen columns (Qiagen, Valencia, CA) and transfected into higher eukaryotic cells by the calcium phosphate procedure (Graham and Eb, 1973
). After 10 days of drug selection, neomycin-resistant colonies were obtained. The presence of the TcMuc-e2 mRNA was detected by RT-PCR, using TcMuc-e2-specific primers. Expression of the protein TcMuc-e2 on the surface of two different transfected cell types (Vero cells and primary culture of porcine hepatocytes) was ascertained by immunohistochemistry, using three polyclonal antibodies obtained (1) from a rabbit immunized with the synthetic peptide KP2T8KP2; (2) from a rabbit immunized with a recombinant protein having five T8KP2 tandem repeats and the conserved TcMuc C-terminus fused to Schistosoma mansoni glutation-S-transferase; and (3) from a serum obtained from a mouse immunized with the plasmid pCTcMuce2/IgEsp. Vero cells expressing TcMuc-e2 were named E2-Vc. A tagged version of a T. cruzi mucin homologous to TcMuc-e2 has been expressed in Vero cells and has shown to yield a highly O-glycosylated mucin (Di Noia et al., 1996
). Because our present objective was the study of immunomodulation, the presence of an immunogenic tag not belonging to the original TcMuc product was avoided in these experiments. pC-Vc and NT-Vc were used as controls throughout the experiments.
Preparation of T cells from PBMCs
Fresh blood drawn from healthy donors using heparinized syringes was centrifuged through Histopaque-1077 (Sigma Diagnostics, St. Louis, MO). The PBMCs collected at the interface were washed twice and resuspended in DMEM. Enriched T cell populations were purified by using nylon wool columns described elsewhere (Agrawal et al., 1996). Cell viability, determined by trypan blue exclusion, was above 90%.
Mixed xenogeneic Vero cellslymphocytes culture
Vero cells, both TcMuc-e2 transfected or controls, were used as stimulators and human lymphocytes as responder cells. One hundred microliters of a suspension of T cells (1.5 x 106) from healthy individuals were incubated with 100 µl of a suspension of cobalt-irradiated (1200 Rads) Vero cells. Culture media were supplemented with 20% human fresh serum from a pool of healthy donors. Every experiment with lymphocytes from the same donor was done by triplicate and PBMCs from three different donors were used. Proliferation assessment of human lymphocytes was done at days 3, 4, and 5. [3H]Thymidine (1 µCi, New England Nuclear, Boston, MA) was added to each well 6 h before culture withdrawal. Cells were harvested on filter paper and cpm determined in a liquid scintillation counter. In a different experiment after the initial culture, lymphocytes exposed to TcMuc-e2 Vero cells were treated with human IL-2 (Sigma Diagnostics). After addition of IL-2 (50 U/ml), the well plate cultures were set up as described previously, and proliferation assessment was done.
Detection of IFN- by ELISA
Nunc enzyme-linked immunosorbent assay (ELISA) plates coated with mouse anti-human IFN- (Genzyme, Cambridge, MA) were used according to manufacturer instructions. The plates were read in an ELISA reader in the kinetic mode.
Detection of apoptosis
Lymphocytes were washed (phosphate buffered saline [PBS], pH 7.2) and fixed with neutral buffer formaldehyde for 24 h. Antigen recuperation was done in a microwave oven. Endogenous peroxidases were blocked with PBS:H2O2 (70:30), and protein blockade was done with normal calf serum. TUNEL assay (Apoptag peroxidase kit, Intergen, Oxford, UK) was utilized according to the instructions supplied by the manufacturer.
Flow cytometry
Analyses were done on an Ortho Cytoron Flow Cytometer (Ortho Diagnostics, Raritan, NJ). To evaluate the expression of CD25, CD69 antigens on CD3(+) lymphocytes, relative fluorescence intensity was measured using anti-CD25 (Immunotech, Westbrook, ME) and anti-CD69 (Becton-Dickinson, Mountain View, CA) antibodies.
Recombinant protein expression and purification
The insert from a genomic clone of TcMuc gene isolated from an expression library (Reyes et al., 1994) was cloned in pGEX-1
T vector (Pharmacia Biotech, Uppsala, Sweden). The resulting recombinant protein contained the hypervariable region, the central amino acid repeats and the conserved C-terminus as C-terminal fusion with glutathione-S-transferase. The glutathione-S-transferase fusion protein was induced and purified by affinity chromatography on glutathione-sepharose beads as described (Di Noia et al., 1996
). Purity was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and quantified using Bradford reagent (Bio-Rad Laboratories, Richmond, CA).
O-syaloglycoprotease and sialidase treatment
Transfected Vero cells (106) were treated with OSGPE (8 µg/ml, Cedarlane Laboratories, Hornby, Ontario, Canada) in DMEM for 1 h at 37°C. The supernatant was collected and cells washed with PBS. Human lymphocytes (106) were added to the treated or mock-treated monolayers and cocultured for 3 days, after which T cells were harvested and prepared for flow cytometry as indicated. The same procedure was done with Sialidase I from C. perfringens (Glyco, Novato, CA).
Statistical analysis
Numerical data were processed using the Primer of Biostatistics statistical package (Stanton D. Glantz, McGraw-Hill, 1992) and expressed as percent or mean with the corresponding standard deviation. The comparative statistical level of significance was calculated accordingly. In every experiment we considered an alpha of 0.05 and a power of 0.8.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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