The Trypanosoma cruzi trans-sialidase is a T cell-independent B cell mitogen and an inducer of non-specific Ig secretion

Wenda Gao, Henry H. Wortis and Miercio A. Pereira

Parasitology Research Center, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA

Correspondence to: M. A. Pereira; E-mail: maperrin{at}yahoo.com


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Polyclonal lymphocyte activation and hypergammaglobulinemia characterize the acute phase of many parasitic diseases, including Chagas' disease, a debilitating condition caused by Trypanosoma cruzi. Polyclonal lymphocyte activation correlates with disease susceptibility inT. cruzi infection. Thus, identifying factors that drive such reactivities should provide insight into mechanisms of parasite evasion from host immunity and of disease pathogenesis. Sensitization of mice with small doses of T. cruzi trans-sialidase (TS) turns the mice into highly susceptible hosts to T. cruzi. In addition, TS heterologously expressed in Leishmania major greatly enhances virulence of the parasite to mice. In attempt to study the mechanism of TS-induced virulence, we found that TS and its C-terminal long tandem repeat (LTR) are T-independent polyclonal activators for mouse B cells. While B cells deficient/defective in L-6, CD40 or Toll-like receptor-4 are similarly activated by TS as compared to wild-type cells, B cells from Bruton's tyrosine kinase-defectiveX-linked immunodeficient mice are remarkably insensitive to TS activation. TS-induced B cell activation in vitro is accompanied by Ig secretion independent of T cells. Furthermore, administration of TS into normal mice leads to non-specific Ig secretion that peaks 4–6 days after injection. Thus TS, through its LTR, induces abnormal polyclonal B cell activation and Ig secretion, which could explain in part its virulence-enhancing activity.

Keywords: Bruton's tyrosine kinase, neuraminidase, polyclonal B cell activator, protozoa, tandem repeats, Trypanosome


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Polyclonal lymphocyte activation is a general feature of many viral, bacterial and parasitic diseases (1). This type of response is thought to constitute an immune evasion mechanism of infectious agents because it masks/deviates the specific responses against pathogens. Such immune deregulation appears to be associated with acute phase immunosuppression and chronic autoimmune reactivities seen in these infections, and becomes a major hindrance for developing effective vaccine strategies (1).

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas' disease, which afflicts millions of people in the Americas (2). T. cruzi infection causes profound immunological disturbances to the host. Abnormality of the B cell compartment is manifested by both polyclonal B cell proliferation (3,4) and non-specific Ig secretion, which lead to hypergammaglobulinemia (5). The continuous and long-lasting B cell stimulation may have important consequences for the pathogenesis of Chagas' disease (6). This notion is supported by the observations that suppressed polyclonal lymphocyte activation correlates with resistance to infection and pathology development (7,8). However, not much is known about the molecular basis responsible for antigen-non-specific mitogenic responses that might directly undermine host immunity against T. cruzi. Proline racemase and a 24-kDa parasite antigen recently identified in T. cruzi can activate mouse B cells polyclonally (9,10), although it has not been determined yet whether these molecules can function as virulence factors in Chagas' disease.

T. cruzi expresses a developmentally regulated neuraminidase (sialidase) located on the cell surface of the parasite (11,12). The neuraminidase is also readily shed into the extracellular milieu (11,12). The enzyme is capable of binding to {alpha}-2,3-linked sialic acid and transferring the carbohydrate to ß-Gal acceptors, and hence it is also a trans-sialidase (TS) (13,14). TS consists of an N-terminal catalytic domain (CD) and a long C-terminal domain of 12-amino-acid (DSSAHS/GTPSTPV/A) tandem repeat (LTR) (15). TS is implicated in facilitating adhesion and invasion of T. cruzi (16,17), as well as conferring the parasite resistance to complement activation (18).

Previous experiments showed that injecting naive mice with small doses (0.5 µg/kg) of TS prior to T. cruzi infection greatly increased parasitemia and animal mortality (19). In addition, TS heterologously expressed in Leishmania major turned the parasites into highly virulent organisms for BALB/c and C57BL/6 mice (20). One way for TS to enhance virulence would be to directly stimulate polyclonal lymphocyte activation prior to the development of specific immune responses. To test this hypothesis, we determined whether TS is capable of inducing polyclonal activation in normal murine lymphocytes. The results show that TS is a T-independent B cell mitogen, and a stimulator of Ig secretion in vitro and in vivo. Thus, TS could facilitate progression of T. cruzi infection in acute Chagas' disease by functioning as a B cell mitogen.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Mice
C3H/HeJ mice were used in the experiments unless otherwise stated. IL-6-/- and CD40-/- mice (both in C57BL/6 background) were purchased from the Jackson Laboratory (Bar Harbor, ME), and used at 6–8 weeks of age. All the mice, including the xid-BALB/c colony, were maintained at the Tufts Animal Facility and were free of T. cruzi infection.

Reagents
The recombinant CD of TS was provided by Dr Marina Chuenkova (21). Purified recombinant trypomastigote surface antigen (TSA)-1 protein was provided by Dr Jerry Manning (22). Culture supernatants containing CD40 ligand (CD40L)–CD8 fusion protein or anti-CD8 antibody were from Dr Thomas Rothstein (Boston University School of Medicine). Lipopolysaccharide (LPS; 0127:B8) was purchased from Sigma (St Louis, MO). The C3H/HeJ-derived bone marrow macrophage cell line, BMDM-3, was a gift from Dr David Monner (National Research Center for Biotechnology, Braunschweig, Germany) (23).

Expression and purification of TS, recombinant TS and the LTR domain
TS was purified from the supernatants of T. cruzi-infected Vero cells by immunoaffinity chromatography as described (24). Recombinant TS protein was purified from bacteria expressing the entire gene of TS, clone 19y of the T. cruzi strain Silvio X-10/4 (15), using the same method for TS purification. The recombinant LTR protein was produced in insect Sf9 cells infected with engineered baculovirus (24). To remove LPS, purified proteins were passed through AffinityPack Detoxi-Gel (Pierce, Rockford, IL) following the instructions of the manufacturer. TS activity was determined as previously reported (19). One unit of TS activity is defined as the ability to catalyze the incorporation of 1.0 µmol of sialic acid into N-acetyllactosamine/min.

Cell aggregation assay
Splenocytes, bone marrow cells and thymocytes were prepared from C3H/HeJ mice and seeded at 5x106 cells/ml, in 0.15 ml DMEM/10% FBS, in 96-well plates. Cells were treated with vehicle medium, TS (4.0 µg/ml), LTR (2.0 µg/ml) or CD (2.0 µg/ml) at the beginning of culture. After 16 h, cell aggregation was observed under an IX70 Olympus reversed microscope (Olympus Optical, Tokyo, Japan) at x40 magnification.

[3H]Thymidine incorporation
Mouse splenocytes depleted of erythrocytes were seeded into flat-bottom 96-well plates (3.0x105/well) and cultured in 0.2 ml DMEM medium (Gibco/BRL, Gaithersburg, MD), supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.05 mM 2-mercaptoethanol, 1% pyruvate, 1% non-essential amino acids and 10% heat-inactivated FCS (Intergen, Purchase, NY) at 37°C in 5% CO2. Cells were treated with various concentrations of TS or its domains. For the last 2–6 h of culture, cells were pulsed with 0.5 µCi [3H]thymidine (New England Nuclear, Boston, MA)/well. Counts per minute of triplicate cultures were measured by an automated ß plate counter (TopCount; Packard, Meriden, CT). Background incorporation (usually <2000 c.p.m.) has been subtracted from presented values.

Flow cytometry
Splenocytes from normal C3H/HeJ mice were depleted of erythrocytes and stained with CD5–phycoerythrin (PE) (1.0 µg/ml) for 20 min on ice in buffer containing FcBlocker (PharMingen, San Diego, CA). After washing, cells were subject to sorting (MoFlow; Becton Dickinson, San Jose, CA) and harvested CD5- cells were >99% pure. Of these, >90% were B220+ B cells, with <0.3% contaminating CD4+ and CD8+ T cells. CD5+ B cells were reduced by 33-fold in the final population (<0.2%).

A total of 5x105 splenocytes or FACS-sorted B cells of each sample from the proliferation assay were stained with fluorochrome-labeled antibodies (B220–PE/RA3-6B2, CD5–PE/53-7.3, CD4–FITC/GK1.5 or CD8{alpha}–FITC/53-6.7) (PharMingen), according to the manufacturer's protocol. Stained cells were then washed twice and 15,000–20,000 cells were analyzed on a FACSCalibur (Becton Dickinson). Histographs were generated using FlowJo Software (TreeStar, San Carlos, CA). To track cell division, splenocytes or B cells were incubated with 2.5 µM of carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) in PBS, at 37°C for 10 min, followed by washing with ice-cold 10% FBS/DMEM 3 times. CFSE-labeled cells were then incubated with test reagents for various periods of time. At the end of incubation, the cells were stained with B220–PE and analyzed as above.

Cytokine ELISA
C3H/HeJ splenocytes from normal mice were seeded at 5x106/ml in 1.0 ml 10% FBS/RPMI in 48-well plates and stimulated with TS. Polymixin B (10 µg/ml) was added at the start of culture. Supernatants were collected at 48 h. IL-2, IL-4, IL-5, IL-6, IL-12, tumor necrosis factor (TNF)-{alpha}, IFN-{gamma} and granulocyte macrophage colony stimulating factor (GM-CSF) were assayed by ELISA (Endogen, Woburn, MA). Alternatively, splenocytes (5.0x105/96-well) were stimulated with TS or LPS, and supernatants were assayed for IL-10 by ELISA at 48, 72 and 96 h.

In vitro and in vivo stimulation of antibody secretion
Splenocytes or sorted B cells (3.5x105 cells/96-well) from normal C3H/HeJ mice were incubated with the indicated doses of TS for 72–96 h. The supernatants were removed and kept at –20°C for later titration of secreted antibodies. For in vivo stimulation, 1.0 µg TS in 0.2 ml PBS was injected (i.p.) into normal C3H/HeJ mice (two per group) daily for 3 days. The same amount of BSA was injected into a control group. Sera were collected from tail blood on days 4, 6 and 8. Total Ig in the supernatants or in the sera was measured by sandwich ELISA. Briefly, plates were coated with goat anti-mouse IgG (which also reacts with the light chains of IgM and IgA; Southern Biotechnology Associates, Birmingham, AL) (5.0 µg/ml) in PBS, pH 7.2, at 4°C overnight. For detecting TS-reactive antibodies, purified TS (4.0 µg/ml) was coated in a similar fashion. After blocking the wells with 2% BSA in PBS, pH 7.2, 50 ml samples (in 10-fold serial dilutions from 1:102–1:105) were added and incubated at 37°C for 1.5 h. Purified mouse IgG was applied as standard. Goat anti-mouse IgG (H + L)–alkaline phosphatase (Southern Biotechnology Associates) was used as detecting antibody. The color reaction was developed with p-nitrophenyl phosphate (1.0 mg/ml, in 0.1 M glycine, 1.0 mM MgCl2, 1.0 mM ZnCl2, pH 10.4) as substrate. Absorbance was determined at 405 nm on a spectrophotometric microplate reader (BioRad, Hercules, CA). Ig titers in the pre-immune sera were subtracted from measured values.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
TS and LTR induce cell aggregation, cytokine secretion and thymidine uptake in mouse splenocytes and bone marrow cells
To assess the cellular response to TS and its N- and C-domains, CD and LTR respectively, we treated bone marrow cells, splenocytes and thymocytes from naive C3H/HeJ mice with vehicle medium, TS, CD and LTR. TS strongly aggregated bone marrow cells and, to a lesser extent, splenocytes, but not thymocytes (Fig. 1AGo). Because TS binds to sialic acid, this phenomenon was thought to be mediated by the carbohydrate-binding CD fragment of the neuraminidase. Surprisingly, aggregation was not induced by CD, but, rather, by the non-catalytic domain LTR (Fig. 1AGo). TS-induced cell aggregation was accompanied by selective secretion of cytokines, in particular IL-6, in bone marrow cells (Fig. 1BGo) and IL-6 plus IFN-{gamma} in splenocytes (Fig. 1CGo). Other cytokines (IL-2, IL-4, IL-5, IL-10, IL-12, TNF-{alpha} and GM-CSF) were not detected by ELISA in TS-treated splenocytes and bone marrow cells (Fig. 1CGo). IFN-{gamma} was not detected in the supernatants of TS-treated bone marrow cells (data not shown). Similar to TS, LTR but not CD stimulated IL-6 secretion from bone marrow cells (data not shown) and from a C3H/HeJ-derived macrophage cell line, BMDM-3 (Fig. 1BGo insert), consistent with previous findings in human endothelial cells and peripheral blood mononuclear cells (24). Furthermore, TS induced [3H]thymidine incorporation in splenocytes (Fig. 1DGo) and bone marrow cells (data not shown) from LPS-resistant C3H/HeJ mice. Similar levels of incorporation were also observed in TS-treated splenocytes from normal BALB/c (Fig. 1DGo) and C57BL/6 mice (data not shown).



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Fig. 1. TS-stimulated cell aggregation, cytokine secretion and [3H]thymidine uptake. (A) Bone marrow cells, splenocytes and thymocytes from normal C3H/HeJ mice were treated for 16 h with PBS (Medium), TS (4.0 µg/ml), LTR (2.0 µg/ml) and CD (2.0 µg/ml). Original magnification: x40. (B) IL-6 levels in the supernatants of TS-treated bone marrow cells at 48 h. Insert shows IL-6 induction by LTR, but not CD, in a C3H/HeJ-derived bone marrow macrophage cell line, BMDM-3. Results are expressed as means ± SD. (C) Cytokine levels in the supernatants of TS-treated splenocytes (5x106/48-well). Fold induction is expressed as TS-induced cytokine level divided by that of medium background (41.8 pg/ml for IL-6 and 2.3 ng/ml for IFN-{gamma}). (D) [3H]thymidine uptake by TS-treated splenocytes from normal C3H/HeJ and BALB/c. Representative data of two different experiments are shown.

 
TS and LTR increase the ratio of B/T in cultured splenocytes
We used FACS to determine the splenocyte population that responded to TS. For this, splenocytes were treated with TS, and the cells were stained for markers specific to B (B220) and T (CD4 or CD8) cells. Compared with a medium control, TS increased the ratio of B220+CD4(8)-/B220-CD4(8)+ in the total live splenocytes (Fig. 2AGo), suggesting that TS increases the ratio of B cells/T cells. This phenomenon was dose and time dependent, with minimal effective concentrations of TS at 1.0–2.0 µg/ml (Fig. 2BGo), and optimal time between 72 and 96 h (Fig. 2CGo). Interestingly, a similar result could be achieved by the LTR domain of TS (Fig. 2DGo). In contrast, CD was ineffective even at equivalent or higher TS activity than the tested active range of TS. However, LTR was less effective than intact TS in increasing the B220+CD4-/B220-CD4+ ratio (Fig. 2DGo). Such differential quantitative activity was also evident in [3H]thymidine incorporation response to TS and LTR (data not shown).



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Fig. 2. TS stimulated the expansion of a B220+CD4-CD8- splenocyte population. (A) C3H/HeJ splenocytes were treated with TS (4.0 µg/ml) for 72 h. Cells were stained with B220–PE and CD4–FITC or CD8–FITC respectively. The live lymphocyte populations were gated, based on forward and side scatter. (B) Dose–response. (C) Time courses of TS effect depicted in (A). (D) Effects of TS, LTR and CD on the expansion of the B220+ population. Staining and FACS analysis were carried out as in (A). The specific TS activities of TS and CD were 63.6 and 107.1 U/mmol respectively, whereas LTR had no detectable activity. Representative data of two different experiments are shown.

 
TS and LTR, but not CD, induce B cell proliferation
Next, we tried to determine whether the increase in the B/T ratio following TS treatment was a result of activation and proliferation of B cells (B220+), an up-regulation of B220 antigen or differential loss of B220- cells. To this end, we used the cell-labeling reagent CFSE to monitor the proliferation of specific cell populations, as the green fluorescent intensity of CFSE-labeled cells reduces by half after each cell division (25). Indeed, TS induced the appearance of an activated cell population (FSChiSSCmed), in which most of the cells are proliferating B220+ B cells (Fig. 3AGo). No proliferation was detected in the resting cell population (FSCmedSSClo) for either B220+ or B220- cells (data not shown). In addition, LTR, but not CD, reproduced TS in inducing B cell proliferation in a dose-dependent manner (Fig. 3BGo). The effect of TS was specific, as another member of the TS superfamily, TSA-1, was without effect (Fig. 3BGo). Recombinant TS was as active as parasite-derived TS in stimulating B cell proliferation (data not shown), indicating that the TS action was not due to contaminants, such as glycosylphosphatidylinositol (26) or the proline racemase (9), that might have been co-purified with TS isolated from T. cruzi trypomastigotes.



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Fig. 3. TS- and LTR-stimulated B cell proliferation. (A) Upper panel: expansion of the activated splenocyte population 72 h after TS (2.0 µg/ml) treatment. Numbers represent the percentage of gated population in total splenocytes. Lower panel: reduction of CFSE intensity of B cells (B220+) in the gated population after TS treatment. (B) Dose–responses of TS, LTR, CD and TSA-1 on B cell proliferation. Numbers represent the concentrations of reagents in µg/ml that were used to stimulate cells. CFSE histograms were plotted for activated B220+ cells, gated as in (A). Medium control, shaded area; reagent-treated, bold line. Representative data of two different experiments are shown.

 
TS is a T-independent mitogen for B-2 cells
Contrary to its mitogenic effect on B cells, TS failed to directly induce proliferation of T cells (B220-) in cultures of splenocytes (Fig. 3AGo). However, TS could require T cells for optimal activation of B cells. To determine whether this is true or not, we depleted splenocytes of T cells by FACS sorting based on their high CD5 expression. T cell contamination in the depleted cell population was <0.3%, as determined by FACS analysis of CD4 and CD8 expression (Fig. 4AGo). We found that TS stimulated proliferation of T cell-depleted B cells to an extent comparable to that of splenocytes containing B cells and T cells (Fig. 4BGo). Thus, TS is a T-independent B cell mitogen.



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Fig. 4. TS is a T-independent mitogen for B-2 cells. (A) FACS analysis of the C3H/HeJ splenocyte population before and after enrichment of CD5- B-2 cells by negative sorting. (B) Dose–responses of TS on unfractionated splenocytes (upper) and sorted B-2 cells (lower). CFSE histograms were plotted for activated B220+ cells at 72 h after TS treatment. Medium control, shaded area; TS-treated, bold line.

 
Moreover, the sorted B cell population contained an overwhelming majority (>90%) of the conventional CD5- B-2 cells, with >33-fold reduction of CD5+ B-1a cells (<0.2%) (Fig. 4AGo). The above result also indicates that TS mainly stimulates B-2 cells. A similar finding was obtained with splenocytes depleted of T cells by antibody-mediated lysis. TS preferentially stimulated the proliferation of CD5- B-2 cells, with little mitogenic effect on CD5+ B-1a cells (data not shown).

B cell-stimulating activity is not due to LPS contamination
To rule out the possibility that the B cell-stimulating activities of TS and LTR were due to LPS contamination, we used splenocytes from the mouse strain C3H/HeJ in the above experiments. Because of a genetic mutation in the LPS receptor gene [Toll-like receptor (TLR)-4], these mice are highly resistant to stimulation by LPS (27). While LPS induced little proliferation of C3H/HeJ, compared to BALB/c or C57BL/6 B cells, TS- and LTR-induced proliferation was not affected (Fig. 5AGo and data not shown). Thus, TS does not utilize the LPS receptor TLR-4 (27) to activate B cells. Further supporting this conclusion, TS and LPS exhibited different patterns of induced cytokines, such as IL-10. At 72 h, TS (2.0 µg/ml) induced stronger B cell proliferation in BALB/c splenocytes than LPS (2.0 µg/ml) (Fig. 5AGo), yet unlike LPS, which induced significant amounts of IL-10, TS failed to induce detectable levels of IL-10 for up to 96 h (Fig. 5BGo).



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Fig. 5. TS- and LPS-mediated cell proliferation and IL-10 secretion in normal C3H/HeJ and BALB/c splenocytes. (A) B cell proliferation at 72 h stimulated by TS (2.0 µg/ml) and LPS (2.0 µg/ml). CFSE histograms were plotted for activated B220+ cells. Medium control, shaded area; TS- or LPS-treated, bold line. (B) IL-10 secretion in the supernatants of BALB/c splenocytes (5x105/96-well) activated by TS or LPS. Cytokine was measured by ELISA.

 
TS-stimulated B cell proliferation does not require IL-6 or CD40
Because TS and LTR induce the secretion of IL-6 (24), a multifunctional cytokine that can support B cell proliferation (28), we asked whether TS-induced B cell proliferation was IL-6 dependent. However, we found that TS-induced proliferation of IL-6-/- B cells was indistinguishable from that of wild-type B cells (Fig. 6Go), although we could detect IL-6 in TS-treated splenocyte cultures (Fig. 1CGo). Thus, IL-6 is dispensable for TS-induced B cell proliferation. Likewise, IL-6-/- and IL-6+/+ mice exhibited similar levels of splenomegaly and spontaneous ex vivo B cell proliferation during acute T. cruzi infection (data not shown), suggesting that IL-6 is not required for the infection-induced polyclonal B cell activation.



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Fig. 6. TS-stimulated proliferation of B cells deficient in IL-6 or CD40. CFSE histograms were plotted for activated B220+ cells. Dose–responses of TS-induced proliferation of wild-type, IL-6-/- and CD40-/- C57BL/6 splenocytes at 72 h. Medium control, shaded area; TS-treated, bold line. Representative data of two different experiments are shown.

 
CD40 is a TNF receptor family member that is involved in B cell activation and differentiation, T cell–antigen-presenting cell (APC) interaction, IL-6 induction and many other effector functions of APC (29). To determine whether TS-triggered B cell activation requires signaling through CD40, we compared the response of CD40-/- splenocytes with wild-type counterparts. No reduction of proliferation was observed in CD40-/- B cells stimulated over a broad concentration range of TS (0.2–2.0 µg/ml) during a 3-day period (Fig. 6Go). In a separate experiment, TS synergized with CD40L in stimulating B cell proliferation (data not shown). Therefore, TS activates B cells through a CD40–CD40L-independent pathway, which further supports TS being a T-independent B cell mitogen.

TS cannot induce proliferation of X-linked immunodeficient (xid) B cells
In attempt to identify the signaling requirements for TS action, we compared the responses to TS of B cells from wild-type and xid mice, which harbor a defect in Bruton's tyrosine kinase (Btk) (30). In contrast to wild-type B cells, xid B cells failed to proliferate when stimulated with TS (Fig. 7Go) or LTR (data not shown) for up to 4 days. In contrast, xid B cells proliferated as well as wild-type B cells in response to CD40 ligation by CD40L (Fig. 7Go). This suggests that the TS-mediated signaling pathway in B cells might require Btk.



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Fig. 7. TS-induced proliferation of wild-type, but not Btk-defective xid B cells. Splenocytes from BALB/c and xid-BALB/c were labeled with CFSE and treated with TS or CD40L for 72 h. Culture supernatant containing CD40L–CD8 fusion protein was used at 1:10 (v/v) as a source of CD40L, which was further cross-linked by anti-CD8 (supernatant used at 1:80, v/v). CFSE histograms were plotted for activated B220+ cells. Medium control, shaded area; TS- or CD40L-treated, bold line. Representative data of three different experiments are shown.

 
TS stimulates non-specific Ig secretion
To characterize the effect of TS on Ig secretion, we measured total Ig in the supernatants of TS-treated splenocytes and found that TS induced Ig secretion in a dose-dependent manner (Fig. 8AGo). TS-induced Ig secretion was independent of T cells, as FACS-sorted B cells secreted Ig in response to TS to an extent similar to that of unfractionated splenocytes (Fig. 8AGo insert). To determine whether TS augments Ig secretion in vivo, we measured serum Ig in the sera of mice injected (i.p.) with TS. Minute amounts of TS (1.0 µg/mousex3) stimulated significant Ig production in vivo over an 8-day observation period, compared to control BSA (Fig. 8BGo). The TS-induced Ig did not react with TS (Fig. 8CGo), showing that TS stimulated antibodies of unknown specificity (i.e. non-specific). The kinetics of the response showed that Ig secretion peaked 4–6 days after the first TS injection (Fig. 8BGo). This response would be too rapid for the generation of antigen-specific antibodies through T–B interaction (31). In addition, TS was injected into the mice without adjuvants. Therefore, the results are consistent with the idea that TS-induced polyclonal Ig secretion is T cell independent.



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Fig. 8. TS-induced polyclonal Ig secretion in vitro and in vivo. Splenocytes or animals of C3H/HeJ strain were used. (A) Dose curve of TS-induced Ig secretion from splenocytes treated with TS in vitro for 96 h. Insert shows Ig induction over medium background in a separate experiment in which FACS-sorted B cells (dotted) and unfractionated splenocytes (solid) were treated with 0.5 or 1.0 mg/ml TS for 72 h. (B) Time course of TS-induced total serum Ig in vivo. An aliquot of 1.0 µg TS, or BSA, in 0.2 ml PBS was injected (i.p.) into mice daily for 3 days. Sera were collected from tail blood on days 4, 6 and 8. Ig titers in the pre-immune sera have been subtracted from the presented values. (C) TS-induced non-specific Ig secretion in vivo. The Ig titers of day 6 sera from (B) were shown. Results are expressed as means ± SD. Representative data of two different experiments are shown.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Faced with infectious agents encoding a myriad of different antigens, the mammalian immune system is not always efficient in eliminating the pathogens, particularly in the acute phase of the infection. It is thought that polyclonal amplification of immune responses by microbial mitogens or superantigens serves to mask/deviate the specific reactions against pathogens (1). Indeed, polyclonal lymphocyte activation has been observed in infections with many types of microbes (1) and in T. cruzi infection, such polyclonal activation (3,5,32) correlates with disease progression (7,8). Very little is known about the identity and mechanism of action of T. cruzi molecules that direct polyclonal activation (9,10).

We have found that the TS of T. cruzi trypomastigotes can greatly enhance virulence in animal models. Sensitization of mice with small doses (0.5 µg/kg) of TS prior to T. cruzi infection increased virulence, as assessed by parasitemia and animal mortality (19), as did heterologous TS expression in L. major (20). In pursuing this phenomenon, we now find that TS is a polyclonal B cell activator and non-specific Ig inducer. The mitogenic effect of TS is independent of T cells, as TS could induce proliferation and Ig secretion in highly purified B cells to the same extent as in unfractionated splenocytes (Fig. 4Go). Supporting this conclusion, TS induced undiminished B cell proliferation in the absence of CD40 (Fig. 6Go). CD40 is known to be a key molecule to deliver T cell help during T–B cell cognition (29). We hypothesize that, as a T-independent B cell mitogen, TS can alter host immune regulation before the onset of adaptive responses (19).

A possible link between B cell expansion and virulence enhancement has been reported in models of Leishmania infection. For instance, continual administration of anti-IgM antibody, which causes B cell depletion, enhanced resistance to L. tropica and L. mexicana in BALB/c mice (33). On the other hand, administration of IL-7, a B cell hematopoietic factor, was shown to markedly increase B cell number and exacerbate L. major infection (34). Similarly, co-transfer of B cells converted T cell-reconstituted L. major-resistant, C.B-17 scid mice into a susceptible phenotype (35). In this context, our observations that TS-transgenic L. major were highly virulent (20) and that TS-sensitized mice became more susceptible to T. cruzi infection (19) are very likely related to TS being a B cell mitogen.

Although B cells are required for the immunity against T. cruzi (36,37), deregulated activation of B cells that secrete Th2-promoting cytokines (e.g. IL-10) might tip the Th1/Th2 balance toward a Th2-dominant one. As Th1-, but not Th2-mediated responses are crucial for effective immunity against T. cruzi (38,39), a B cell mitogenic response might correlate with immune deviation of a proper type of Th response (34). Another possibility is that polyclonal activation of B cells, whose specificities are not against parasite, could mask and blunt the parasite-specific responses. These alterations should conceivably allow T. cruzi to establish infection and underlie autoimmune responses in chronic Chagas' disease.

Interestingly, the C-terminal LTR, but not the N-terminal CD of TS, is the active moiety that causes cell aggregation, B cell activation and IL-6 secretion (24), although the specific activity of LTR is much lower than that of TS. There are two possibilities for the lower activity of LTR. First, in TS, the sialic acid-binding CD domain may help enrich LTR at the cell surface. Second, the binding of CD to cell surface sialic acid may also help LTR cross-link its receptor to a higher degree. Thus, without CD, the effective concentration of LTR at the cell surface and the ability to cross-link its receptor would be much lower than those of TS.

It was postulated that the highly antigenic tandem repeats in TS and in certain other parasite proteins function as molecular bait to attract host antibody attack, thus deviating the humoral response away from domains important for infection (40). As a result, many previous studies focused on the immune responses triggered by TS or other TS family members that function as T cell-dependent antigens during infection or immunization (41–43). However, we show here that the tandem repeats of TS can directly modulate immune responses of unprimed cells and thus TS is not just a bystander antigen being passively recognized as an immunodominant protein by host immune cells. Our present findings suggest that parasitokines (parasite-derived mimetics of host cytokines/growth factors) can cause immune deregulation in a T cell-independent manner (W. Gao and M. A. Pereira, in press).

Clearly, it will be fruitful to direct future efforts to identifying the receptor for LTR in order to elucidate the mechanism of TS action. Although TS does not utilize TLR-4 to stimulate B cells, it is worth investigating the involvement of other TLR family members, which play important roles in the innate immunity against pathogens (44). On the other hand, identifying the signaling requirements for TS will help pinpoint TS-targeted receptors. In the present study, compared with wild-type B cells, xid B cells were severely dampened in response to TS and LTR (Fig. 7Go and data not shown). xid mice have an R28C mutation in Btk (30) and lack CD5+B-1a cells (45). There are many reasons why the relatively immature xid B cells do not respond to TS. This phenomenon is unlikely, however, due to the absence of CD5+ B-1a cells, because TS preferentially activated conventional CD5- B-2 cells (Fig. 4Go). It is more likely that Btk mediates the activation of B-2 cells in response to TS.

Btk is known to be a downstream effector of the IL-5 receptor (46), CD38 (47) and the BCR (4849). In preliminary experiments measuring Ca2+ mobilization, we found that while anti-IgM F(ab')2 induced a strong Ca2+ influx within 1 min, TS at concentrations that induce vigorous B cell proliferation failed to trigger any Ca2+ response for up to 10 min nor did it modify the anti-IgM-induced Ca2+ response (data not shown). Further experiments need to be carried out to determine whether TS can induce BCR-dependent Ca2+ influx at later time points. Nevertheless, because TS and its LTR activate cells that do not express membrane Ig, such as endothelial cells, epithelial cells (24) and macrophages (Fig. 1BGo insert and data not shown), receptors other than BCR that transmit Btk-dependent cell activation signals are potential TS targets.

Finally, it is unlikely that deregulation of the immune responses by TS would be confined only to the B cell compartment during infection. This is because activated B cells are efficient APC in co-stimulating T cell activation (50,51). Therefore, TS might potentiate in vivo T cell response to antigen-specific or non-specific stimuli through activating APC and secreting cytokines (such as IL-6), as suggested in an in vitro study (52). Thus, TS may play a pivotal role in acute phase polyclonal B and T cell activation in T. cruzi infection, potentially contributing to the virulence-enhancing activity of the enzyme. As the level of B cell activation and proliferation induced by the LTR is an order of magnitude lower than TS, inducing a specific and protective immunity after vaccination with the LTR might be achieved without the risk of triggering non-specific responses. The efficacy of such a strategy to curtail TS-mediated polyclonal lymphocyte activation warrants further investigation.


    Acknowledgments
 
We thank Drs David Monner for the C3H/HeJ macrophage cell lines (BMDM-3 and -4), Marina Chuenkova for CD protein, Jerry Manning for TSA-1 protein and Isabel Tussie-Luna for the recombinant TS construct. We also thank Drs Thereza Imanishi-Kari and Thomas Rothstein for reagents, Allen Parmelee for FACS sorting, and Lei Jin for measuring the Ca2+ influx. This work was supported by NIH grant AI 18102 (to M. A. P.).


    Abbreviations
 
APC antigen-presenting cell
Btk Bruton's tyrosine kinase
CD catalytic domain
CFSE carboxyfluorescein diacetate succinimidyl ester
GM-CSF granulocyte macrophage colony stimulating factor
LTR long tandem repeat domain
LPS lipopolysaccharide
PE phycoerythrin
TLR Toll-like receptor
TNF tumor necrosis factor
TS trans-sialidase
TSA trypomastigote surface antigen
xid X-linked immunodeficiency

    Notes
 
Transmitting editor: E. Clark

Received 2 August 2001, accepted 3 December 2001.


    Reference
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 

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