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Keywords:

  • Monocytes;
  • Macrophages;
  • Parasitic-protozoan;
  • Cytokines;
  • Lipopolysaccharide

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Secretion of proinflammatory mediators by activated macrophages plays an important role in the immune response to Trypanosoma cruzi. We have previously reported that AgC10, a glycosylphosphatidylinositol-anchored mucin from T. cruzi, inhibits TNF secretion by activated macrophages (de Diego, J., Punzon, C., Duarte, M. and Fresno, M., Alteration of macrophage function bya Trypanosoma cruzi membrane mucin. J. Immunol. 1997. 159: 4983–4989). In this report we have further investigated the molecular mechanisms underlying this inhibition. AgC10 inhibited TNF, IL-10 and cyclooxygenase-2 (COX-2) synthesis by macrophages activated with LPS or LPS plus IFN-γ in a dose-dependent manner. AgC10 did not affect other aspects of macrophage activation induced by LPS, such as inducible nitric oxide synthase (iNOS) expression. AgC10 also had no effect on TNF or COX-2 transcription or the induction of their promoters but inhibited the stability of TNF and COX-2 mRNA, which are regulated post-transcriptionally by the mitogen-activated protein kinase (MAPK) p38 pathway. AgC10 was found to inhibit both the activation and the activity of p38 MAPK, since MAPK activated protein kinase-2 (MAPKAP-K2 or MK-2) phosphorylation was also strongly inhibited. This led to TNF and COX-2 mRNA destabilization. In contrast, AgC10 did not affect p38 activation induced by TNF. Furthermore, AgC10 inhibition must lie upstream in the MAPK activation pathway by LPS, since this mucin also inhibited extracellularly regulated kinase (ERK) and Jun kinase (JNK)activation.

Abbreviations:
COX-2:

Cyclooxygenase-2

ERK:

Extracellularly regulated kinase

iNOS:

Inducible nitric oxide synthase

JNK:

c-jun terminal kinase

MAPK:

Mitogen-activated protein kinase

MAPKAP-K2:

MAPK-activated protein kinase-2

MKK:

MAPK kinase

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, which affects several million people in South and Central America 1. Clinically, T. cruzi infection proceeds in two phases, the acute phase in which circulating blood trypomastigotes and local inflammation at the sites of infection are observed, and the chronic phase, where parasitemia does not exist but progressive tissue damage occurs in esophagus, colon and heart 1. At least three morphologically different forms appear in its life cycle: epimastigote, which replicates in the insect gut; metacyclic and blood trypomastigote, the forms able to infect mammalian cells; and amastigote, the replicative form inside the host cells, including macrophages 2.

Macrophages also play a crucial role in the elimination of this parasite. Activation of monocytes by cytokines released by Th1 cells is a key process in controlling infection "in vitro" as well as »in vivo" 3. TNF and IFN-γ are the most important cytokines involved in the killing of intracellular T. cruzi through an NO-mediated, L-arginine-dependent killing mechanism [4, 5. On the other hand, parasites have evolved sophisticated systems to evade the immune system 6, and T. cruzi is probably one of the best examples of adaptation to the host. The entry and replication of T. cruzi inside macrophages is required for its dissemination in the host 7, but T. cruzi has the capacity to evade the protective immune response produced by macrophages by escaping from the phagolysosome to the cytoplasm and by altering the profile of secreted cytokines such as TNF or IL-10 8, 9.

T. cruzi mucins comprise a heterogeneous group of surface molecules required for the interaction with host cells during invasion 10. Specifically, they play a role in invasion and survival inside host cells and in the modulation of macrophage functions 8, 11. There are at least two sets of mucin-like molecules in T. cruzi. One group includes mucins present in the insect forms (metacyclic and epimastigotes) that migrate in the 35–55 kDa range 8, 12. The other group includes mucins from tissue-derived or blood trypomastigotes that migrate as a broad band of 60–200 kDa 10. In all cases the protein portions of these mucins are encoded by a heterogeneous gene family 13, which appears to be differentially expressed in the different stages 14. The carbohydrate structure and the lipid nature of their GPI anchors, which define their biological activity, also differ between the different stages 10.

AgC10, a 40–50 kDa T. cruzi mucin purified from epimastigotes and metacyclic trypomastigotes, is able to inhibit TNF and IL-12 secretion after macrophage activation 8. In this manuscript we have characterized the molecular mechanisms by which AgC10 deactivates macrophages, leading to TNF and cyclooxygenase-2 (COX-2) synthesis inhibition. Our results show that AgC10 destabilizes TNF and COX-2 mRNA by inhibiting activation of p38 mitogen-activated protein kinase (MAPK) and its substrate MAPK-activated protein kinase-2 (MAPKAP-K2). Macrophage stimulation with LPS activates these kinases, which have been previously described as important regulators of TNF and COX-2 mRNA stability 15, 16. These results represent a previously undescribed mechanism in which a parasite surface molecule is able to avoid the release of proinflammatory mediators that play a crucial role in the elimination of T. cruzi.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 AgC10 inhibits TNF and COX-2 protein expression in activated macrophages

We had previously shown that AgC10 inhibits LPS-induced TNF secretion by human monocytes 8. To further analyze the molecular basis of this effect, we used Raw mouse macrophage cells as a model. We observed that the morphological changes induced by LPS or LPS plus IFN-γ were inhibited by AgC10 (Fig. 1). AgC10 treatment in the absence of stimuli neither produced morphological change of Raw cells nor affected cell viability (data not shown). We then tested whether AgC10 affects TNF and COX-2 synthesis upon stimulation. Unstimulated Raw cells did not produce amounts of TNF detectable by ELISA in supernatants, while substantial amounts of TNF were detected 6 h after stimulation with LPS, and levels were five to six times higher in supernatants obtained after 16 h of stimulation. As previously observed with human macrophages 8, AgC10 inhibited TNF release by Raw cells in a dose-dependent manner. Interestingly, this inhibition was more pronounced at longer times after stimulation (60% at 10 μg/ml of AgC10 after 16 h stimulation versus 10% after 6 h stimulation) (Fig. 2A). IL-10 induction was also inhibited by AgC10 (Fig. 2B).

COX-2 protein induction after macrophage activation was also inhibited by AgC10, while the synthesis of inducible nitric oxide synthase (iNOS), another proinflammatory molecule induced in macrophages by LPS or LPS plus IFN-γ stimulation, was not affected by AgC10 (Fig. 3A). Similar inhibition of COX-2 protein induction (Fig. 3B) and TNF (not shown) was observed with LPS-activated mouse peritoneal macrophages treated with AgC10, indicating that its inhibitory effect on Raw cells was not due to a peculiar feature of those cells.

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Figure 1. Inhibition of LPS-induced cell morphological changes in Raw cells by AgC10. Raw cells (105 cells/ml) in RPMI/2% FCS were left to adhere for 2 h. Cells were then stimulated with LPS (2 μg/ml) alone or in combination with IFN-γ (10 U/ml) for 16 h and/or treated with AgC10 (15 μg/ml). Activated cells are larger and adhere stronger to the plastic. AgC10 was added to the culture 45 min before the stimuli, but similar results were obtained when AgC10 was added at the same time as the stimuli (data not shown). Representative fields at the optic microscope (×10) are shown.

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Figure 2. Effect of AgC10 on TNF and IL-10 secretion by Raw cells in response to LPS. (A) Raw cells were stimulated with LPS (2 μg/ml) for 6 h or 16 h and treated with the indicated dosis of AgC10. TNF concentration was measured in the supernatants. (B) IL-10 concentration in supernatants after 6 h of stimulation. Values show the mean of duplicates ± standard deviation of a representative experiment of three performed.

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Figure 3. Effect of AgC10 on COX-2 and iNOS protein expression in activated macrophages. Macrophages were stimulated for 16 h with LPS (2 μg/ml) alone or in combination with 10 U/ml IFN-γ and treated with AgC10 (15 μg/ml) where indicated. Protein expression was determined by Western blot analysis using anti-COX-2 and anti-iNOS in Raw cells (A) or mouse peritoneal macrophages (B). Anti-β-actin was used as a loading control. Numeric values indicate the relative absorbance of the specific proteins. A representative experiment of three performed is shown.

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2.2 AgC10 inhibition of TNF and COX-2 synthesis takes place at a post-transcriptional level

The above results suggested that AgC10 inhibition on macrophage activation was not taking place at early steps of LPS activation. To address the step affected by AgC10, we first analyzed its effect on TNF and COX-2 promoter driven transcription in transiently transfected Raw cells. LPS induced a substantial activation of a TNF promoter driven luciferase reporter, whereas the activation of a COX-2 promoter was less pronounced. None of those inductions was significantly altered by AgC10 (Fig. 4A). Run-on assays were performed to investigate if TNF or COX-2 RNA de novo synthesis was affected by AgC10. LPS-stimulated de novo TNF or COX-2 transcription, which was comparable in the presence or absence of AgC10 (Fig. 4B). Those results suggested an effect of AgC10 on TNF and COX-2 post-transcriptional regulation.

TNF mRNA accumulation in Raw cells after activation with LPS or with LPS plus IFN-γ was inhibited by AgC10, in the same way that protein accumulation was (Fig. 5A). TNF mRNA induction by LPS in mouse peritoneal macrophages was also inhibited by AgC10 in a dose-dependent manner (Fig 5B). TNF mRNA was already expressed at high levels 1 hr after LPS activation, reaching a plateau at 3 h that was maintained up to 6 h and diminishing thereafter (Fig. 5C). However, AgC10 did not significantly inhibit the early TNF mRNA induction after 1 h of LPS stimulation. It required at least 3 h of stimulation to detect a significant inhibition, which was maintained after longer activation times (Fig. 5C). AgC10 also inhibited COX-2 mRNA accumulation induced in response to LPS in a dose-dependent manner in both Raw cells (Fig. 5D) and mouse peritoneal macrophages (Fig. 5E).

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Figure 4. AgC10 does not affect TNF and COX-2 transcription. (A) Raw cells were transiently transfected with the human TNF (pTNFluc) or COX-2 (pCOX-2luc) promoters coupled to luciferase, stimulated with LPS (2 μg/ml) and treated with AgC10 (15 μg/ml) where indicated for 16 h. Luciferase activity was quantified and normalized by protein concentration in each sample. Data shown represent the mean ± standard deviation of three different experiments. (B) Raw cells were stimulated and treated with AgC10 as in (A), and nuclei were analyzed for de novo-transcribed TNF, COX-2 or β-actin by nuclear run-on experiments, where pcDNA3 was used as a control plasmid. A representative experiment is shown. Numeric values shown indicate the densitometric scanning of the spots.

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Figure 5. AgC10 inhibits TNF and COX-2 mRNA accumulation in activated macrophages. (A) Raw cells were stimulated for 6 h with LPS (2 μg/ml) alone or combined with IFN-γ (10 U/ml) and treated with AgC10 (15 μg/ml) where indicated. RNA was isolated, run on agarose gels and transferred to nylon membranes that were hybridized with TNF and β-actin probes. The obtained bands were scanned in a densitometer, and RNA was quantified. Bars under the corresponding gels represent TNF absorbance units with respect to the β-actin control. (B) Mouse peritoneal macrophages were stimulated for 6 h with LPS (2 μg/ml) alone or with AgC10 (2 μg/ml or 15 μg/ml). TNF and β-actin RNA were detected by RT-PCR. (C) Raw cells were stimulated with LPS (2 μg/ml) for the indicated times and treated with AgC10 (15 μg/ml) where indicated, and mRNA was analyzed as above. (D) Raw cells were stimulated for 6 h with LPS (2 μg/ml) alone or with AgC10 (1, 5, 15 and 20 μg/ml). COX-2 and β-actin RNA were amplified by RT-PCR. (E) Mouse peritoneal macrophages were stimulated and treated with AgC10 as described for Raw cells in (D) and RNA was detected by RT-PCR. Representative experiments of the three performed for each assay are shown.

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2.3 AgC10 destabilizes TNF and COX-2 mRNA

TNF and COX-2 expression are also regulated through mRNA stability 17. Thus, a suitable hypothesis compatible with the above results was that AgC10 affects TNF and COX-2 at the level of mRNA stability. To test this, we stimulated Raw cells with LPS and then stopped cellular transcription with Actinomycin D in the presence or absence of AgC10. AgC10 diminished the half-life of TNF mRNA as compared with control values of β-actin mRNA. This was detected both by Northern blot and semiquantitative RT-PCR analysis (Fig. 6). A similar effect was observed when COX-2 mRNA values were assayed by semiquantitative RT-PCR (Fig. 6B).

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Figure 6. AgC10 destabilizes TNF and COX-2 mRNA. Raw cells were stimulated with LPS (2 μg/ml) for 1 h and treated with AgC10 (20 μg/ml) where indicated. Cell transcription was stopped at 10, 20, 30 or 40 min after stimulation with actinomycin D (2 μg/ml, added to the culture 45 min before the indicated times). TNF mRNA expression was analyzed in (A) by Northern blot (performed as explained in Fig. 5A), or in (B) by RT-PCR (as explained in Fig. 5B), together with COX-2 mRNA expression and β-actin as control. The obtained bands were scanned in a densitometer and RNA was quantified. The graph represents TNF absorbance units with respect to its β-actin control.

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2.4 AgC10 inhibits p38 and MAPKAP-K2 activation

In response to LPS and various proinflammatory stimuli, MAPK p38 is activated and phosphorylated by upstream MAPK kinases (MKK3 or MKK6) 18, 19. In turn, p38 phosphorylates and activates MAPKAP-K2 20. This signal transduction cascade is required for the expression of several proinflammatory genes, including TNF and COX-2, and exerts its effects, at least in part, through mRNA stability 15, 16. To test if AgC10 affects p38 activation, we performed Western blot analysis of activated macrophages using anti-phosphorylated p38 antibody that recognizes the phosphorylated and active forms of this kinase. As expected, LPS induced p38 activation in Raw cells, as shown by a strong increase in the amount of phosphorylated p38 (Fig. 7A). AgC10 completely inhibited this induction. The total amount of p38 was not affected by LPS treatment or AgC10. Similar results were obtained using mouse peritoneal macrophages, where AgC10 strongly inhibited LPS-induced p38 phosphorylation without affecting total p38 levels (Fig. 7B). The phosphorylation of MAPKAP-K2, detected with an anti-phospho-MAPKAP-K2 antibody, was also induced in Raw and peritoneal macrophages after LPS stimulation (Fig. 7C, D). Moreover, treatment of macrophages with AgC10 strongly inhibited phosphorylation of MAPKAP-K2 in both cell types, confirming that AgC10 suppresses p38 activity induced by LPS. The observed effects on the p38 MAPK pathway were prevented when AgC10 was neutralized with the specific monoclonal antibody mAbC10 8, confirming the specificity of AgC10 inhibition on p38 phosphorylation. mAbC10 treatment itself, or in combination with AgC10, did not affect p38 phosphorylation. The effect of AgC10 seems to be specific for the TLR4/LPS signaling pathway, since no effect of AgC10 on induced p38 phosphorylation was observed when cells were stimulated with TNF (Fig. 7E). Furthermore, the observed AgC10 inhibitory effects on p38 activation lie further upstream where the different MAPK activation pathways converge, since the activation of extracellularly regulated kinase (ERK) and c-jun terminal kinase (JNK) induced by LPS in Raw macrophages and detected by Western blot analysis using anti-phosphorylated JNK and ERK was also inhibited by AgC10 (Fig. 8).

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Figure 7. AgC10 inhibits MAPK p38 activation and activity upon macrophage stimulation by LPS. Raw cells (A, C, E) or mouse peritoneal macrophages (B, D) were stimulated where indicated with LPS (2 μg/ml; at 10 min in A and E, and at 20 min in D) or during 20 min with TNF (100 U/ml) in the presence or absence of AgC10 (20 μg/ml) and/or mAbC10 (15 μg/ml). Cells were harvested, and Western blotting was performed using antibodies specific for phosphorylated p38 (A, B, E), phosphorylated MAPKAP-K2 (C, D) and total p38 as loading control. Numeric values indicate the relative absorbance of the specific proteins. Representative experiments of the three performed for each assay are shown.

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Figure 8. Effect of AgC10 on the MAPK JNK and ERK activation. Raw cells were stimulated for 10 min with LPS (2 μg/ml) and treated with AgC10 (20 μg/ml) where indicated. Cells were harvested, and Western blotting was performed using antibodies specific for phosphorylated JNK (A) or phosphorylated ERK (B). β-actin antibody was used as loading control. Numeric values indicate the relative absorbance of the specific proteins. Representative experiments of the two performed for each assay are shown.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Induction or inhibition of cytokine production by T. cruzi plays a very important role in the regulation of both the resistance to and the pathology of the infection caused by this parasite 3. Several parasite molecules responsible for those processes have been identified. We previously identified AgC10, a T. cruzi surface mucin abundantly expressed at the cell surface of epimastigotes, metacyclic trypomastigotes and amastigotes that can be released during T. cruzi infection 21, as a molecule capable of inhibiting the function of human macrophages by altering TNF and IL-12 production in response to LPS 8. Those cytokines play a very important role in resistance to T. cruzi infection 6, 22. The important role of this mucin in T. cruzi-mediated macrophage deactivation was reflected by the fact that adding neutralizing antibodies to AgC10 before infecting human macrophages substantially decreased the inhibition of parasite infection on cytokine production 8. These data suggested that interaction of AgC10 with its host cell surface receptor modulates TNF and IL-12 synthesis in infected cells.

Here we have characterized the molecular mechanism by which AgC10 exerts this inhibition. AgC10 inhibits both the morphological changes associated with macrophage activation and the synthesis of TNF and COX-2 upon LPS activation. Interestingly, AgC10 affected the mRNA stability, but not the transcription, of TNF and COX-2. TNF and COX-2 promoter induction and de novo RNA synthesis were not affected, as measured by the run-on assays, supporting the conclusion that AgC10 does not affect LPS-induced transcription but does, in fact, affect post-transcriptional steps. Moreover, we found that the effect of AgC10 on TNF release and TNF mRNA, which was barely detectable at early times after activation, became more pronounced over time. Experiments performed with Actinomycin D confirmed that the stability of TNF and COX-2 mRNA is indeed affected by AgC10. This fits with the fact that the synthesis of these two molecules is post-transcriptionally regulated in a similar, if not identical way. The effect of AgC10 on TNF release seems to be direct and not due to a putative induction of anti-inflammatory cytokines such as IL-10, which is able to inhibit TNF induction 23 and was also inhibited by AgC10.

mRNA levels depend on the ratio of the rates of synthesis and degradation. Therefore, mRNA stability regulation is an important means of modulating TNF and COX-2 gene expression 17. TNF mRNA stability is mediated by cis-acting adenosine/uridine-rich elements (ARE) within the 3′UTR. Deletion of ARE from the mouse genomic TNF locus results in elevated basal and LPS-induced TNF expression, chronic inflammatory arthritis and inflammatory Bowel disease 24. COX-2 mRNA contains 3′UTR similar to those of TNF and is post-transcriptionally modulated in a similar way 17. The signal transduction pathways leading to regulation of TNF and COX-2 mRNA stability have been recently elucidated in myeloid cells activated by LPS 15, 25; after LPS stimulation, p38 MAPK is activated through phosphorylation by upstream MKK3 or MKK6. Phosphorylated p38 MAPK in turns phosphorylates MAPKAP-K2, which is thought to mediate the effects of p38 on TNF mRNA stability. Pharmacological inhibition of p38 activity by SB203580 inhibits this process, blocking in turn MAPKAP-K2 phosphorylation and TNF and COX-2 mRNA stability (data not shown and 26). AgC10 suppresses LPS-induced p38 activation, measured as p38 phosphorylation, without affecting total p38 levels. As a result of this inhibition, AgC10 treated cells are unable to phosphorylate MAPKAP-K2.

How AgC10 affects the LPS-induced pathways in macrophages, leading to p38 inhibition, is unknown and is currently under investigation. AgC10 is clearly not a general deactivating agent and its target on macrophages should not be proximal to LPS receptor engagement, since AgC10 did not inhibit other LPS-induced events such as transcription of TNF and COX-2 genes or iNOS synthesis. TNF 27, iNOS 28 and COX-2 29 have NF-κB sites in their promoters, which are able to bind the NF-κB transcription factor once it is liberated from the inhibitory molecule IκB after its degradation due to LPS activation 30. Preliminary experiments indicate that AgC10 does not inhibit NF-κB activation or IκB degradation (data not shown). This fits with the lack of an effect on LPS-induced TNF and COX-2 transcription. On the other hand, the post-transcriptional regulation of iNOS seems to be different from that of TNF and COX-2 mRNA 31. Moreover, the p38/MAPKAP-K2 pathway is not involved in LPS-induced iNOS expresion in Raw macrophages 32. Thus, the divergent results of the AgC10 effect on iNOS and COX-2 strongly support a rather specific effect of AgC10 on the p38 pathway leading to TNF and COX-2 mRNA destabilization. In addition, the p38 upstream effector affected by AgC10 must be common to the p38, JNK and ERK activation pathways, since they are also strongly inhibited by AgC10. AgC10 inhibition of p38 MAPK seems to be specific for the LPS stimulation pathway, because no inhibition was observed when cells were stimulated with TNF. This result, apart from confirming the specificity of AgC10, also poses several questions regarding the molecular target of AgC10. Thus, AgC10 may inhibit a common inducer of the three MAPK pathways upon LPS stimulation that is different from the one induced by TNF. In this regard, receptor proximal events of the two pathways are known to be different in macrophages 33. Thus, a putative candidate will be an MKK kinase, such as Cot/Tpl-2, which has been shown to be induced by LPS, since Cot/Tpl-2 is involved in the post-transcriptional regulation of TNF 34 and COX-2 35 and is able to activate all three MAPK (p38, JNK and ERK). The inhibitory effects of AgC10 on macrophage morphology (spreading, adherence, etc.) induced by LPS are also likely due to inhibition of MAPK pathways.

We previously demonstrated that AgC10 is able to bind human macrophages through L-selectin 8, although those results did not exclude the existence of another receptor. The mannose receptor could be another receptor in macrophages, since AgC10 contains many mannose residues. However, none of those receptors are significantly expressed in Raw cells. Experiments are in progress to elucidate the receptor.

Manipulation of host cell MAPK pathways may represent a strategy evolved by several pathogens to evade the immune response generated against them, since these kinases control expression of many genes involved in the innate and adaptive immune responses 36. However, very few examples of pathogens subverting this pathway have been described. In this regard, it has been shown that intracellular pathogens such as Yersinia, which infect macrophages, have virulence factors that deactivate macrophages through p38 inhibition, preventing TNF secretion 37. Bacillus antracis releases the anthrax lethal toxin, a major virulence factor that proteolytically cleaves MKK1 and MKK2, inactivating them and leading to inhibition of the ERK signal transduction pathway in macrophages 38. Moreover, Leishmania donovani evade the activation of p38, JNK and ERK during infection of naive macrophages 39. However, none of these effects have been linked to a decrease in TNF mRNA stability.

On the other hand, glycoinositolphospholipids from insect forms are able to reduce TNF secretion by human macrophages stimulated with LPS 40. We have not directly addressed the role of the glycosylphosphatidylinositol anchor, but the fact that AgC10 can be released by the enzyme phosphoinosytol-phospholipase C (PI-PLC) from the T. cruzi membrane and this supernatant was also suppressive (data not shown) is suggestive that this is not the case. In contrast with our results, others have shown that mucins from T. cruzi blood trypomastigotes are potent stimulators of macrophage proinflammatory activity 10. It is noteworthy that AgC10 is present in the insect infective metacyclic forms and not in blood trypomastigotes 8, in sharp contrast with the immunostimulatory mucins present in blood trypomastigotes 14. How these different outcomes are related to survival and/or replication in macrophages of metacyclic versus blood trypomastigotes "in vivo" is presently unclear. It is likely that metacyclic trypomastigotes must evade macrophage activation to gain entry into the mammalian host; activation of the MAPK pathways may lead to an undesirable host response such as secretion of proinflammatory cytokines, which are detrimental for the parasite. Thus, evasion of this pathway by AgC10 represents a novel virulence strategy that may contribute to the entry and dissemination of the parasite in the mammalian host. In addition, at the early steps of T. cruzi infection, there is a clear indication that the cytokines produced by macrophages dictate the subsequent type of immune response 3. To develop a protective immune response, macrophages must secrete cytokines, such as TNF, that are involved in protection against the parasite 6, 22. By altering the ability of macrophages to secrete proinflammatory cytokines through the abundant surface molecule AgC10, T. cruzi modifies the protective immune response to its own benefit, enabling its persistence in the infected host.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Antibodies and reagents

Anti-iNOS and anti-COX-2 antibodies were from BD-Transduction; anti-phosphorylated p38, anti-ERK and anti-MAPKAP-K2 were from Cell Signaling; and anti-β-actin and the phosphorylated JNK were from Santa Cruz Biotechnology. Monoclonal mAbC10 specific for AgC10 was obtained in our laboratory as previously described 21. The human TNF promoter construct (pTNFluc, from position –1311) and the COX-2 promoter (pCOX-2luc, between –1796 and +104), both coupled to luciferase, were previously described 41, 42.

4.2 Isolation and culture of peritoneal macrophages

Peritoneal macrophages were isolated by peritoneal lavage from BALB/c mice 4 days after a single peritoneal injection of 10% thioglycolate solution (1 ml; Difco Laboratories). Cells (1.5×106/well) were left to adhere for 1 h in 12-well flat-bottomed plates and covered with fresh RPMI/0.5% FCS in the presence or absence of the indicated amount of LPS (026.B6 E. coliserotype, Sigma) and/or AgC10.

4.3 AgC10 purification

AgC10 was purified from T. cruzi stationary-phase epimastigotes and metacyclic trypomastigotes as described 8. Briefly, 1012 parasites were washed with PBSand extracted twice with three volumes of chloroform/methanol/water (10:20:8 v/v/v) at room temperature. Supernatants were dried and partittioned in 1-butanol/H2O (2:1 v/v). Aqueous phaseswere lyophilized and resuspended in ammonium acetate (200 mM) containing 5% 1-propanol to be loaded onto a column of octyl Sepharose CL4B equilibrated in the same buffer. A gradient of 1-propanol was applied to elute the column. Collected fractions were tested by ELISA or Western blot with mAbC10. Positive fractions were lyophilized and resuspended in PBS. The procedure leads to 98% pure AgC10. AgC10 was used at 15 μg/ml in most experiments, which corresponds roughly to 2×107 parasites/ml 21.

4.4 Cell culture and transfection

Raw 264.7 cells (1.5×106) maintained in RPMI 1640 medium containing 5% FCS (GIBCO Laboratories, Grand Island, NY) were transfected with 0.5 μg pCOX2luc or pTNFluc using LipofectAMINEPLUSTM Reagent (Invitrogen) following the manufacturer's instructions and cultured in complete RPMI/2% FCS for 16 h in the presence or absence of LPS (2 μg/ml) and/or AgC10 (15 μg/ml). Cells were lysed with lysis buffer from the "Luciferase Assay System Kit" (Promega), and luciferase activity was measured.

4.5 Western blotting assays

Raw cells or peritoneal macrophages (5×106 cells/well) were cultured in 12-well flat-bottomed plates with LPS (2 μg/ml) and/or IFN-γ (10 U/ml) or TNF (100 U/ml) in the presence or absence of AgC10 (15 μg/ml) for the appropriate times (10, 20 or 30 min to study MAPK activation and 16 h for COX-2 and iNOS expression). Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% Triton X-100, 15 mM NaCl, 0.5% deoxyglicolate, 0.1% SDS and 10 mM NaF; the protease inhibitors aprotinin and leupeptin at 2 μg/ml, 1 μM pepstatin and 1 mM PMSF; and 100 μM of the phosphatase inhibitor Na3VO4) for 30 min at 4°C, and supernatants were collected after centrifugation. The extracts (20 μg) were separated by SDS-PAGE (10% polyacrylamide) and subjected to Western blot with the appropriated antibodies.

4.6 RNA analysis

RNA was isolated by using TRIzol reagent (Life Technologies) following the manufacturer's instructions. For mRNA stability experiments, 3×106 cells were seeded in 12-well plates and treated for 1 h with LPS (2 μg/ml) and/or AgC10 (added 1 h before LPS). After stimulation, cells were treated for 10, 20, 30, or 40 min with ActD (2 μg/ml). mRNA was analyzed by Northernblot or semiquantitative RT-PCR. Total RNA (20 μg) were separated by agarose-formaldehyde, transferred to a nylon membrane and hybridized to radiolabeled cDNA fragments for TNF and β-actin.A 0.9 Kb NarI-BglII genomic probe containing part of the first exon of mTNF gene was used to detect TNF mRNA, and a 0.9 Kb mouse Pst β-actin fragment was used as a quantitative control. The sense and anti-sense primers used to detect TNF, COX-2 and β-actin by RT-PCR were synthesized as follows: TNF sense (5′-3′): CTC CAG CTG GAA GAC TCC TCC CAG; TNF anti-sense (5′-3′): GAT CTC AAA GAC AAC CAA CTA GTG; β-actin sense (5′-3′): CTC TTT GAT GTC ACG CAC GAT TTC; β-actin anti-sense (5′-3′): GTG GGC CGC TCT AGG CAC CAA; COX-2 sense (5′-3′): TTC AAA AGA AGT GCT GGA AAA GGT; COX-2 anti-sense (5′-3′): GAT CAT CTC TAC CTG AGT GTC TTT. The number of PCR cycles for each mRNA was adjusted to the linear phase of the curve to allow for variations in mRNA levels.

4.7 Cytokine production

Raw cells or peritoneal macrophages (1.5×106 cells/well) were cultured with LPS (2 μg/ml) and/or AgC10 for the indicated times and TNF and IL-10 levels in supernatants determined using sandwich TNF (R&D) and IL-10 (Endogen) ELISA.

4.8 Run-on assays

Nuclear run-on assays to measure nascent RNA transcripts were essentially performed as described elsewhere 43. Briefly, Raw cells were stimulated with LPS (2 μg/ml) and/or AgC10 (20 μg/ml) where indicated. Nuclei were isolated from 1×107 cells/well and incubated with 2× reaction buffer (10 mM Tris-HCl, 5 mM MgCl2, 0.3 mM KCl, 10 mM each of ATP, GTP, CTP, 1 mM dithiothreitol and 10 μl [α-32P]UTP-3000 Ci/mmol) at 30°C for 30 min. Labeled RNA was purified by using TRIzol reagent. The probes consisted of 5 μg linearized plasmids containing cDNA corresponding to mouse TNF, COX-2, β-actin or the control plasmid pcDNA3, which were immobilized on nylon filters after denaturation in NaOH. Hybridizations were performed in 0.2 M sodium phosphate buffer pH 7.2, 1 mM EDTA, 7% SDS and 45% formamide for 24 h at 65°C. After hybridization, the filters were washed at 65°C in 40 mM sodium phosphate buffer pH 7.2 containing 1% SDS and autoradiographed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This work has been supported by grants from the following Spanish organizations: Ministerio de Ciencia y Tecnologia, Red RICET, Fondo de Investigaciones Sanitarias, Comunidad Autonoma de Madrid and Fundacion Ramon Areces.

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