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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 1, 2002 as doi:10.1096/fj.02-0477fje. |
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O B. PESQUERO*
Instituto de Biofísica Carlos Chagas Filho, Universidade do Brasil, CCS, Bloco G, Cidade Universitária, CEP 21944–900, Rio de Janeiro, Brazil;
* Departmento de Biofísica, Universidade Federal do Estado de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil;
Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany;
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Denver, Colorado, USA;
Institute for Biochemistry II, University of Frankfurt Medical School, Frankfurt, Germany;
|| Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil; and
Departamento de Biologia Celular e Genética, Universidade do Estado do Rio de Janeiro, Brazil
2Correspondence: Instituto de Biofísica Carlos Chagas Filho, Universidade do Brasil, CCS, Bloco G, Cidade Universitária, CEP 21944–900, Rio de Janeiro, Brazil. E-mail: scharf{at}biof.ufrj.br
SPECIFIC AIMS
In view of evidence that activation of B2 kinin receptors (B2R) increases host cell susceptibility to invasion by the protozoan Trypanosoma cruzi in vitro, we asked whether 1) injection of the pathogen into mice results in triggering of proinflammatory kinin cascades in vivo and 2) trypomastigotes may engage B1 kinin receptors (B1R) to invade cardiomyocytes and activated endothelial cells.
PRINCIPAL FINDINGS
1. Kinin receptors mediate edema induced by trypomastigotes
In a previous study we demonstrated that endothelial cell susceptibility to infection by trypomastigotes is enhanced by the parasites’ ability to release kinin peptides that activate constitutively expressed B2R. We further showed that these host cells are protected from T. cruzi infection by the kinin-degrading activity of ACE/kininase II. To test whether T. cruzi (Dm28c strain) triggers the kinin system in vivo, tissue culture trypomastigotes (TCTs) were injected into the footpad of wild-type (wt) J129 mice and local volume changes were measured. The infected animals developed a prominent edema after systemic injection of ACE inhibitor captopril (Cap) but not in its absence. The time course of edema formation revealed an initial peak 3 h postinfection (p.i.) ("early phase") and a sustained response ("late phase") lasting for > 24 h. Inoculation of an equivalent number of epimastigotes, the avirulent forms of T. cruzi, or of sheep erythrocytes failed to induce a significant edema in Cap-treated wt mice (Fig. 1
A). Thus, T. cruzi’s ability to induce plasma exudation in the mouse paw is linked to the expression of an infective phenotype.
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2. Temporal shifts in the recruitment of kinin receptors in the infected paw
To identify the kinin receptor types involved in the two phases of edema formation, we used specific antagonists and knockout animals. HOE 140, a potent B2R antagonist, blocked plasma efflux at 3 h in Cap-treated mice whereas B9858, a specific B1R antagonist, did not interfere with the early reaction (Fig. 1)
. Consistent with this observation, parasite inoculation in mice where the B2R gene had been ablated (B2R-/-) failed to produce a significant edema at 3 h (Fig. 1A
). Because B1R expression is known to be up-regulated during inflammation, we tested the effects of B9858, a specific antagonist of B1R, on the parasite-induced edema. Application of B9858 in Cap-treated wt mice did not interfere with early phase response, but the late phase reaction (24 h p.i.) was significantly attenuated (Fig. 1B
). Consonant with these data, Cap-treated B1R-deficient mice (B1R-/-) failed to produce a significant edema at 24 h p.i., although the animals responded vigorously at the onset of the infection (Fig. 1B
). Repeated injections of HOE140 abrogated both early and late phase vascular responses in wt mice (Fig. 1B
), suggesting that activation of B2R is a prerequisite for subsequent engagement of the up-regulated B1R pathway.
3. TCTs invade host cells expressing B1R by a Kininase I dependent pathway
Because B1R is up-regulated at the primary site of infection, we asked whether trypomastigotes could invade host cells in vitro more efficiently by stimulating this inducible receptor, as we previously reported for B2R. We applied TCTs at parasite/host cell ratios of 3:1 or 10:1 to monolayers of CHO cells overexpressing B1R (CHO-B1); B2R (CHO-B2) and mock-transfected cells (CHO-mock) served as controls. The interaction proceeded for 3 h in the presence of Ham’s-BSA medium supplemented (or not) with Cap. The ACE inhibitor selectively enhanced the infection index of CHO-B1 (41.0±7.6; 149%) vs. Ham’s-BSA (27.6±2.5; 100%) whereas addition of the B1R antagonist des-Arg9-[Leu8]-BK (15.5±3.0; 56%) nullified the effect of Cap. In the absence of Cap, the "basal" infectivity of T. cruzi for CHO-B1 (26.3±0.8; 100%) was reduced by the kininase I inhibitor MGTA (16.7±1.7; 63%) or by [Leu8] des-Arg9-BK (18.4±0.2; 70%). Given previous evidence that IP3-triggered transients of [Ca2+]i were required for invasion of CHO-B2, we depleted endoplasmic [Ca2+] stores of CHO-B1 and CHO-mock by pretreating them with thapsigargin (0.5 µM). The drug inhibited the invasion of CHO-B1 to nearly the same extent as [Leu8]-des-Arg9-BK (76%) but did not interfere with CHO-mock invasion.
4. Parasite invasion of LPS-treated endothelial cells may involve either B1R or B2R
We used bacterial lipopolysaccharide (LPS) to induce B1R on primary cultures of human umbilical vein endothelial cells (HUVECs) and asked whether the activated cells were efficiently invaded by TCTs. Similar to data obtained with CHO-B1, addition of [Leu8]-des-Arg9-BK or MGTA to Cap-treated LPS-HUVECs drastically reduced the infectivity (70–80%). In contrast, the B1R antagonist failed to attenuate parasite invasion of "resting" HUVECs, whereas HOE140 reduced parasite infectivity for both resting (40%) and LPS-HUVECs (90%) in Cap medium. As with CHO-B1, addition of MGTA drastically reduced parasite invasion of LPS-HUVECs (68%) in Cap medium. In contrast, MGTA stimulated (2.5-fold) infection of resting HUVECs, suggesting that the concentration of B2R-agonist(s) in Cap medium is further increased when kininase I is inactivated. It appears that B2R is productively engaged by the parasites irrespective of the "activation" state of endothelial cells.
5. B1R or B2R are engaged in parasite invasion of neonatal rat cardiomyocytes in vitro
Because B2R is expressed abundantly in the mammalian heart, we reasoned that the kinin-releasing trypomastigotes could exploit this pathway to invade cardiomyocytes. [Leu8]-des-Arg9-BK reduced invasion by 60% (Fig. 2
) and MGTA inhibited (40%, data not shown) to a similar extent, indicating that the parasites depend on kininase I to productively engage B1R of cardiomyocytes. Considering that HOE140 protected these cells to the same extent as [Leu8]-des-Arg9-BK (inhibition of 50%) and that the combined application of these antagonists did not result in additive inhibitory effects (Fig. 2A
), we conclude that the B1R and B2R signaling pathways cannot be independently activated by parasites.
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CONCLUSIONS
Here we have extended to an in vivo context our previous findings that infective forms of T. cruzi (the Dm28c strain) activate the proinflammatory kinin cascade in vitro. To analyze the role of kinins and their receptors on the progression of paw edema, we used wt J129 mice as well as B1R-/- and B2R-/- J129B57 mice in combination with antagonists to B1R and B2R, antibody probes for kininogen, and inhibitors of the major kinin-degrading peptidases (kininase I and ACE). Our data demonstrate that B2R mediates the early phase vascular responses induced by trypomastigotes whereas the up-regulated B1R pathway accounts for the late phase reaction. The vascular reactions in wt mice were consistently mild or even negligible, except for animals purposefully deprived of ACE activity by systemic administration of Cap before parasite inoculation. These results indicate that tissue expression levels of ACE may modulate the intensity of inflammatory responses induced by kinin-releasing strains of T. cruzi. Engagement of B1R appears to depend critically on the activation of B2R, suggesting the two signaling pathways are intertwined. Our finding that avirulent epimastigotes fail to induce edema suggests a link between the pathogen’s ability to infect cells and their ability to trigger the proinflammatory kinin system in vivo.
In an attempt to define B1R activation requirements, we found that infection of CHO-B1 cells was selectively impaired by the kininase I inhibitor MGTA, whereas Cap slightly enhanced parasite infectivity in vitro. These contrasting effects are compatible with the notion that the ACE inhibitor acts primarily by preventing the degradation of both B1R and B2R agonists whereas MGTA blocks the kininase I-mediated conversion of bradykinin and kallidin into the corresponding B1R agonists, i.e., [desArg9]-bradykinin and [desArg10]-kallidin, respectively. An intriguing finding was that addition of B1R or B2R antagonists to host cells coexpressing these receptors (cardiomyocytes or LPS-HUVECs) inhibited parasite infectivity to a similar extent. Since the combined application of both antagonists had no additive effects in invasion, we deduce that B1R or B2R operate interdependently to meet the [Ca2+]i threshold required for efficient pathogen uptake.
Activation of the kinin cascade may translate into mutual benefits to the host-parasite equilibrium because T. cruzi may invade cardiovascular cells more efficiently by activating B2 and/or B1 kinin receptors, while formation of vasodilating kinins may locally relieve ischemia, thus limiting myocardial injury (Fig. 3
).In some patients, changes in the tissue expression of kinin-degrading peptidases (e.g., ACE/kininase II or CPM/kininase I) may offset the delicate host-parasite equilibrium established in the indeterminate stage of the disease. For example, parasite-induced activation of B1R by the kininase I-dependent pathway may represent an alternative pathway for parasite invasion and intracellular growth in the inflamed tissues. In the chronic stage, immunopathology may be further exacerbated due to engagement of the up-regulated B1R, a pathway that drives leukocyte recruitment to sites of infection (Fig. 3)
. Hence, we hypothesize that fluctuations in the regulatory activity of kininases may play an important role in the pathogenesis of Chagas’ disease.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0477fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0477fje 
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