There correlation between focal immunopathology and the presence of parasite remnants in tissues has been extensively docuneted over the years (Andrade ZA, this volume). Ribeiro dos Santos and Hudson (1980) were the first to propose that the passive adsorption of T. cruzi antigens onto certain types of non-infected host cells may renderi them susceptible to by-stander antibody-dependent cellular cytotoxicity (ADCC). In addition to being possibly involved in the peripheral neuropathy which occurs in the acute phase (Koberle et al. 1968), ADCC may be possibly involved in the microangiopathy observed in acutely infected dogs (Andrade et al. 1994). Although the molecular basis of microvascular pathology is still unknown, recent studies on the cysteine-proteinases of the cruzipain family (Cazzulo et al. 1989, Murta et al. 1990, Eakin et al. 1992, Meirelles et al. 1992, McKerrow et al. 1995) may shed light on such processes. Originally identified because of their antigenic properties in chagasic patients (Scharfstein et al. 1983, 1986, Murta et al. 1990, Gazzinelli et al. 1990, Arnholdt et al. 1993, Morrot et al. 1997), the members of the cruzipain family consist of a heterogeneous group of closely related isoforms (Lima et al. 1994) encoded by about 130 genes (Campetella et al. 1992). The expression of these proteinases is upregulated when the invading trypomastigotes transform into amastigotes (Tomas & Kelly 1996), this process being accompanied by increased cell surface expression of the enzymes. It is thus conceivable that these amastigotes, once released into interstitial spaces, may degrade the adjacent extracellular matrix (ECM) in adjacent tissues (unpublished observations). By doing so, the parasites may prevent or disrupt ECM-interactions with TNF-a, chemokines and other inflammatory mediators involved in the recruitment and/or activation of immune cells T cells (Gillat et al. 1996). Cruzipain (and/or other minor isoforms) are worth studying in this context, because they were recently identified as antigenic deposits in sites of myocardial inflammation in autopsies from patients with severe chronic cardiomyopathy (Morrot et al. 1997). Given that cruzipain (and/or other isoforms) are stable and enzymatically active in neutral-acid pH, we reasoned that their half-life in extracellular tissues would likely depend on the levels of host proteinase inhibitors which permeate inflammatory sites. The immunological implications of cruzipain interactions with a2-Macroglobulin, a non-specific plasma proteinase inhibitor were previously studied in our laboratory (Morrot et al. 1997). In view of previous evidences indicating that CD4+ T cells from chagasic patients promptly respond to cruzipain (Arnholdt et al. 1993), we investigated if functional inactivation by a2M could influence the efficiency of antigen presentation and processing by human monocytes. Our studies revealed that monocytes engage a highly endocytic scavenger receptor expressed by monocytes (CD91 or LRP/a2MR) to rapidly internalize a2M-cruzipain complexes. The enhanced endocytic uptake of cruzipain-complexes ultimately favor increased intracellular processing and presentation of cruzipain peptides to CD4+ T cells (Morrot et al. 1997). Because a2M binds to cell surface proteinases from amastigotes (Coutinho et al. 1997), it is conceivable that the multifunctional LRP/a2MR scavenger receptor may promote the uptake of amastigotes coated with a2M ligated to cruzipain, in ways that are reminiscent of mechanisms recently described for the macrophages' mannose receptor (Khan et al.1995). In both cases, the efficiency of antigen-presentation of amastigotes epitopes to T cells may be potentiated. In short, our studies illustrate how interactions between parasite proteinases and host inhibitors may integrate elements of innate defense systems with those in charge of acquired immunity.
As a follow-up from the studies involving a2M, we turned our attention to kininogen, a member of the cystatin superfamily of cysteine proteinase inhibitors (Barret et al. 1986). In addition to the cystatin-like domains, these multifunctional glycoproteins can participate in the activation of the intrinsic pathway of coagulation (high molecular weight kininogen) and modulate platelet activation by thrombin. Important to our discussion, kininogens (high or low molecular weight) is the parent substrate molecule from which bradykinin or lysil-bradykinin is released, upon proteolytic cleavage either by plasma or tissue kallikrein (Bhoola et al. 1992). Kinins are short-lived peptides engage distinct subtypes of G-coupled kinin receptors in a wide spectrum of biological processes, such as modulation of neuronal activity (Higashida et al. 1990), cell proliferation and vascular permeability (Bhoola et al. 1992), smooth muscle contraction or relaxation (Monbouli & Vanhoutte 1995). Depending on the host cell, kinin signalling is transduced by receptors that are either expressed constitutively (B2) or are induced (B1) during anoxia or noxious stimuli (Burch & Kyle 1992, Regoli 1980).
The initiative to investigate the relationship of T. cruzi with the kinin system was initially motivated by enzymatic specificity studies carried out with cruzipain 2 (Lima et al. 1994), a minor isoform whose recombinant form was only recently expressed in S. cerevisae. After noting that its substrate specificity somewhat resembled that of tissue kallikrein, we were able to demonstrate that these isoforms could act as kinin-releasing enzyme (Del Nery et al. 1997), although with different efficiency. On a first look, cruzipain's ability to process kininogen seemed paradoxical because this protease is highly sensitive to inhibition by the tight-binding cystatin domains displayed in both high or low molecular weight forms (Stoka et al. 1995, Scharfstein et al. 1995). Clues to understand the mechanism of kinin-release emerged when the Ki's obtained for kininogen were determined for different isoforms of cruzipain: interestingly, we observed that r-cruzipain 2 was markedly less sensitive to inhibition by kininogen as compared to cruzain (Lima et al. in preparation) the archetype from the cruzipain family (McKerrow et al. 1995). Our data suggest that the kininogenase activity of T. cruzi cysteine-proteinases might have evolved due to structural diversification of the catalytic site of some isoforms, such as cruzipain 2. Notably, in the same study (Del Nery et al. 1997) we also showed that cruzipain could generate bradykinin indirectly, that is, by converting plama prekallikrein in active kallikrein. In a parallel study, we used intravital microscopy to verify if the topical application of purified cruzipain on the hamster cheek pouch could stimulate increases in vascular permeability (Svensjo et al. 1997). Potent responses were indeed observed, suggesting that the hemoflagellate may be able to use cruzipain (and/or isoforms) to generate kinins upon contact with endothelial cells. Ongoing studies in mice with targeted deletion of kinin-receptors should reveal if these kinin-releasing proteinases may facilitate T. cruzi migration across non-fenestrated capillaries.
The realization that T. cruzi may use cruzipain isoforms to generate pro-inflammatory kinins upon contact with kinin-receptor bearing cells (Fig. 2) raised questions regarding their role in pathogenesis. More recently, its was shown that bradykinin stimulation of the B2 subtype of kinin-receptor from guinea pig macrophages leads to stimulation of superoxide radical, arachidonic acid and PGE2 (Bockmann & Paegelow 1998). It will be interesting to know if kinins released by T. cruzi may trigger PGE2 production by macrophages. As discussed earlier in this article, factors leading to cyclic AMP accumulation, eg. PGE2, may stimulate IL-10 synthesis in macrophages that had been exposed to tGPI-mucins (Procopio et al. 1999).
The role of T. cruzi-induced kinin release in the pathogenesis of chronic heart disease is also worth exploring, the main assumption being that microvascular lesions caused by infection-associated events (Andrade et al. 1994) may slowly accumulate over the years, thereby creating conditions for the exacerbation of chronic inflammation and fibrosis (Morris et al. 1990, Rossi 1990). In a recent study, Higuchi et al. (1999) have reaffirmed the potential importance of microvascular pathology: in a detailed analysis of post-necropsy heart specimens from chronic chagasics by three-dimensional confocal microscopy, these authors observed abnormalities of the cardiac microcirculation as well as in the interstitial matrix patterns of myocardial tissues. Peculiar lesions, often manifested as arteriolar dilatation and capillary vessel tortuosity, were described, being thus far vaguely attributed to fibrosis and/or to as yet uncharac-terized lesions induced by the parasites. Apart from initial studies looking at effects of desialilation by TS enzymes (Libby et al. 1986), little is known about the biochemical basis of T. cruzi interaction with endothelial cells (Morris et al. 1990). As discussed earlier in this section, the realization that cruzipain-isoforms are capable of releasing kinins offers a new experimental framework to investigate the pathophysiological consequences of long-term stimulation of myocardial capillaries by vasoactive kinins (Fig. 2). Our studies in the cheek pouch suggest that cruzipain molecules released/leaked from merely a few parasites may suffice to generate vasoactive kinins, thereby provoking plasma exudation across the nearest capillaries (Fig. 1, right panel). Similar processes, perhaps involving NO released by activated endothelial cells, may underlie the lesions observed earlier in infection, when the parasites are released next to kinin-receptor bearing cells, situated in the proximity of autonomous nervous system ganglia.
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