Apoptosis in parasites and parasite-induced apoptosis in the host immune system: a new approach to parasitic diseases

Apoptosis, a form of programmed cell death (PCD), has been described as essential for normal organogenesis and tissue development, as well as for the proper function of cell-renewal systems in adult organisms. Apoptosis is also pivotal in the pathogenesis of several different diseases. In this paper we discuss, from two different points of view, the role of apoptosis in parasitic diseases. The description of apoptotic death in three different species of heteroxenic trypanosomatids is reviewed, and considerations on the phylogenesis of apoptosis and on the eventual role of PCD on their mechanism of pathogenesis are made. From a different perspective, an increasing body of evidence is making clear that regulation of host cell apoptosis is an important factor on the definition of a host-pathogen interaction. As an example, the molecular mechanisms by which Trypanosoma cruzi is able to induce apoptosis in immunocompetent cells, in a murine model of Chagas' disease, and the consequences of this phenomenon on the outcome of the experimental disease are discussed.

Chronic experimental Chagas' disease: functional syngeneic T-B-cell cooperation in vitro in the absence of an exogenous stimulus.

We have investigated CD4+ T-cell autoreactivity to normal syngeneic B cells in vitro in chronic experimental Chagas' disease. Resting B cells induced an intense proliferative response and lymphokine secretion by splenic CD4+ T cells from Trypanosoma cruzi-infected (8 months or more of infection) donors, compared to much lower responses by uninfected controls. On the other hand, lipopolysaccharide-activated B cells induced syngeneic CD4+ T-cell activation in both control and infected groups. The observed syngeneic T-B-cell cooperation was bidirectional. In the absence of any exogenous stimulus, CD4+ T cells from T. cruzi-infected animals induced much higher production of all tested immunoglobulin (Ig) isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) by syngeneic B cells, compared to T cells from uninfected donors. When lipopolysaccharide-treated B cells were used, CD4+ T cells from either control or infected donors enhanced IgG1 and IgG3 production, but only CD4+ T cells of infected origin induced IgG2a production in this system without addition of exogenous gamma interferon. Enhanced T-cell proliferation and Ig production were also observed with highly purified CD4+ T cells and in serum-free medium. Both proliferation and Ig production could be blocked with anti-major histocompatibility complex class II monoclonal antibodies. Enhanced reactivity and help for Ig production were seen only in response to syngeneic BALB B cells and not in response to allogeneic B10 B cells. These results indicate that chronic infection with T. cruzi results in increased CD4+ T-cell reactivity towards syngeneic B cells, which leads to spontaneous Ig production. These autoreactive T cells might play a role in polyclonal autoantibody production in chronic Chagas' disease.

Trypanosoma cruzi: both chemically induced and triatomine-derived metacyclic trypomastigotes cause the same immunological disturbances in the infected mammalian host.

Infection of BALB/c mice with chemically induced metacyclic forms of Trypanosoma cruzi clone Dm28c led to characteristic changes of experimental Chagas' disease, with protracted but marked parasitemia, intense splenomegaly, and splenic T cell hyporeactivity to TcR;CD3-dependent stimulation. Infection of BALB/c mice with either chemically induced or triatomine-derived Dm28c metacyclic forms led to comparable parasitemias, a synchronous increase in the number of splenic large lymphocytes, and a similar reduction in T cell responsivity to immobile anti-CD3 antibody. A marked and selective reduction in the level of CD8 expression per cell was also seen in mice infected with either form of metacyclic parasites. Large inflammatory mononuclear cell infiltrates were present in the hearts of mice infected with either chemically induced or insect vector-derived metacyclic forms, at both acute and chronic stage, with predominance of CD8 over CD4 T cells in the lesions, in both cases. These results indicate that infection with chemically induced metacyclic forms of T. cruzi can be a useful model of Chagas' disease, resembling infection caused by the insect vector.

Functional inactivation of primary T-cells stimulated in vitro in the presence of cyclosporine.

Cyclosporine (CsA) blocks in vitro polyclonal activation of primary murine T-cells in a complex manner. This cannot be completely reversed by exogenous IL2, and leads to a partial blockade in expression of the IL2 receptor (p55 chain) and, more intensely, in CD69. In proliferation assays, T-cells recovered from CsA-treated cultures and washed free from CsA were markedly refractory to restimulation in the presence of fresh accessory cells. In cell titration restimulation assays, CsA-treated, but not control T-cells, were also markedly unresponsive to accessory cell-independent stimuli provided by immobilized anti-CD3 antibody or rIL2, combined to phorbol ester. CsA-treated, but not control activated T-cells, undergo progressive cell death after drug removal and reculturing. In contrast, primary T-cells activated by a CsA-resistant pathway (rIL2 plus phorbol ester) and treated with CsA, did not develop unresponsiveness, compared to controls. When primary T-cells were stimulated with rIL2 plus phorbol ester in the presence of the calcium ionophore ionomycin, treatment with CsA resulted in marked unresponsiveness of the T-cells, compared to untreated controls. The data indicate that primary activation of T-cells in vitro in the presence of CsA induces an unresponsive state which lasts independent of the presence of CsA, and results in progressive cell death. We suggest that these effects could characterize one additional mechanism of CsA action in vivo.

Trypanosoma cruzi-induced immunosuppression: blockade of costimulatory T-cell responses in infected hosts due to defective T-cell receptor-CD3 functioning.

A model of experimental Trypanosoma cruzi murine infection with chemically induced metacyclic forms (opossum clone Dm28c) showed a marked state of T-cell unresponsiveness during acute phase, but lacked evidence of suppressor cell activity. Spleen cells from infected mice were suppressed in vitro in responses to T-cell activators concanavalin A, anti-Thy1 monoclonal antibody (MAb), and anti-CD3 MAb compared with spleen cells from control littermates. Activation with accessory cell-independent stimulus provided by immobilized anti-CD3 was defective in splenic CD4-positive T cells from infected mice, but not in such cells from control mice. No evidence of splenic suppressor cell activity was found in cell-mixing experiments using nylon-passed T cells from control and infected donors. Kinetic experiments showed that there was a discrete stage in infection when T cells were already suppressed in response to anti-CD3 but still responded to anti-CD69 MAb. In these T cells, immobilized anti-CD3 failed to enhance simultaneous CD69 responses, although anti-CD3 enhanced CD69 responses in control T cells from uninfected donors. These results demonstrate an intrinsic defect in T-cell receptor-mediated T-cell activation, which could be a mechanism generating T-cell suppression during infection by T. cruzi.

Interactions between CD3 and Thy1 T cell activation pathways: blockade of CD3-mediated T lymphocyte activation induced by immobilized anti-Thy1 antibodies.

Resting murine T cell activation induced by either CD3 complexes or Thy1 molecules was investigated in vitro, using surface-bound anti-CD3 mAb as the stimulus. One mitogenic anti-Thy 1 mAb (G7) lost mitogenicity when presented to T cells immobilized on a plastic surface, even in the presence of phorbol ester. Moreover, T cell activation induced by immobilized anti-CD3 was potently blocked by coimmobilized anti-Thy 1 mAb. Nonmitogenic anti-Thy 1 mAb also blocked CD3-induced activation when coimmobilized with anti-CD3. Control experiments showed that anti-Thy 1 specifically blocked T cell activation, even in the presence of measurable and functional concentrations of plastic-bound anti-CD3. Coimmobilized anti-Thy 1 potently blocked IL2 secretion stimulated by anti-CD3. Addition of exogenous rIL2 completely prevented anti-Thy 1-mediated blockade. On the other hand, while completely blocking T cell proliferation, immobilized anti-Thy 1 only partially blocked secretion of IL3-like activity by the T cells. One IgM anti-Thy 1 mAb (2A3) induced secretion of IL3-like activity by T cells when immobilized in the absence of bound anti-CD3. These results indicate that extensive aggregation of Thy 1 molecules delivers a potent negative signal which antagonizes CD3-mediated T cell activation and growth.

Triggering of thymocyte function by IL-2 as the only exogenous stimulus; analysis of two distinct modes of IL-2-induced thymocyte proliferation and IL-3 secretion in vitro.

Addition of recombinant interleukin-2 (rIL-2) to normal adult murine thymocytes in vitro as the only exogenous stimulus leads to a dose-dependent mitogenic response characterized by two distinct dosage kinetic components. The high-affinity IL-2 thymocyte response is mounted by in vivo-activated (IL-2 receptor light chain positive) thymocytes, while the low-affinity IL-2 response, of larger amplitude, is carried out by resting thymocytes. Addition of IL-2 to thymocytes also triggers intense IL-3 secretory responses with both high and low IL-2 affinity components. Addition of high IL-2 dosages to thymocyte bulk cultures results in a dramatic increase in IL-2 responsiveness for both proliferation and IL-3 secretion on a per viable cell basis and with tightly coupled temporal kinetics. The low-affinity component of IL-2-proliferative and IL-3-secreting responses is carried out by resting mature CD4+ thymocytes, as assessed by negative selection with monoclonal antibodies (mAb) plus complement. The mechanism of resting thymocyte activation by high doses of IL-2 is partially characterized. Depletion of endogenous thymus-adherent cells abolished both proliferation and IL-3 secretion, and addition of splenic accessory cells or peritoneal macrophages to depleted thymocytes restored IL-2 responsiveness. Mature CD4+ thymocytes spontaneously form rosettes with adherent accessory cells, while CD8+ thymocytes do so with much less efficiency. Rosette formation of CD4+, but not of CD8+ thymocytes, can be blocked by anti-CD4 mAb GK1.5. At the same dosage as it prevents rosette formation, mAb GK1.5 also blocks the low-affinity thymocyte response to IL-2. The high-affinity IL-2 response is completely resistant to the action of cyclosporin A (CsA), but the low-affinity IL-2 response, although of much larger amplitude, can be almost completely suppressed by CsA. Together, these results demonstrate that resting CD4+ thymocytes can be induced to proliferation and lymphokine secretion by IL-2 alone in a process that is dependent on interaction with accessory cells, involves CD4 adhesion molecules and triggers activation through a CsA-sensitive pathway. In addition, the results demonstrate that IL-2 alone is able to enhance thymocyte IL-2 responsiveness and IL-3 secretory responses in vitro. The ability of IL-2 to induce and maintain thymocyte function is discussed in the light of these results.

Functional heterogeneity in the process of T lymphocyte activation; barium blocks several modes of T cell activation, but spares a functionally unique subset of PHA-activable T cells.

The effects of the alkaline earth divalent cation Barium (Ba2+) were studied in in-vitro murine polyclonal T cell activation induced with a panel of T cell mitogens consisting of the plant mitogens concanavalin A (ConA), jacalin and phytohaemagglutinin (PHA), a mitogenic anti-Thy1 monoclonal antibody (MoAb), and an anti-murine CD3 MoAb combined with phorbol ester. All modes of T cell activation, except PHA-induced mitogenesis, were blocked in a reversible and dose-related manner by Ba2+. Blockade was evident only if Ba2+ was added within the first 6 h of stimulation, was totally reversed in a competitive fashion by addition of Ca2+ to the medium, and selectively affected interleukin 2 (IL-2) production, without interfering with expression of IL-2 receptor light chains, nor with late IL-2-dependent activated T cell growth. On the other hand, PHA-induced responses stimulated by optimal mitogen doses were resistant to the effects of Ba2+. Ba2+-resistance of PHA responses was due to IL-2-dependent activation and growth of a Ba2+-resistant T cell subset since: (i) limiting dilution analysis demonstrated that this PHA response had a much lower precursor cell frequency than control PHA responses; (ii) proliferation was blocked by anti-IL2 agents, such as cyclosporin A and anti-IL-2 receptor light chain MoAbs, which were much less effective in blocking control PHA responses. Thus, pharmacological use of Ba2+ reveals the existence of a pathway of T cell activation, induced by PHA, with differential interleukin requirements.

Murine polyclonal T-lymphocyte activation induced by phytohemmagglutinin; differential lymphokine requirements of two unusual activation pathways defined by resistance to blockade by barium and by cyclosporin A.

We have recently demonstrated that polyclonal T-cell activation induced by PHA defines an activation pathway which is resistant to blockade by barium (Ba2+) ions. Other modes of T-cell activation, including ConA-induced responses, are completely blocked by Ba2+, which seems to affect an early Ca2+-dependent step of T-cell activation, as determined by kinetic and competition experiments. In the present study, we have analysed the lymphokine requirements of Ba2+-resistant pathway of PHA-induced T-cell activation by means of functional blocking experiments with monoclonal antibodies (mAbs) directed against mouse IL-2 (mAb S4B6) and against mouse IL-4 (mAb 11B11). We found that Ba2+-resistant T-cell activation can be blocked by either S4B6 or 11B11. Thus, both IL-2 and IL-4 participate in Ba2+-resistant T-cell growth induced by PHA. In addition, we found that cyclosporin A (CsA) completely blocks T-cell activation induced by either ConA or by PHA plus Ba2+, but not T-cell activation induced by PHA in the absence of Ba2+, which is reduced by less than 50% in most experiments. This CsA-resistant proliferative component of the PHA response is, thus, distinct from the Ba2+-resistant response, and is carried out by proliferating T-cells. Although mAbs S4B6 and 11B11 are potent blockers of ConA-induced responses, they failed to block CsA-resistant T-cell growth induced by PHA. At the doses of CsA employed, no IL-2 and/or IL-4 activity could be detected in the supernatants of CsA-treated, PHA-stimulated T-cell cultures. The data indicate that this CsA-resistant pathway is both IL-2 and IL-4-independent. The lymphokine involved in this T-cell activation pathway remains to be identified.

Do self-heart-reactive T cells expand in Trypanosoma cruzi-immune hosts?

Anti-heart T-cell activity was evaluated by a lymph node cell proliferative assay in isogenic strains of mice immunized with several Trypanosoma cruzi epimastigote and trypomastigote antigenic preparations. In addition, chronically infected animals were boosted with trypomastigote antigens and their lymph node cells were tested by in vitro proliferative responses. Our results indicated that (i) use of allogeneic sources of heart antigens may induce alloreactive responses in T. cruzi-immune T cells, (ii) specific autoimmune T-cell reactivity against self-heart constituents could not be demonstrated after immunization of the host with T. cruzi, and (iii) a proportion of chronically infected mice showed a small but detectable level of auto-anti-heart T-cell reactivity. These results argue against the notion that T. cruzi epitopes cross-reactive with self-heart tissue play a role in initiating T-cell-mediated autoimmunity. Anti-heart autoreactive T cells, generated in a proportion of the animals, may result from heart lesions associated with the infection process.

Stage-specific distinctions in potassium channel blocker control of T-lymphocyte activation.

Effects of four known blockers of T-lymphocyte potassium channels [verapamil, quinine, 4-aminopyridine (4-AP) and tetraethylammonium (TEA)], were studied on polyclonal T-cell activation induced by two plant mitogens (phytohemmaglutinin; PHA and concanavalin A; ConA), a mitogenic anti-Thy 1.2 monoclonal antibody (mAb G7) and phorbol ester (phorbol myristate acetate; PMA). Potassium channel blockers blocked all four modes of T-cell activation in a dose-dependent fashion with the same rank order of potency (verapamil greater than quinine greater than 4-AP greater than TEA). Kinetic studies of the timing of potassium channel blocker effect, indicated that, while 4-AP and TEA interfere only with early events of T-cell activation, verapamil and quinine can also interfere with later steps of T-cell mitogenesis. This notion was confirmed by studies of interleukin 2(IL-2)-directed activated T-cell growth. Verapamil and quinine blocked this late step in different types of activated T-cells with the same potency they blocked induction of resting T-cell mitogenesis. On the other hand, 4-AP and TEA, at maximal inhibitory doses for resting T-cells, showed little or no effect at IL-2-directed growth. Kinetic studies of the timing of quinine effect showed that the target of quinine action on activated T-cells is critically involved in IL-2 signalling within the first 2-4 h of IL-2 addition. These studies suggest that, besides the voltage-gated potassium channel previously described, a second target for verapamil and quinine action controls IL-2-derived signals to activated T-cells.

The syngeneic mixed leukocyte reaction: an autoreactive self-renewing cellular network for mature T lymphocytes.

The functional properties of responding T lymphocytes and the nature of the antigenic stimulus in the guinea pig Syngeneic Mixed Leukocyte Reaction (SMLR) are reviewed. The evidence presented here indicates that the SMLR is a polyclonal T cell proliferative response of several distinct antigen-specific clones by virtue of their reactivity with self Ia (I-region-encoded antigens) molecules expressed by accessory cells. Clonal analysis of SMLR-responding cells indicates that individual T cell colonies proliferate in response to syngeneic, unmodified macrophages, but not in response to I-region-disparate macrophages. This autoreactive response of T cell colonies cannot be ascribed to exposure to any extrinsic antigen, and is completely blocked by monoclonal anti-Ia antibodies. Each individual SMLR-T cell colony recognizes a particular Ia epitope on the autologous stimulating cell. Clonal analysis of extrinsic antigen-specific responses revealed three types of proliferating T cell colonies: antigen-specific, Ia-restricted; autoreactive, Ia-restricted; and antigen-specific/autoreactive, Ia-restricted. The finding of T cell colonies with antigen specificity but that could also be stimulated by Ia alone lends support to the hypothesis that the SMLR is a polyclonal T cell response to self Ia molecules. The possible in vivo attributes of the SMLR, and its main effector and regulatory mechanisms are discussed in terms of these findings.