Damage to myocytes by infiltrating cytotoxic lymphocytes, containing lytic granules and the pore-forming protein, perforin, thereof, probably contribute to the immunological rejection of the transplanted heart, to autoimmune diseases and possibly to congestive heart failure associated with myocarditis. In the present study we investigated whether electrophysiological and morphological changes induced in guinea pig ventricular myocytes by lytic granules extracted from cytotoxic T lymphocytes, are modified by L-type Ca++ channel blockers. The organic blockers, verapamil (2 microM) and nisoldipine (500 microM) were unable to prevent or inhibit any of the deleterious effects of lytic granule on action potential and myocyte morphology, and the granule-induced increase in the membrane current measured at the end of 300-msec clamp pulse. In contrast, the inorganic blockers CoCl2 (3 mM) and NiCl2 (4 mM) provided considerable protection against the granule actions mentioned above, but an equally potent Ca++ blocker, CdCl2 (3 mM) was ineffective. The protective efficacy of CoCl2 (and probably that of NiCl2) was most likely due to its capacity to reduce or block the generation by lytic granules/perforin of large-conductance (approximately 1400 pS) channels responsible for inducing Ca++ overload and cell destruction. We consider these studies of importance because they direct further studies aimed at developing effective means for attenuating cytotoxic T lymphocyte-induced tissue damage, for example during transplant rejection or in autoimmune diseases.
The central role of CTLs in immunopathology accounts for the increasing interest in deciphering the mechanism whereby they kill at the molecular level. Recent studies show that CTLs have two molecularly distinct lytic mechanisms at their disposal. The first involves the direct effect(s) of the pore-forming protein perforin, possibly in conjunction with granzymes. In recent years, experiments conducted in our laboratory led to an alternative pathway, of receptor-mediated mechanism for CTL killing, involving neither the secretion nor the lytic action of the pore-forming protein perforin or of granzymes. By this mechanism, engagement of a CTL membrane ligand and an apoptosis-inducing target cell surface receptor triggers the disintegration of the CTL-bound target cell. Cross-linking of apoptosis-inducing target cell surface molecules (e.g. Fas), induced upon binding of CTL ligands (e.g. Fas-L), may be required and sufficient to trigger target cell apoptosis. Intracellular lethal signals emanating from the cross-linked intracellular death domain of Fas are postulated.
s: Abstracts for the 20th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer (Primary Authors are Italicized): NATURAL KILLER CELLS AND INNATE IMMUNITY
Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC) is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL-TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.
Objective: Involvement of cytotoxic T lymphocytes (CTL) in heart transplant rejection as well as in viral myocarditis is well established, but the precise mechanisms whereby infiltrating CTL damage the myocardium are unknown. The aim of the study was to investigate how CTL derived perforin, the serine protease granzyme A, and the combination of both, damage guinea pig ventricular myocytes. Methods: Action potentials and membrane currents were recorded by means of the whole cell configuration from guinea pig ventricular myocytes. Results: Resembling the effects of CTL derived lytic granules, perforin caused gradual myocyte shortening and contracture, leading to complete loss of the rod shaped morphology and to cell destruction. These changes were preceded by shortening of action potential duration and reduction of resting potential and action potential amplitude, followed by complete inexcitability. Granzyme A alone was ineffective, but accelerated the deleterious effects of perforin on the morphological and electrophysiological properties of myocytes. The effects of perforin were further evaluated by measuring membrane currents by means of the whole cell voltage clamp. Perforin induced discrete changes in membrane current, reminiscent of single ion channels, with large conductance and open time of up to several seconds. Linear regression analysis of the channel I-V relations resulted in a conductance of 890 pS and a reversal potential of −7.6 mV. These results suggest that perforin induces large non-selective channels, which can account for most of the observed adverse effects. Conclusions: As CTL participate in the immunological rejection of the transplanted heart, it is conceivable, but remains to be shown, that part of this damage is inflicted by perforin containing lytic granules. Cardiovascular Research 1994;28:643-649
