Effects of purified perforin and granzyme A from cytotoxic T lymphocytes on guinea pig ventricular myocytes

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;28:643-64

Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses

Ligation of the Fas (CD95) receptor leads to an apoptotic death signal in T cells, B cells, and macrophages. However, human CD34(+)-derived dendritic cells (DCs) and mouse DCs, regardless of their maturation state, are not susceptible to Fas-induced cell death. This resistance correlates with the constitutive expression of the Fas-associated death domain-like IL-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP) ligand. We demonstrate a new role of Fas in DC physiology. Engagement of Fas on immature DCs by Fas ligand (FasL) or by anti-Fas antibodies induces the phenotypical and functional maturation of primary DCs. Fas-activated DCs upregulate the expression of the major histocompatibility complex class II, B7, and DC-lysosome-associated membrane protein (DC-LAMP) molecules and secrete proinflammatory cytokines, in particular interleukin (IL)-1beta and tumor necrosis factor alpha. Mature DCs, if exposed to FasL, produce even higher amounts of IL-1beta. Importantly, it is possible to reduce the production of IL-1beta and interferon (IFN)-gamma during DC-T cell interaction by blocking the coupling of Fas-FasL with a Fas competitor. Finally, during cognate DC-T cell recognition, IL-12 (p70) could not be detected at early or late time points, indicating that Fas-induced, IFN-gamma secretion is independent of IL-12

Apoptosis of Fashigh CD4+ synovial T cells by borrelia-reactive Fas-ligand(high) gamma delta T cells in Lyme arthritis

The function of the minor subset of T lymphocytes bearing the gamma delta T cell antigen receptor is uncertain. Although some gamma delta T cells react to microbial products, responsiveness has only rarely been demonstrated toward a bacterial antigen from a naturally occurring human infection. Synovial fluid lymphocytes from patients with Lyme arthritis contain a large proportion of gamma delta cells that proliferate in response to the causative spirochete, Borrelia burgdorferi. Furthermore, synovial gamma delta T cell clones express elevated and sustained levels of the ligand for Fas (APO-1, CD95) compared to alpha beta T cells, and induce apoptosis of Fashigh CD4+ synovial lymphocytes. The findings suggest that gamma delta T cells contribute to defense in human infections, as well as manifest an immunoregulatory function at inflammatory sites by a Fas-dependent process

Baff Mediates Survival of Peripheral Immature B Lymphocytes

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance

Malarial Hemozoin Is a Nalp3 Inflammasome Activating Danger Signal

BACKGROUND: Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents. METHODOLOGY/PRINCIPAL FINDINGS: We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1beta. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K(+) efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged. SIGNIFICANCE/CONCLUSIONS: The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria

Malarial Hemozoin Is a Nalp3 Inflammasome Activating Danger Signal

BACKGROUND: Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents. METHODOLOGY/PRINCIPAL FINDINGS: We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1beta. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K(+) efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged. SIGNIFICANCE/CONCLUSIONS: The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria