Perforin and Fas Act Together in the Induction of Apoptosis, and Both Are Critical in the Clearance of Lymphocytic Choriomeningitis Virus Infection

Abstract

In this report we questioned the current view that the two principal cytotoxic pathways, the exocytosis and the Fas ligand (FasL)/Fas-mediated pathway, have largely nonoverlapping biological roles. For this purpose we have analyzed the response of mice that lack Fas as well as granzyme A (gzmA) and gzmB (FasxgzmAxB ؊/؊ ) to infection with lymphocytic choriomeningitis virus (LCMV). We show that FasxgzmAxB ؊/؊ mice, in contrast to B6, Fas ؊/؊ , and gzmAxB ؊/؊ mice, do not recover from a primary infection with LCMV, in spite of the expression of comparable numbers of LCMV-immune and gamma interferon-producing cytotoxic T lymphocytes (CTL) in all mouse strains tested. Ex vivo-derived FasxgzmAxB ؊/؊ CTL lacked nucleolytic activity and expressed reduced cytolytic activity compared to B6 and Fas Studies with perforin (perf)-deficient mice have shown that perf is a key element in NK cell-and cytotoxic T-lymphocyte (CTL)-dependent recovery from viral infections (20,28,63). However, these studies did not address the question of whether NK cells and CTL can mediate their exocytosis-mediated function in vivo solely via perf or whether they depend on additional effector molecules for maximal effector function, in particular components of the exocytosis pathway, such as granzymes (gzm) (58), and/or those of other cytolytic processes, such as Fas ligand (FasL) or tumor necrosis factor (TNF) (15,21,58). A signature of all three effector pathways is induction of DNA fragmentation (nucleolysis) in vitro, a marker of apoptosis and target cell death (31, 64).In vitro studies using either purified perf (18, 38, 65) or mice lacking both gzmA and gzmB (45, 46) have shown that NK cells and CTL can lyse target cells-as monitored by the 51 Cr release assay-solely by means of perf. However, it is still unclear whether this in vitro cytotoxicity is of biological relevance for processes underlying NK cell-and CTL-mediated host defenses in vivo. In vitro induction of programmed cell death (termed apotosis) by the granule exocytosis pathway is strictly dependent on the concerted action of perf and functional active gzm (17,42,43,45,46). According to recent studies, gzm, in particular gzmA and gzmB, which are released by NK cells and CTL upon encountering antigens, are bound to and internalized by target cells, are subsequently delivered to the cytosol via perf, and initiate cell death by caspase-dependent (gzmB) and/or caspase-independent (gzmA, gzmB) pathways (7, 12-14, 27, 33, 40, 55, 58).Recent studies with mouse strains deficient in one or more components of the granule exocytosis pathway indicate that the concerted action of perf and gzm is essential for recovery from primary infection with a number of pathogens, such as ectromelia virus (EV) (29), mouse cytomegalovirus (39), herpes simplex virus (36), and Trypanosoma cruzi (30), as well as for the control of certain tumors (34). On the other hand, a number of related studies suggest that gzms and/or the FasL/ Fas pathway is dispensable for perf-mediated control of tumor growth (9,49,50,...

Stored Fas Ligand, a Mediator of Rapid CTL-Mediated Killing, Has a Lower Threshold for Response Than Degranulation or Newly Synthesized Fas Ligand

“…In contrast, when there is a massive virus infection that has infected large numbers of cells, it might be advantageous for the CTL to kill all surrounding cells, sacrificing some noninfected cells to quickly clear the virus. Indeed, recent studies have suggested that FasL may be important for clearing chronic infections (7,8). It would be of interest to investigate the contribution from particular FasL pools (stored versus de novo synthesized) for the antiviral immunity during these infections.…”

“…Initial studies suggested that FasL was largely dispensable for viral clearance in the relatively few systems that were examined (6). However, more recent studies suggest that FasL may be important for clearing persistent infections (7,8) and may contribute, along with the perforin pathway, to the shaping of the diversity of escape variants of influenza (9). Although FasL is not required for clearance of viruses that induce hepatitis in mice, it appears to contribute to viral pathogenesis because of significant bystander killing of hepatic cells (10)(11)(12).…”

Mouse granzyme K has pro-inflammatory potential

“…Aliquots of liver tissues were homogenized and used for determination of virus titers as described elsewhere. 39 Generation of ex vivo CD8 þ cells. On day 8 p.i., CD8 þ cells were positively selected from the spleen using a-CD8-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) with an autoMACS (Miltenyi Biotec) and resuspended in MEM/5% FCS before use in cytotoxic assays as described previously.…”

“…of LCMV-WE according to established protocols. 25,39 IL-1Ra (Anakinra) treatment. Mice were injected 1 day before infection with 10 5 p.f.u.…”

Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes?

“…Splenocytes, mouse granulocytes, and human PBMCs and PMNs were washed with FACS-washing buffer (PBS, 5% FCS, 0.1% NaN 3 ) as previously described 31 and incubated with 10 L mAbs for 20 minutes. ␣Mouse (m) mAb used were fluorescein isothiocyanate (FITC)-and phycoerythrin (PE)-labeled ␣B220 (clone RA3-6B2), PE-labeled ␣CD8 (clone 53-6.7), FITC-and PE-labeled ␣Gr-1 (clone RB6-8C5), FITC-labeled ␣Mac-1 (clone M1/70), FITC-and PE-labeled ␣NK1.1 (clone PK136), and FITC-and PE-labeled ␣Thy1.2 (clone 53-2.1).…”