INTRODUCTION

Excess, autoreactive, or damaged cells have the ability to self-destruct by a process termed apoptosis. When properly controlled, apoptosis maintains tissue homeostasis. However, dysregulation of apoptosis, resulting in the abnormal accumulation or destruction of cells, is involved in the pathogenesis of a variety of disorders including autoimmune disease, cancer, and AIDS (1). The signaling pathways controlling apoptosis are beginning to be defined. One such pathway is triggered by the cell surface receptor Fas, a member of the tumor necrosis receptor family expressed in a wide variety of tissues including thymus, liver, heart, kidney, and lymphoid and nonlymphoid malignancies (2-4). Fas induces apoptosis when ligated by natural Fas ligand, which is found predominantly on activated T cells and natural killer cells, or by Fas agonist antibody (2, 3, 5).

Fas-induced apoptosis plays an important role in modulating the immune response. Mice or humans deficient in Fas or Fas ligand develop lymphoproliferative and autoimmune disorders (6-10), suggesting that Fas plays a physiological role in the elimination of excess and/or autoreactive lymphocytes. Activated or autoreactive T and B cells in the periphery are eliminated via Fas-induced apoptosis (11-15), as are inflammatory cells entering immune privileged sites such as the eye and the testis (16, 17). In addition, cytotoxic T cells and natural killer cells kill their targets via Fas-induced apoptosis (18-20).

The intracellular domain of Fas contains a region of homology with the tumor necrosis factor receptor 1, termed the death domain, which is required for inducing apoptosis (21, 22). Trimerization of Fas by Fas ligand leads to the association of FADD/MORT1 with Fas via their homologous death domains (23-25). Caspase-8, a cysteine protease related to the interleukin 1B-converting enzyme family, then binds to FADD/MORT1 (26, 27). This family of cysteine proteases (caspases) cleave their substrates after aspartate residues (4). Caspases have significant homology to the ced-3 gene of Caenorhabditis elegans, which is required for apoptosis in this nematode (28, 29). Caspases also appear to be required for Fas-induced apoptosis in higher organisms because the cowpox virus crmA gene product, a serpin that inhibits the enzymatic activity of interleukin 1B-converting enzyme and related caspases (30), dramatically inhibits Fas-induced apoptosis in mammalian cells (31). After caspase-8 binds to FADD/MORT1, additional members of the caspase family are sequentially activated and subsequently cleave proteins such as poly(ADP-ribose) polymerase, leading to the characteristic morphologic changes of apoptosis.

Many Fas-expressing cells are not sensitive to Fas-induced apoptosis, suggesting that cellular factors exist that inhibit Fas signaling. Possible mediators of Fas resistance include elevated Bcl-2 or Bcl-xL expression (32-34), production of soluble Fas that blocks Fas ligand binding to Fas (35), or overexpression of FAP-1, a phosphatase that binds to Fas and inhibits apoptosis (36). Another potential inhibitor of Fas signaling is nitric oxide (NO),¹ or related molecules, synthesized by the enzyme NO synthase (NOS), of which there are three major isoforms (37). In previous studies, we demonstrated that low level basal NOS activity inhibits spontaneous apoptosis in certain human B cell lines. Because Fas is a critical regulator of apoptosis, we were interested in whether NOS also modulates Fas-induced apoptosis in human leukocytes. Such a role for NO would strengthen the case for its involvement in the human immune response, which has been difficult to demonstrate.

EXPERIMENTAL PROCEDURES

Cell lines were obtained from the ATCC (Jurkat, H9, 2F7, and 10C9), or from the laboratory of Dr. Elliott Kieff (IB4 and BJAB).

100 µl of BJAB or 10C9 cells that had been fed 1-3 days previously and were growing logarithmically (approximately 500,000 cells/ml) in RPMI 1640 containing 1 mM L-arginine (Sigma) and 10% fetal bovine serum (Life Technologies, Inc.) were plated in 96 well plates. 100 µl of modified RPMI 1640 10% fetal bovine serum (Life Technologies, Inc. SelectAmine) were added to each well to bring the final L-arginine concentration to 0.5 mM. The Fas agonist monoclonal antibody clone CH-11, IgM (Upstate Biotechnology) was added to the appropriate wells at 5-10 ng/ml for BJAB cultures and 5-20 ng/ml for 10C9 cultures. L-NMA (5 mM versus 0.5 mM L-arginine in the medium), L-arginine (2.5-5 mM), or S-nitrosopenicillamine (10-100 µM) was added to the indicated wells. After 24-48 h, the percentage of apoptotic cells was determined by staining the cells with acridine orange as described below.

30 µl of cells from each culture were pelletted, and 10 µl of cell slurry was mixed with 10 µl of acridine orange (5 µg/ml) diluted in phosphate-buffered saline. The percentage of cells with apoptotic morphology (nuclear and cytoplasmic condensation, nuclear fragmentation, membrane blebbing, and apoptotic body formation) was then analyzed on a wet mount slide using a Zeiss Axioskop equipped with epifluorescence.

Cells were stained with propidium iodine as described previously (38). The percentage of apoptotic cells was quantitated using a Facscan Flow Cytometer (Becton Dickinson). Fluorescence data were collected using logarithmic amplification, and necrotic debris and cellular aggregates were eliminated from the data by forward and right angle light scatter gates. Apoptotic cells were distinguished from nonapoptotic intact cells by their decreased DNA content as determined by lower propidium iodine staining intensity.

Whole cell lysates were made by boiling cells in sample buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 20% glycerol, 10% 2-mercaptoethanol). Proteins in the cell lysates were then separated by SDS/polyacrylamide gel (7%) electrophoresis and transferred to nitrocellulose. Rabbit anti-iNOS (used at a 1:500 dilution) and rabbit anti-PARP (Upstate Biotechnology Inc., 1 µg/ml) were used as primary antibodies for Western blots, and horseradish peroxidase-labeled goat-anti-rabbit Ig was used as a secondary antibody (Amersham Corp.). The blots were developed using enhanced chemiluminescence (ECL) as per the manufacturer's instructions (Amersham Corp.).

Cells were incubated in the presence or the absence of 2 µg/ml of Fas monoclonal antibody (clone CH-11, IgM, Upstate Biotechnology Inc.) for 60 min at 4 °C, followed by three washes in ice-cold phosphate-buffered saline containing 0.1% fetal bovine serum and sodium azide. The cells were then incubated with fluorescein-conjugated goat-anti-mouse Ig (Jackson Labs) for 45 min at 4 °C, washed three times, and resuspended in 1% formalin. The percentage of Fas-positive cells was quantitated using a Facscan Flow Cytometer (Becton Dickinson).

RESULTS

To confirm that human leukocytes are capable of synthesizing NO (and related species) intracellularly, iNOS expression (the main NOS isoform expressed in murine leukocytes) was analyzed by Western blot analysis in multiple human leukocytic cell lines. Basal expression of iNOS was detected in all human B cell, T cell, and monocytic cell lines tested (Fig. 1). The effects of NOS on Fas-induced apoptosis was examined initially in BJAB, the human B cell line that in previous studies contained the highest levels of basal NOS activity (39). Fas was ligated on the surface of BJAB cells with anti-Fas monoclonal antibody (5-10 ng/ml clone CH-11, IgM, Upstate Biotechnology) in the presence or the absence of the competitive NOS inhibitor L-NMA. NOS inhibition significantly increased Fas-induced apoptosis (Figs. 2, A and B), suggesting that constitutive NOS activity attenuates Fas-mediated apoptosis. Confirming that the effects of L-NMA were due to a specific inhibition of NOS, the enzyme substrate L-arginine and the NO donors S-nitroso-N-acetylpenicillamine (Fig. 2A) or sodium nitroprusside (data not shown), partially reversed the inhibition. These findings were reproduced in another Burkitt's lymphoma B cell line, 10C9, which is derived from a patient infected with human immunodeficiency virus, type 1 (HIV-1). That is, inhibition of NOS by L-NMA significantly increased Fas-induced apoptosis in 10C9 cells (Fig. 2C), and the effects were reversed by both L-arginine and the NO donors S-nitrosopenicillamine (Fig. 2C) or sodium nitroprusside (data not shown).

We next analyzed whether our findings in human B lymphoma cell lines were generalizable to other human cell lines of hematopoietic lineage. L-NMA increased Fas-induced apoptosis in the T lymphoma cell line H9, the T leukemia cell line Jurkat, the promonocytic cell line U937, the Epstein-Barr virus (EBV)+ B lymphoblastoid cell line IB4, and in the EBV + AIDS-related Burkitt's lymphoma B cell line 2F7 (Fig. 3). Thus intracellular NOS activity may be a general mechanism by which transformed cells of hematopoietic lineage inhibit Fas-induced apoptosis.

Significant inhibitory effects of NOS on platelets and smooth muscle derive from increases in cGMP levels due to activation of soluble guanylyl cyclase (40, 41). However, the inhibitory effects of NOS on Fas-induced apoptosis appear to be cGMP-independent: the cell permeable cGMP analog, 8-bromo-cGMP (0.1-1 mM) failed to overcome the effects of L-NMA (Fig. 4). Although the guanylyl cyclase inhibitors ¹H-[1,2,3]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and LY 83583 (10-100 µM) increased spontaneous as well as Fas-induced apoptosis, the mechanism did not appear to be due to a specific inhibition of guanylyl cyclase, because the apoptosis was not prevented by 8-bromo-cGMP (0.1-1 mM) (Fig. 4).

One mechanism by which NOS could inhibit Fas-induced apoptosis is to decrease Fas expression levels on cells. To test this hypothesis, 10C9 B cells or H9 T cells were grown in the presence or the absence of the NOS inhibitor L-NMA, and Fas expression was analyzed by immunofluorescent staining and flow cytometry. L-NMA had no effect on Fas expression at concentrations that increased Fas-induced apoptosis (Fig. 5), indicating that other mechanisms are involved in the inhibitory effects of NOS.

Because NOS did not affect Fas expression levels, we began to map the step(s) in the Fas signaling pathway that might be inhibited by NOS. Members of the caspase family of cysteine proteases are activated by Fas ligation and cleave PARP to its signature 85-kDa fragment (4, 42). Caspase activation can be monitored by analyzing the extent of PARP cleavage in cells. To determine whether caspase activation is inhibited by NOS, 10C9 or H9 cells were grown in the presence or the absence of Fas agonist antibody and L-NMA, and at various time intervals the extent of PARP cleavage was assessed by Western blot analysis using an anti-PARP polyclonal antibody. NOS inhibition resulted in increased levels of Fas-induced PARP cleavage (Fig. 6), suggesting that NOS inhibits caspase activation.

DISCUSSION

Our results indicate that NOS inhibits Fas-induced apoptosis in human B cell, T cell, and monocytic cell lines via a cGMP-independent mechanism. NOS appears to inhibit Fas-induced caspase activation and PARP cleavage without altering levels of Fas expression. Although studies have shown that NO or related molecules can exert either pro- or anti-apoptotic effects depending on the cell type and stimulus (39, 43-49), the emerging picture is one where the predominant physiologic effect of NOS is inhibition of apoptosis. For instance, NO inhibits apoptosis in murine splenic B cells, human B cell lines, rat ovarian follicles, and human eosinophils (39, 47-49). The cellular factors and target proteins that are responsible for the specificity of NO-related effects on apoptosis remain to be determined.

The cGMP-independent actions of NOS (such as inhibition of Fas-induced apoptosis in human leukocytes) appear to occur through S-nitrosylation of proteins or modulation of redox state. Both mechanisms may operate to inhibit Fas signaling. Specifically, several studies show a role for oxidative stress in Fas-induced apoptosis (50-52). In addition, NO inhibits many enzymes, including cysteine proteases, by S-nitrosylation of active site thiols (53, 54). S-Nitrosylation of the caspase family of cysteine proteases is suggested by our finding that NOS decreases PARP cleavage.

The observation that NOS inhibits Fas-induced apoptosis may explain a previously described association between NO and Fas in studies of the MRL lpr/lpr mouse. The MRL lpr/lpr mouse carries a spontaneous Fas mutation that impairs Fas transcription (6, 55). These mice develop arthritis and nephritis, presumably as a result of impaired Fas-mediated apoptosis of autoreactive lymphocytes. However, other strains of mice carrying the same Fas mutation do not develop autoimmune disease, suggesting that additional factors are involved in the phenotype (56). Interestingly, NOS expression and activity are increased in MRL lpr/lpr mice but not in B6 lpr/lpr mice (who do not develop arthritis and glomerulonephritis); the glomerulonephritis and arthritis can be reduced in MRL lpr/lpr mice using NOS inhibitors (57). The mechanism by which NOS induces glomerulonephritis and arthritis in these mice has not been elucidated. Our data raise the possibility that NO production may unmask the lpr phenotype by inhibiting already low levels of Fas-mediated apoptosis of autoreactive lymphocytes.

Likewise, failure of cells to undergo apoptosis is a factor contributing to the development of some forms of cancer (1). Resistance to Fas-mediated apoptosis may contribute to the pathogenesis of lymphomas because many primary lymphoid neoplasms express Fas (58) without undergoing apoptosis. Elevated levels of NO-related species may contribute to Fas resistance in some of these tumors. In support of this hypothesis, HIV-1-infected patients have a markedly increased incidence of non-Hodgkin's lymphoma (59, 60) and elevated levels of nitrates and nitrites (the stable end products of NO synthesis) in their blood (61-63). Thus, elevated NO production may inhibit apoptotic death of lymphoma cells in AIDS patients, thereby contributing to lymphomagenesis. NO-based therapies may be useful in the treatment of these malignancies and other Fas-associated diseases.