CD8+ T cells appear to play an important pathophysiologic role in many inflammatory lung diseases. The primary effector function of this T-cell subset is cytolysis of virus-infected cells, and it is widely believed that there are two primary molecular mechanisms by which this occurs: the perforin/granzyme-mediated pathway of cytolysis, and the Fas ligand (FasL)–Fas (CD95/APO-1) pathway of induction of target-cell apoptosis. This conclusion is based primarily on data obtained with hematopoetic cell lines as target cells. There is also a growing body of evidence that Fas is involved in the transduction of apoptotic signals in a variety of inflammatory disease states, particularly involving the liver and the lung. In the study reported here we took advantage of a novel in vitro assay to directly assess the effector mechanisms employed in CD8+ T-cell–mediated cytolysis of alveolar epithelial cells. We present evidence that FasL-induced, Fas-mediated apoptosis does not directly contribute to T-cell–mediated cytolysis of alveolar epithelial-derived cells, even though Fas is expressed and functional on these cells. We also demonstrated that the perforin-independent cytolytic activity of CD8+ T cells against alveolar epithelial-derived cells is explained entirely by tumor necrosis factor-α (TNF-α), which is expressed on CD8+ T cells. Furthermore, we show that bystander cytolysis of alveolar epithelial-derived cells by antiviral CD8+ T cells is entirely perforin-independent. This activity is mediated exclusively by TNF-α. Both alveolar epithelial-derived cells and primary murine type II cells show susceptibility to apoptosis triggered by soluble TNF-α, without the need for transcriptional or translational inhibition. We also confirmed the resistance of alveolar type II cells to FasL in vivo by performing adoptive transfer of perforin-deficient antiviral CD8+ T cells into transgenic mice expressing a target antigen in type II epithelial cells. Significant lung injury developed in the transgenic CD8+ T-cell recipients, whether or not Fas was expressed in these animals. Furthermore, preincubation of the T cells with antibody to TNF-α completely abolished the injury. These results suggest that alveolar epithelial cells are relatively sensitive to T cell-triggered, TNF-α–mediated apoptosis, and resistant to apoptosis triggered by FasL. These observations may have important ramifications for understanding of the pathophysiology of interstitial and inflammatory lung diseases.
A number of inflammatory lung diseases are presumed to be T-cell mediated, and are characterized by the accumulation of T cells in the alveolar space or in the interstitium (1-4). The role(s) that CD8+ T cells play in the pathogenesis of interstitial lung disease is unclear, as is the nature of the effector function(s) expressed at the sites of inflammatory activity. CD8+ T cells express potent cytotoxic activity, which appears to function primarily in the clearance of virus infection (5-11). In vitro analysis of antiviral activity of CD8+ T cells strongly suggests that cytolytic activity is both necessary and sufficient to effect the lysis of virus- infected target cells (12-14). Several groups have shown that CD8+ T cells utilize two primary molecular mechanisms to induce target cell death: the perforin/granzyme-mediated pathway of cytolysis and the Fas ligand (FasL)– Fas (CD95) pathway of induction of target-cell apoptosis. In the absence of both of these mechanisms, there is no cytolysis of conventional target cells, which are generally of hematopoetic origin (12, 13, 15-19). The molecular basis of T-cell–mediated cytolysis of respiratory cells has not been directly addressed, but the issue is an important one, since the respiratory tract is a frequent target of virus infection.
Initial interest in Fas focused on its role in maintenance of the lymphoid compartment (20-25). However, over the past several years, there has been intensified interest in extralymphoid expression of Fas and its potential participation in tissue inflammation and injury. In particular, numerous reports have suggested a role for Fas in chronic inflammatory liver disease (26-33), thyroiditis (34, 35), experimental diabetes (36), and lung disease. The literature on participation of Fas in lung disease is replete with demonstrations of expression of Fas in clinical (37-40) and experimental specimens (41, 42). In addition, several intriguing studies have involved the experimental induction of pneumonitis (43) and type II cell apoptosis by ligation of Fas by agonist antibodies (44, 45). There is so far no direct evidence that Fas-mediated apoptosis plays a role in lung injury mediated by FasL-expressing T cells. Several lines of evidence have, however, suggested that tumor necrosis factor (TNF)-α has a primary or secondary role in pulmonary inflammation (46-48), though not specifically in injury mediated directly by T cells. TNF-α is a minor contributor to T-cell effector function, and has not been shown to participate in acute cytolysis of conventional target cells (49). In the present study, we took advantage of a novel in vitro assay to present evidence that alveolar epithelial-derived cells are susceptible to CD8+ T-cell–triggered apoptosis induced by TNF-α, but are resistant to apoptotic signals triggered by FasL. We also showed that perforin-independent, T-cell–mediated cytotoxicity of alveolar epithelial-derived cells is mediated entirely and exclusively by TNF-α. Furthermore, using a transgenic model of T-cell–mediated lung injury (50, 51), we showed in vivo that Fas-deficient animals are not protected against injury by activated CD8+ T cells, even when the T cells are derived from perforin-deficient animals, whereas pretreatment of T cells with antibody to TNF-α abolishes lung injury induced by perforin-deficient T cells.
MLE-15 cells (52) were transfected with the class I major histocompatibility complex (MHC) molecule, Kd (construct provided by Dr. Michael Bevan, University of Washington, Seattle, WA). The transfectants were stained with SF1.1 (anti-Kd; American Type Culture Collection [ATCC], Rockville, MD) and TIB95 (anti-Kq, the native haplotype of the parental line; ATCC), and sorted by flow cytometry according to their level of Kd expression with a FACScan instrument using CellQuest software (Becton-Dickinson, Palo Alto, CA). The cells were then cloned by limiting dilution, and were passaged twice a week.
The CD8+ T-cell clones used in our experiments were generated by limiting dilution as previously described (53), from either wild-type or perforin-deficient mice (18). The clones were restimulated weekly in vitro with irradiated syngeneic splenocytes that were infected with A/Japan/57 influenza virus, and were placed into fresh Iscove's complete medium supplemented with 10 IU/ml of interleukin (IL)-2. On Day 5 after in vitro stimulation, T cells were tested with a 51Cr release assay for cytolytic activity against target cells infected with A/Japan/57 or against target cells loaded with 10−9 M synthetic peptide representing the 210–219 epitope of the A/Japan/57 hemagglutinin (HA) (50). The target cells used were MLE-Kd cells and L121+ cells, the latter of which are transfected with a Fas- overexpression construct (54). In some experiments, the anti-Fas monoclonal antibody MFL3 was added to wells at a final concentration of 5 μg/ml, or anti-TNF-α antiserum (IP400, Genzyme, Boston, MA) was added at a final dilution of 1:100. Cytotoxicity assays were conducted for 6 h in 96-well plates with a final volume of 0.2 ml/well of target and effector cells in culture medium, after which 0.1 ml was harvested and counted on a gamma counter (Isomedic; ICN Biomedicals, Inc., Costa Mesa, CA). Percent specific 51Cr release was calculated according to the formula: (test cpm − spontaneous release cpm)/(total cpm − spontaneous cpm) × 100. Spontaneous release from targets incubated with medium alone was always less than 10%. Specific lysis values represent the mean percent specific 51Cr release from four replicate wells.
MLE-Kd cells were labeled with tritiated thymidine for 6 h and were then incubated for 24 h with 0.01 μg/ml Jo-2 antibody (PharMingen, San Diego, CA) or with an isotype- matched control hamster antibody (MFL3), with or without 20 μg/ml protein G (Sigma, St. Louis, MO). The cells were then lysed and harvested. L1210+ and L1210− cells, which are transfected with a Fas-overexpression or antisense construct, respectively (54), were similarly treated and were used as controls. In some experiments, soluble TNF-α (Genzyme, Boston, MA) was added to MLE-Kd cells at varying concentrations for 6 h prior to harvesting (5 × 107 U/mg). Percent DNA fragmentation was calculated as described (55) according to the formula: (total cpm [immediately after labeling] − test cpm)/total cpm × 100. All values represent the mean of four replicate wells.
Primary alveolar type II cells were prepared according to a modification of a previously published method (56). Briefly, BALB/c mice were anesthetized and exsanguinated by severing the inferior vena cava and left renal artery. The tracheae were exposed and cannulated, and the lungs were perfused with 10–20 ml sterile saline via the pulmonary artery until visually free of blood. Dispase (Collaborative Research, Inc., Bedford, MA) was instilled into the lungs via the tracheal catheter, and was followed by 1% low melting agarose, warmed to 45°C. The lungs were immediately covered with ice and incubated for 2 min to gel the agarose. Lungs were then dissected free of the animal, put in a culture tube containing an additional 1 ml of Dispase, and incubated for 45 min at room temperature. Lungs were then transferred to a culture dish containing deoxyribonuclease (DNAse) I (Sigma, St. Louis, MO) and the tissue was gently teased away from the airways. The cell suspension was successively filtered and then pelleted. Crude cell suspensions were added to culture dishes coated with anti-CD45 and anti-CD32 antibodies (PharMingen) and incubated for 1–2 h. Plates were removed from the incubator and gently “panned” to free settled type II cells, which were resuspended in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Purity of the type II cell preparations used for these studies was 92 ± 1% according to morphologic criteria (56), and cell viability determined by Trypan blue exclusion after overnight incubation was > 60%. (Purity for more than 100 separate preparations was consistently > 90%.) Cells were plated in 96-well plates and labeled overnight with tritiated thymidine. The wells were washed to remove unincorporated label and nonadherent cells, and the remaining cells were then incubated for 6 h with TNF-α. The percent DNA fragmentation was calculated as described (55), and represents the mean of four replicate wells.
SPC-HA transgenic mice (H-2d) expressing the A/Japan/ 305/57 influenza HA under the transcriptional control of the surfactant protein-C (SP-C) promoter were used in these studies (50). Transgene-negative littermates were used as control recipients. Animals subjected to adoptive transfer were used at 10–12 wk of age (18–22 g). Fas-deficient (lpr) animals in the H-2d haplotype (generously provided by Dr. Philip Cohen of the University of North Carolina, Chapel Hill, NC) were bred with the SPC–HA transgenic mice for use in some experiments. The offspring were screened for the HA transgene as described (50), and for the lpr mutation with a three-primer polymerase chain reaction (PCR) method as described (57). The CD8+ cytolytic T-cell clone 40-2 was restimulated in vitro with irradiated syngeneic splenocytes that were infected with A/Japan/57 influenza virus. On Day 5 after stimulation, clones were separated from stimulators by density gradient centrifugation and were injected via the tail vein into appropriate recipient animals. In some instances, T cells were preincubated with anti-TNF-α antibody (1:100, IP400; Genzyme) for 30 min at 4°C and were then washed twice before adoptive transfer. In order to quantitatively assess the physiologic dysfunction induced by T-cell transfer, we measured carbon monoxide (CO) uptake and inspiratory capacity (IC) 4 d after cell transfer, as previously described (51). Briefly, after tracheal intubation, the IC of each animal was estimated by inflation with air to a pressure equivalent to 20 cm H2O (58, 59). The lungs were then inflated with a volume equal to the IC of a gas mixture containing 0.3% CO, 0.3% methane, 20% oxygen, and the balance consisting of nitrogen. The gas was allowed to dwell for a 10 s breathhold, after which one half of the sample was drawn off and discarded as dead space and the other half was drawn off as an alveolar sample (60). Three measurements were made for each animal, and the CO uptake was determined as the ratio of CO loss to methane loss (59, 61).
Significant differences were determined with Student's unpaired t test or through one-way analysis of variance (ANOVA).
In order to explore the molecular basis of CD8+ T-cell– mediated injury to alveolar epithelium, we developed an in vitro system for measuring T-cell cytolysis of alveolar epithelial-derived cells. The MLE-15 cell line, which maintains phenotypic characteristics of type II alveolar epithelial cells (52), was transfected with the class I MHC molecule Kd (the haplotype of the transgenic animals and the T-cell clones used in these studies). The level of expression of Kd was comparable to the expression level of the endogenous MHC class I molecule, Kq, as determined by flow cytometry (not shown). We have previously shown that CD8+ T cells recognize and lyse transfectant MLE-Kd cells if the target cell is infected with the appropriate strain of influenza virus or if exogenous peptide loading of Kd occurs with the appropriate Kd-restricted epitope, but that no such recognition of untransfected MLE-15 cells occurs (51). As shown in Figure 1, the recognition and cytolysis of peptide-loaded MLE-Kd cells by the CD8+ cytolytic T-cell clone 40-2 was completely unaffected by the anti-FasL antibody MFL3 (at 5 μg/ml). In order to demonstrate that this antibody (5 μg/ml) was capable of completely blocking FasL-dependent lysis by clone 40-2, we used the paired target cells L1210+ and L1210−, which are transfected with sense and antisense Fas overexpression constructs, respectively. These cells are (not surprisingly) either very sensitive or insensitive, respectively, to FasL-triggered apoptosis (19, 54). Cao and colleagues, using the identical T-cell clone 40-2, have previously shown that T-cell recognition of foreign antigen triggers both the perforin and the FasL/ Fas pathways of cytotoxicity. In contrast, recognition of a “self antigen” triggers cytolysis mediated specifically and exclusively by FasL, with no utilization of the otherwise intact perforin-mediated pathway of cytotoxicity (54). Therefore, this target cell pair used in our study provides a very useful readout of perforin-independent cytotoxicity. As shown in Figure 2, there was minimal inhibition by the anti-FasL antibody MFL3 of recognition and cytolysis of target cells (L1210+ or L1210−) by 40-2 cells in the presence of the HA210-219 peptide, indicating significant residual perforin-mediated lysis. However, in the absence of peptide antigen (i.e., in the presence of self antigen only), there was no cytolysis of L1210− cells, and yet there was significant lysis of L1210+ cells, indicating perforin-independent (FasL-mediated) cytolysis. Furthermore, the cytotoxicity of 40-2 cells toward L1210+ cells in the absence of peptide was completely inhibited by the MFL3 antibody at 5 μg/ml. Therefore, this concentration of the antibody was adequate to completely inhibit the FasL-mediated cytotoxic activity of clone 40-2. Importantly, as shown in Figure 1, blockade of FasL-mediated activity by antibody did not inhibit the lysis of MLE-Kd cells, indicating the insensitivity of these alveolar type II cells to Fas-dependent lysis.
In order to further explore the mechanisms involved in the T-cell–mediated cytotoxicity of alveolar epithelial-derived cells, we used an alternative strategy, utilizing CD8+ T-cell clones from perforin-deficient animals (18). Bulk HA-specific CD8+ T cells were generated from perforin-deficient animals (bred into the H-2d haplotype) that were immunized with A/Japan/57 influenza virus. We then produced individual CD8+ T-cell clones from the bulk cultures by limiting dilution (53). These T cells exhibited no cytolysis of Fas-deficient L1210− cells, but did exhibit lysis of peptide-loaded L1210+ cells, and this perforin-independent cytolytic activity was completely inhibited by antibody to FasL (MFL3; not shown). We therefore tested the sensitivity of MLE-Kd cells to perforin-independent cytolysis, using perforin-deficient T cells. As shown in Figure 3, the CD8+ T-cell clone PKO-GV effected cytotoxicity at levels similar to those of wild-type T cells in the presence of antigenic peptide, but showed no effect of MFL3 antibody. Interestingly, cytolysis of MLE-Kd cells by PKO-GV cells was completely inhibited by antibody to TNF-α, which has been shown to be expressed on CD8+ T cells, predominantly as a membrane-bound molecule (49). Several other, independently evaluated perforin-deficient CD8+ T-cell clones exhibited the same phenotype (not shown). These data indicate that FasL-insensitive MLE-Kd cells are unusually sensitive to TNF-α-mediated cytotoxicity by CD8+ T cells. The sensitivity of MLE-Kd cells to TNF-α is a very uncommon phenotype: it is very difficult to demonstrate this phenomenon in other target cells without the addition of inhibitors of transcription or protein synthesis (62-64) and/or without testing for cytotoxicity over a considerably longer assay period, usually 24 h or more (19, 49). Furthermore, the inhibition of perforin-independent cytolysis by anti-TNF-α antibody confirms that MLE-Kd cells are resistant to apoptotic signals delivered by FasL expressed on CD8+ T cells.
In order to rule out the possibility that FasL-induced apoptosis of alveolar epithelial-derived cells plays a role in “bystander” injury, as has been suggested with other cell types (65, 66), we investigated the relationship between antigen recognition of and cytotoxicity to neighboring cells, using a transfectant cell line (BHA) as antigen-presenting cells (P815 cells expressing full length A/Japan/57 HA). Like most cells that have been studied with regard to apoptosis (62), P815 (and BHA) cells are resistant to TNF-mediated apoptosis in the absence of protein synthesis inhibitors, and anti-FasL antibody completely blocks all cytolysis of these target cells by perforin-deficient T cells (not shown). Figure 4 shows the results of a representative experiment in which BHA cells (unlabeled) were used as the antigen-presenting cells and 51Cr-labeled MLE-Kd cells (without virus infection or peptide loading) were used as bystander cells. Recognition of “cold” BHA cells by PKO-GV cells resulted in cytolysis of “hot” MLE-Kd cells, but in minimal lysis in the absence of the BHA cells. The level of cytolysis was almost identical to that produced by peptide treatment of the MLE-Kd cells (in the absence of BHA cells). This phenomenon is clearly the result of a specific recognition event, since untransfected (parental) P815 cells do not activate the T cells to effect bystander cell lysis. These results also clearly indicate that the capacity to induce bystander-cell cytotoxicity is not a constitutive phenomenon (i.e., after in vitro activation), since specific antigen recognition in the assay is required. Importantly, the cytolysis of bystander MLE-Kd cells induced by BHA-triggered PKO-GV cells was completely inhibited by anti-TNF-α antibody.
We further investigated TNF-mediated bystander injury with the wild-type CD8+ T-cell clone 40-2 as the effector cells and with A/Japan/57 influenza-infected MLE-Kd cells as the triggering cells. In these experiments, the bystander targets were either 51Cr-labeled MLE-Kd cells or 51Cr-labeled parental MLE cells (both without virus infection or peptide loading). As shown in Figure 5, there was no significant cytolysis when 40-2 cells were incubated with either the labeled uninfected MLE-Kd cells or with labeled uninfected parental (untransfected) MLE-15 cells, and there was no effect of anti-TNF antibody in either circumstance. However, the addition of unlabeled influenza-virus-infected MLE-Kd cells (at a 1:1 ratio to labeled uninfected bystanders cells) resulted in significant bystander cytotoxicity, which was independent of Kd expression by the bystander cell. As in the previous experiment, bystander-cell cytolysis was completely inhibited by anti-TNF antibody. These results suggest that this effect represents true bystander injury (i.e., cytotoxicity unrelated to peptide transfer and cross-presentation). These data also suggest that perforin does not participate in bystander injury to alveolar epithelial-derived cells, in contrast with the primary role played by perforin in direct cytolysis of virus-infected MLE-Kd cells by wild-type T cells (data not shown).
Although others have demonstrated Fas expression by the parental (MLE-15) cell line (45, 67, 68), our results as described earlier could be explained by the loss of Fas expression that might have occurred during transfection, selection, and cloning of the MLE-Kd cell. Other investigators have reported induction of apoptosis of alveolar epithelial cells, including MLE-15 cells, with the agonistic anti-Fas antibody Jo-2 (44, 45). In order to confirm that MLE-Kd cells express functional Fas, we investigated whether Jo-2 could induce apoptosis in MLE-Kd cells. We were unable to detect cytolysis of MLE-Kd cells induced by Jo-2 antibody in standard 6-h 51Cr-release assays, although this was not surprising in view of our observations with other cell lines (even those that are functionally Fas-sensitive) in standard 51Cr-release assays (unpublished results). Therefore we took advantage of a more sensitive DNA fragmentation assay (55) that has been used previously to measure Fas-mediated cell death (54). This assay has the advantage of detecting earlier events in apoptosis, occurring before the membrane disruption necessary for 51Cr-release (55). As shown in Figure 6, MLE-Kd cells exhibited significant DNA fragmentation by 6 h after crosslinking with the Jo-2 antibody. This effect in MLE-Kd cells was not enhanced by the addition of protein G, even though significant enhancement of DNA fragmentation occurred in L1210+ cells upon addition of protein G. Spontaneous apoptosis (medium alone) was less than 5% by 6 h. An isotype-matched control hamster antibody (MFL3) had no effect, at a variety of concentrations, with or without protein G (not shown). Similarly, protein G alone had no effect. These data strongly suggest that Fas is present and capable of triggering the apoptotic cascade in MLE-Kd cells, although the functional significance of this remains unclear.
Although anti-TNF antibody completely inhibited the cytolysis of MLE-Kd cells by perforin-deficient CD8+ T cells, the data do not reveal whether this was due to membrane-bound or secreted TNF-α. To investigate whether MLE-Kd cells are susceptible to apoptosis triggered by soluble TNF-α, we performed DNA fragmentation assays after 6 h of treatment with varying doses of TNF-α. As shown in Figure 7A, soluble TNF-α induced significant DNA fragmentation in MLE-Kd cells as compared with media controls, and the effect reached a plateau at 1,000 U/ml. In order to confirm that the susceptibility of the MLE-Kd cells to cytotoxicity mediated by soluble TNF is not unique to this type II alveolar cell line, but rather reflects an intrinsic property of untransformed lung epithelial cells, we isolated primary murine type II pneumocytes for use in correlative studies. As shown in Figure 7B, soluble TNF-α (1,000 U/ml) induced significant apoptosis in primary type II cells as compared with controls (P < 0.05). This suggests that the TNF-sensitivity of MLE-Kd cells is not a unique feature of this particular transformed cell line.
We have previously reported that wild-type, influenza-virus– specific CD8+ T cells induce significant lung injury upon being adoptively transferred into transgenic mice expressing influenza HA in their type II alveolar epithelial cells (50, 51). This injury is characterized by significant loss of alveolar epithelial cells, as well as by restrictive physiology, significant impairment of gas exchange, and death (51). We also demonstrated that the impairment in gas exchange, as determined by CO uptake, correlated especially strongly with the histologic and clinical abnormalities observed in these mice (51). In order to determine the relative contribution of the perforin-independent mediators FasL and TNF-α to T-cell cytotoxicity and injury in vivo, we performed adoptive transfer of the perforin-deficient (FasL+, TNF-α+), HA-specific CD8+ T-cell clone PKO-GV into three recipient groups: wild-type HA+ mice, Fas-deficient/HA+ mice, and HA− control littermates. As shown in Figure 8, there was a significant decrement in CO uptake in HA+ transgenic recipients as compared with their HA− littermates (which was similar to that measured in animals that had not received cells; data not shown). Interestingly, there was no abrogation of injury by PKO-GV T cells in the absence of Fas expression in the Fas-deficient recipients (lpr/HA+ mice). However, preincubation of PKO-GV T cells with anti-TNF antibody resulted in complete abrogation of lung injury, further suggesting the Fas-independent nature of this (perforin-independent) CD8+ T-cell–mediated injury process. Histologically, the lung sections harvested at Day 4 from lpr/HA+ animals that received anti–TNF-antibody-treated T cells were indistinguishable from the lungs of HA− recipients (data not shown); there was minimal perivascular and peribronchiolar cuffing without significant alveolar infiltration, resembling that previously described (50, 51). No difference was observed histologically between the HA+ and the lpr/HA+ recipients of PKO-GV T cells, and both showed significant infiltration of the alveolar walls and spaces, resembling that previously described (50, 51). Prior to T-cell transfer, lpr/HA+ mice were indistinguishable from normal (HA−) mice both histologically and physiologically (data not shown). The 4-d time point was chosen on the basis of previous data (51) as well as from the tempo of progression of clinical illness observed in these animals.
Many interstitial lung diseases are in part characterized by varying degrees of CD8+ T-cell (in addition to other inflammatory cell) infiltration of the alveolar septae, spaces, or both. We have previously shown that antigen-specific CD8+ T-cell recognition by a single clonal population and of a single epithelial “autoantigen,” is in and of itself sufficient to trigger an inflammatory cascade that results both histologically and physiologically in the clinical picture of interstitial pneumonia (50, 51). Adoptive transfer of 107 T cells resulted in a complete loss of alveolar epithelial cells, resembling the epithelial denuding seen in disease states associated pathologically with diffuse alveolar damage (such as acute interstitial pneumonia). T cells express a range of cytotoxic and noncytotoxic effector functions, which have been intensively investigated in the context of viral infection. CD8+ T cells are particularly associated with cytotoxic activity when they recognize class I MHC-restricted peptide epitopes, such as viral antigens. The major mechanisms involved in CD8+ T cell cytotoxicity have been elucidated in vitro, and several groups have clearly shown that the perforin/granzyme pathway and the Fas– FasL pathway account for all of these cells' in vitro cytolytic effect as observed in cytotoxicity assays using conventional target cells, which are generally of hematopoetic origin (12, 13, 15-18). The role of Fas in T-cell suicide and fratricide is well described (20-25, 69), although Fas expression has recently been reported in a number of nonhematopoetic tissues, such as liver (26-33) and lung (37-40). The role of Fas in the expression of immune effector activity (or injury) in these tissues is unclear, although it has been suggested that Fas may play a role in immunity to virus infection (37-40). Several studies have suggested that Fas may participate in immune injury in chronic inflammation of the liver (28-30) and lung (38, 40). There is so far no direct evidence that Fas-mediated apoptosis plays a role in lung injury caused specifically by FasL-expressing T cells.
Experimentally, alveolar epithelial-derived cells may be triggered to undergo apoptosis upon ligation of Fas with Jo-2 antibody (44, 45), and intratracheal administration of this antibody in mice results in significant lung injury (43). We have confirmed Fas expression in the lungs of HA+ transgenic mouse lines by reverse transcription– PCR, and in the alveolar epithelium of these animals by in situ hybridization (unpublished observations). It was therefore somewhat surprising to observe that the absence of Fas expression in lpr/HA+ transgenic mice did not abrogate the injury produced by T-cell transfer, even in the absence of perforin. It was also surprising to observe that MLE-Kd cells were totally insensitive to the effects of FasL expressed by activated T cells, whereas neutralizing antibody to FasL completely inhibited cytotoxicity to Fas-sensitive L1210+ target cells by wild-type CD8+ T cells of clone 40-2 (without peptide) and by T-cells of the perforin-deficient clone PKO-GV (with peptide). In this experiment, anti-FasL antibody completely eliminated all FasL-mediated cytotoxicity by both 40-2 cells and PKO-GV cells against L1210+ cells, but had no effect on the cytolysis of MLE-Kd cells. Interestingly, neutralizing antibody to TNF-α completely inhibited all cytotoxicity to MLE-Kd cells by the perforin-deficient T-cell clone PKO-GV. The lung injury observed in vivo in lpr/HA+ mice after transfer of perforin-deficient T cells, and its abrogation by pretreatment of such T cells with antibody to TNF-α, further support the hypothesis that alveolar epithelial cells may be insensitive to FasL-triggered apoptosis. This in vivo result is consistent with our in vitro observation that perforin-independent type II cell cytotoxicity is mediated exclusively by TNF-α (though we cannot formally exclude the possibility that the carryover of a small amount of anti-TNF may also have blocked TNF-α secreted in vivo from non–T-cell [host] sources despite the washing of T cells after preincubation). Nevertheless the effect of pretreatment with this antibody, as well as the significant injury observed in Fas-deficient lpr/HA+ mice, argue against an important effect of FasL on the injury process.
MLE-Kd cells were also susceptible to cytotoxicity triggered by soluble TNF-α, although our data do not address the quantitative importance of this contribution. This observation nevertheless reflects a qualitatively unusual phenotype, which may have important implications in the lung. TNF receptor engagement simultaneously triggers several parallel signaling pathways, including the proapoptotic activation of the caspase cascade, but also the induction of transcription of antiapoptotic gene products mediated primarily by nuclear factor-κB (70). In nearly all cells examined, induction of apoptosis by TNF requires treatment with actinomycin D or cycloheximide in order to inhibit the transcription or translation of antiapoptotic gene products, especially in short-term assays (62). In order to confirm this phenotype in untransformed type II pneumocytes, we tested the sensitivity of primary alveolar type II cell isolates for sensitivity to apoptosis triggered by soluble TNF-α, and observed a similar effect. As noted earlier, our data do not address the quantitative physiologic significance of this effect, since we used a saturating dose of TNF-α. However, the qualitative effect was quite evident. Not unexpectedly, there was a moderate degree of spontaneous apoptosis in primary type II cells that occurred over a period of time after they were removed from their normal anchorage to basement membrane.
It has been reported for other cell types that the level of Fas expression does not correlate with biologic responsiveness as determined by induction of apoptosis with Jo-2 antibody (71). This strongly suggests that mechanisms may exist to tightly regulate programmed cell death in most cell types. Though MLE-Kd cells, upon exposure to FasL, are relatively resistant to Fas-mediated apoptosis, these cells clearly express functional Fas, as indicated by the induction of DNA fragmentation by the Jo-2 antibody in a more sensitive assay. These unknown regulatory mechanisms that tightly control programmed cell death may have important ramifications with regard to inflammatory lung disease, as may the unusual sensitivity of alveolar epithelial-derived cells to TNF-α expressed by CD8+ T cells. Numerous other factors may contribute to the regulation of alveolar epithelial-cell susceptibility to Fas-mediated apoptosis. For example, other cell types, such as T cells, appear to be resistant to Fas-induced apoptosis during the S phase of the cell cycle (72), and this resistance may correlate with the inability to recruit caspase-8 to the death-inducing signal complex (DISC) early after T cell stimulation (73). There are also suggestions in other cell types that Fas may participate in transducing signals for nonapoptotic cellular functions, such as proliferation in keratinocytes (74) and angiogenesis (75), as is the case with other members of the TNF receptor family (76).
The findings in this study lead to two conclusions: (1) that MLE-Kd cells are relatively resistant to FasL-mediated Fas-dependent cytotoxicity affected by CD8+ T cells, in contrast to the effect of TNF-α; and (2) that TNF-α expressed by CD8+ T cells accounts entirely for the perforin-independent cytotoxic effect of MLE-Kd cells. We have also shown that alveolar epithelial-derived cells, as well as primary type II pneumocytes, are sensitive to the apoptotic signal generated by soluble TNF-α. Data obtained after adoptive transfer of perforin-deficient CD8+ T cells into Fas-deficient (lpr/HA+) mice expressing HA exclusively in type II cells in vivo (50) are also consistent with these observations, suggesting that the phenotype of MLE-Kd cells may reflect that of alveolar type II cells in general. The sensitivity of alveolar cells to TNF expressed (or produced) by CD8+ T cells may have greater importance in bystander than in direct cytotoxicity, since we have shown that bystander-cell lysis is perforin-independent (and therefore TNF-dependent). The mechanisms underlying the unusual sensitivity of alveolar epithelial cells to TNF remain to be elucidated, but the observations described earlier raise the possibility that although perforin-mediated apoptosis may represent a primary antiviral effector mechanism of T cells, TNF-α may play a role in the lung injury that occurs during (and possibly after) a viral respiratory infection.
There is a further potentially important consequence of the preferential sensitivity of alveolar epithelial cells to TNF-α as compared with FasL. In contrast to ligation of Fas, ligation of TNF receptors results in activation of proinflammatory signaling, leading to transcription of a host of cytokines and chemokines (62). For example, human alveolar epithelial cell lines have been reported to express IL-8, a potent neutrophil chemoattractant, upon exposure to TNF-α (48). This raises the possibility that alveolar epithelial cells may be predisposed to nonspecific amplification of inflammatory processes mediated by CD8+ T cells, as we have observed in vivo in our model of T-cell–mediated lung injury (51). We previously demonstrated that from 4 to 5 d after the initial T-cell–mediated insult to alveolar cells, the inflammatory infiltrate consists primarily of host macrophages. These cells are also capable of producing TNF-α (as well as other cytokines), which may serve to further amplify the inflammatory cascade. Detailed investigation will be required to dissect the mechanisms controlling the amplification of inflammation in the lung, which may be critical in understanding the pathogenesis of interstitial lung diseases.
This work was performed in conjunction with the Academic Enhancement Program for Gene Transfer in the Cardiovascular System at the University of Virginia, whose support the authors gratefully acknowledge. They also gratefully acknowledge the critical assistance of Dr. Dudley F. Rochester for many helpful discussions and much advice. The work was supported by research grants HL58660 (R.I.E.), HL33391, and AI15608 (T.J.B.) from the United States Public Health Service, as well as by grants from the American Lung Association of Virginia and the American Heart Association of Virginia. The continuing support of the Beirne B. Carter Foundation is gratefully acknowledged.
|1.||Costabel U., Teschler H., Guzman J.Bronchiolitis obliterans organizing pneumonia (BOOP): the cytological and immunocytological profile of bronchoalveolar lavage. Eur. Respir. J.51992791797|
|2.||Katzenstein, A. 1997. Katzenstein and Askin's Surgical Pathology of Non-Neoplastic Lung Disease. W.B. Saunders, Philadelphia.|
|3.||Kradin R. L., Divertie M. B., Colvin R. B., Ramirez J., Ryu J., Carpenter H. A., Bhan A. K.Usual interstitial pneumonitis is a T-cell alveolitis. Clin. Immunol. Immunopathol.401986224235|
|4.||Leatherman J., Michael A., Schwartz B., Hoidal J.Lung T cells in hypersensitivity pneumonitis. Ann. Intern. Med.1001984390396|
|5.||Lukacher, A. E., L. A. Morrison, V. L. Braciale, and T. J. Braciale. 1986. T lymphocyte function in recovery from experimental viral infection: the influenza model. In Mechanisms of Host Resistance to Infectious Agents, Tumors, and Allografts. R. M. Steinman, editor. Rockefeller University Press, New York. 233–254.|
|6.||Yap K. L., Braciale T. J., Ada G. L.Role of T-cell function in recovery from murine influenza infection. Cell. Immunol.431979341351|
|7.||Wells M. A., Albrecht P., Ennis F. A.Recovery from a viral respiratory infection: I. Influenza pneumonia in normal and T-deficient mice. J. Immunol.126198110361041|
|8.||Couch R. B., Kasel J.Immunity to influenza in man. Annu. Rev. Microbiol.371983529549|
|9.||Ada G., Jones P.The immune responses to influenza infection. Curr. Top. Microbiol. Immunol.1281986154|
|10.||Ada, G., and P. Jones. 1987. The cell-mediated and humoral responses to influenza virus infection in the mouse lung. Adv. Exp. Med. Biol. 216B: 1033–1042.|
|11.||Andrew M. E., Braciale V. L., Henkel T. J., Hatch J. A., Braciale T. J.Stable expression of cytolytic activity by influenza virus-specific cytotoxic T lymphocytes. J. Immunol.135198535203523|
|12.||Henkart, P. 1993. Cytotoxic Cells. Birkhauser, Boston. 9–45.|
|13.||Kojima H., Shiohara N., Hanaoka S., Shirota Y., Takagaki Y., Ohno H., Katayama T. S., Yagita H., Okumura K., Shinkai Y., Alt F., Matsuzawa A., Yonehara S., Takayama H.Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. Immunity11994357364|
|14.||Braciale T. J., Mojcik C. F., Hauptfeld V.Target-cell recognition by cloned lines of influenza virus-specific cytotoxic T lymphocytes: selective inhibition by a monoclonal H-2-specific antibody. Immunogenetics1519824152|
|15.||Henkart P.Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity11994343346|
|16.||Kagi D., Vignaux F., Ledermann B., Burki K., Depraetere V., Nagata S., Nengartner H., Golstein P.Fas and perforin as major mechanisms of T cell-mediated cytotoxicity. Science2651994528530|
|17.||Lowin B., Hahne M., Mattmann C., Tschopp J.Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature3701994650652|
|18.||Walsh C., Matloubian M., Lui C., Udea R., Kurahara C., Christiansen J., Huang M., Young J., Ahmed R., Clark W.Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA9119941085410858|
|19.||Walsh C. M., Glass A. A., Chiu V., Clark W. R.The role of the Fas lytic pathway in a perforin-less CTL hybridoma. J. Immunol.153199425062514|
|20.||Alderson M. R., Tough T. W., Davis S. T., Braddy S., Falk B., Schooley K. A., Goodwin R. G., Smith C. A., Ramsdell F., Lynch D. H.Fas ligand mediates activation-induced cell death in human T lymphocytes. J. Exp. Med.18119957177|
|21.||Brunner T., Mogil R. J., LaFace D., Yoo N. J., Mahboubi A., Echeverri F., Martin S. J., Force W. R., Lynch D. H., Ware C. F., Green D. R.Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas. Nature3731995441444|
|22.||Crispe I.Fatal interactions: Fas-induced apoptosis of mature T cells. Immunity11994347349|
|23.||Dhein J., Walczak H., Baumler C., Debatin K., Krammer P. H.Autocrine T-cell suicide mediated by APO-1 (Fas/CD95). Nature37341995438448|
|24.||Ettinger R., Panka D., Wang J., Stanger B., Ju S., Rothstein A.Fas ligand-mediated cytotoxicity is directly responsible for apoptosis of normal CD4+ T cells responding to a bacterial superantigen. J. Immunol.154199543024308|
|25.||Lynch D. H., Ramsdell F., Alderson M. R.Fas and FasL in the homeostatic regulation of immune responses. Immunol. Today161995569574|
|26.||Seino K., Kayagaki N., Takeda K., Fukao K., Okumura K., Yagita H.Contribution of Fas ligand to T cell-mediated hepatic injury in mice. Gastroenterology113199713151322|
|27.||Pensati L., Costanzo A., Ianni A., Accapezzato D., Iorio R., Natoli G., Nisini R., Almerighi C., Balsano C., Vajro P., Vegnente A., Levrero M.Fas/Apo1 mutations and autoimmune lymphoproliferative syndrome in a patient with type 2 autoimmune hepatitis. Gastroenterology113199713841389|
|28.||Luo K. X., Zhu Y. F., Zhang L. X., He H. T., Wang X. S., Zhang L.In situ investigation of Fas/FasL expression in chronic hepatitis B infection and related liver diseases. J. Viral Hepatol.41997303307|
|29.||Hayashi N., Mita E.Fas system and apoptosis in viral hepatitis. J. Gastroenterol. Hepatol.121997S223S226|
|30.||Galle P., Hofmann W., Walczak H., Schaller H., Otto G., Stremmel W., Krammer P., Runkel L.Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J. Exp. Med.182199512231230|
|31.||Takehara T., Hayashi N., Mita E., Kanto T., Tatsumi T., Sasaki Y., Kasahara A., Hori M.Delayed Fas-mediated hepatocyte apoptosis during liver regeneration in mice: hepatoprotective role of TNF-alpha. Hepatology27199816431651|
|32.||Schneider P., Holler N., Bodmer J. L., Hahne M., Frei K., Fontana A., Tschopp J.Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med.187199812051213|
|33.||Chandler J. M., Cohen G. M., MacFarlane M.Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver. J. Biol. Chem.27319981081510818|
|34.||French L. E., Tschopp J.Thyroiditis and hepatitis: Fas on the road to disease. Nature Med.31997387388|
|35.||Giordano C., Stassi G., De Maria R., Todaro M., Richiusa P., Papoff G., Ruberti G., Bagnasco M., Testi R., Galluzzo A.Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis (see comments). Science2751997960963|
|36.||Itoh N., Imagawa A., Hanafusa T., Waguri M., Yamamoto K., Iwahashi H., Moriwaki M., Nakajima H., Miyagawa J., Namba M., Makino S., Nagata S., Kono N., Matsuzawa Y.Requirement of Fas for the development of autoimmune diabetes in nonobese diabetic mice. J. Exp. Med.1861997613618|
|37.||Gochuico B. R., Miranda K. M., Hessel E. M., De Bie J. J., Van Oosterhout A. J., Cruikshank W. W., Fine A.Airway epithelial Fas ligand expression: potential role in modulating bronchial inflammation. Am. J. Physiol.2741998L444L449|
|38.||Kazufumi M., Sonoko N., Masanori K., Takateru I., Akira O.Expression of bcl-2 protein and APO-1 (Fas antigen) in the lung tissue from patients with idiopathic pulmonary fibrosis. Microsc. Res. Tech.381997480487|
|39.||Spinozzi F., Fizzotti M., Agea E., Piattoni S., Droetto S., Russano A., Forenza N., Bassotti G., Grignani F., Bertotto A.Defective expression of Fas messenger RNA and Fas receptor on pulmonary T cells from patients with asthma. Ann. Intern. Med.1281998363369|
|40.||Tomokuni A., Aikoh T., Matsuki T., Isozaki Y., Otsuki T., Kita S., Ueki H., Kusaka M., Kishimoto T., Ueki A.Elevated soluble Fas/APO-1 (CD95) levels in silicosis patients without clinical symptoms of autoimmune diseases or malignant tumours. Clin. Exp. Immunol.1101997303309|
|41.||Nomoto Y., Kuwano K., Hagimoto N., Kunitake R., Kawasaki M., Hara N.Apoptosis and Fas/Fas ligand mRNA expression in acute immune complex alveolitis in mice. Eur. Respir. J.10199723512359|
|42.||Hagimoto N., Kuwano K., Nomoto Y., Kunitake R., Hara N.Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice. Am. J. Respir. Cell Mol. Biol.16199791101|
|43.||Hagimoto N., Kuwano K., Miyazaki H., Kunitake R., Fujita M., Kawasaki M., Kaneko Y., Hara N.Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen. Am. J. Respir. Cell Mol. Biol.171997272278|
|44.||Gochuico B. R., Williams M. C., Fine A.Simultaneous in situ hybridization and TUNEL to identify cells undergoing apoptosis. Histochem. J.291997413418|
|45.||Fine A., Anderson N. L., Rothstein T. L., Williams M. C., Gochuico B. R.Fas expression in pulmonary alveolar type II cells. Am. J. Physiol.2731997L64L71|
|46.||Shanley T. P., Schmal H., Friedl H. P., Jones M. L., Ward P. A.Role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in acute lung injury in rats. J. Immunol.154199547934802|
|47.||Peschon J. J., Torrance D. S., Stocking K. L., Glaccum M. B., Otten C., Willis C. R., Charrier K., Morrissey P. J., Ware C. B., Mohler K. M.TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J. Immunol.1601998943952|
|48.||Standiford T. J., Kunkel S. L., Basha M. A., Chensue S. W., Lynch J. P. D., Toews G. B., Westwick J., Strieter R. M.Interleukin-8 gene expression by a pulmonary epithelial cell line: a model for cytokine networks in the lung. J. Clin. Invest.86199019451953|
|49.||Ratner A., Clark W. R.Role of TNF-alpha in CD8+ cytotoxic T lymphocyte-mediated lysis. J. Immunol.150199343034314|
|50.||Enelow R., Stoler M., Srikiatkhachorn A., Kerlakian C., Agersborg S., Whitsett J., Braciale T.A lung-specific neo-antigen elicits specific CD8+ T cell tolerance with preserved CD4+ T cell reactivity. J. Clin. Invest.981996914922|
|51.||Enelow R. I., Mohammed A. Z., Stoler M. H., Young J. S., Lou Y. H., Braciale T. J.Experimental T cell-mediated lung disease: structural and functional consequences of alveolar cell recognition by CD8+ T lymphocytes. J. Clin. Invest.102199816531661|
|52.||Wikenheiser K., Vorbroker D., Rice W., Clark J., Bachurski C., Oie H., Whitsett J.Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc. Natl. Acad. Sci. USA9019931102911033|
|53.||Graham, M., T. Braciale, and V. Braciale. 1996. Use of antiviral T-lymphocyte clones to characterize antigen presentation and T-lymphocyte subsets. Methods: A Companion to Methods in Enzymology 9:439–444.|
|54.||Cao W., Tykodi S. S., Esser M. T., Braciale V. L., Braciale T. J.Partial activation of CD8+ T cells by a self-derived peptide. Nature3781995295298|
|55.||Matzinger P.The JAM test: a simple assay for DNA fragmentation and cell death. J. Immunol. Methods1451991185192|
|56.||Corti M., Brody A. R., Harrison J. H.Isolation and primary culture of murine alveolar type II cells. Am. J. Respir. Cell Mol. Biol.141996309315|
|57.||Maldonado M. A., Eisenberg R. A., Roper E., Cohen P. L., Kotzin B. L.Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I. J. Exp. Med.1811995641648|
|58.||Hartsfield, C., D. Lipke, Y. Lai, D. Cohen, and M. Gillespie. 1997. Pulmonary mechanical and immunologic dysfunction in a murine model of AIDS. Am. J. Physiol. 272 (Lung Cell Mol. Physiol. 16):L699–L706.|
|59.||Takezawa, J., F. Miller, and J. O'Neil. 1980. Single-breath diffusing capacity and lung volumes in small laboratory mammals. J. Appl. Physiol.: Respir. Environ. Exerc. Physiol. 48:1052–1059.|
|60.||Sabo J., Kimmel E., Diamond L.Effects of the Clara cell toxin, 4-ipomeanol, on pulmonary function in rats. J. Appl. Physiol.541983337344|
|61.||Depledge M., Collis C., Barrett A.A technique for measuring carbon monoxide uptake in mice. Int. J. Radiat. Oncol. Biol. Phys.71981485489|
|62.||Ashkenazi A., Dixit V. M.Death receptors: signaling and modulation. Science281199813051308|
|63.||Binder C., Binder L., Kroemker M., Schulz M., Hiddemann W.Influence of cycloheximide-mediated downregulation of glucose transport on TNF alpha-induced apoptosis. Exp. Cell Res.2361997223230|
|64.||Sanchez-Alcazar J. A., Ruiz-Cabello J., Hernandez-Munoz I., Pobre P. S., de la Torre P., Siles-Rivas E., Garcia I., Kaplan O., Munoz-Yague M. T., Solis-Herruzo J. A.Tumor necrosis factor-alpha increases ATP content in metabolically inhibited L929 cells preceding cell death. J. Biol. Chem.27219973016730177|
|65.||Smyth M. J., Krasovskis E., Johnstone R. W.Fas ligand-mediated lysis of self bystander targets by human papillomavirus-specific CD8(+) cytotoxic T lymphocytes. J. Virol.72199859485954|
|66.||Kojima H., Eshima K., Takayama H., Sitkovsky M. V.Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL. J. Immunol.159199727282734|
|67.||Fine A., Anderson N.Expression, function and regulation of alveolar type II cell Fas. Am. J. Respir. Crit. Care Med.1531996A537. (Abstr.)|
|68.||Pang H., Miranda K., Fine A.Sp3 regulates fas expression in lung epithelial cells. Biochem. J.3331998209213|
|69.||Lynch D. H.Biology of Fas. Circulatory Shock4419946366|
|70.||Wang C. Y., Mayo M. W., Korneluk R. G., Goeddel D. V., Baldwin A. S.NF-κB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science281199816801683|
|71.||Owen-Schaub L. B., Radinsky R., Kruzel E., Berry K., Yonehara S.Anti-Fas on nonhematopoietic tumors: levels of Fas/APO-1 and bcl-2 are not predictive of biological responsiveness. Cancer Res.54199415801586|
|72.||Dao T., Huleatt J. W., Hingorani R., Crispe I. N.Specific resistance of T cells to CD95-induced apoptosis during S phase of the cell cycle. J. Immunol.159199742614267|
|73.||Peter M. E., Kischkel F. C., Scheuerpflug C. G., Medema J. P., Debatin K. M., Krammer P. H.Resistance of cultured peripheral T cells towards activation-induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death-inducing signaling complex. Eur. J. Immunol.27199712071212|
|74.||Freiberg R. A., Spencer D. M., Choate K. A., Peng P. D., Schreiber S. L., Crabtree G.R., Khavari P.A.Specific triggering of the Fas signal transduction pathway in normal human keratinocytes. J. Biol. Chem.27119963166631669|
|75.||Biancone L., Martino A. D., Orlandi V., Conaldi P. G., Toniolo A., Camussi G.Development of inflammatory angiogenesis by local stimulation of Fas in vivo. J. Exp. Med.1861997147152|
|76.||Beutler B., Van Huffel C.Unraveling function in the TNF ligand and receptor family. Science2641994667668|
Abbreviations: Fas ligand, FasL; hemagglutinin, HA; tumor necrosis factor-α, TNF-α.