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GROWTH, DIFFERENTIATION, AND APOPTOSIS
induces barrier destabilization and apoptosis in renal proximal tubular epithelium1Division of Physiology, Department of Physiology and Medical Physics, and 2Department of Therapeutic Radiology and Oncology, Innsbruck Medical University, Innsbruck, Austria
Submitted 27 March 2007 ; accepted in final form 14 November 2007
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ABSTRACT |
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, are major immune response regulators produced and released by activated macrophages, dendritic cells, and virus-infected cells. Due to their immunomodulatory functions and their ability to induce cell death in tumors and virus-infected cells, they are used therapeutically against cancers, viral infections, and autoimmune diseases. However, little is known about the adverse effects of type I IFNs on nondiseased tissue. This study examined the effects of IFN- on cell death pathways in renal proximal tubular cells. IFN- induced apoptosis in LLC-PK1 cells, characterized by the activation of caspase-3, -8, and -9, DNA fragmentation, and nuclear condensation. IFN- also caused mitochondrial depolarization. Effector caspase activation was dependent on caspase-8 and -9. In addition to apoptosis, IFN- exposure also decreased renal epithelial barrier function, which preceded apoptotic cell death. Caspase inhibition did not influence permeability regulation while significantly attenuating and delaying cell death. These results indicate that IFN- causes programmed cell death in nondiseased renal epithelial cells. IFN--induced apoptosis is directed by an extrinsic death receptor signaling pathway, amplified by an intrinsic mitochondrial pathway. Caspase-dependent and -independent apoptotic mechanisms are involved. These findings reveal a novel aspect of IFN- actions with implications for normal renal function in immune reactions and during IFN- therapy. cytokines; interferon; cell death; transepithelial electrical resistance; caspase
, play a major role as regulators of immune functions. IFNs are produced and released mainly by activated monocytes/macrophages, plasmacytoid dendritic cells (also known as "natural IFN-producing cells"), and virus-infected cells. They recruit and activate macrophages and natural killer cells, promote the differentiation and activation of dendritic cells, and induce T helper cell type 1 cytokine release. Type I IFNs thus act as a bridge system linking innate and adaptive immunity (8). Concomitant with their immunomodulatory functions, type I IFNs also directly affect target cells by preventing virus replication, inducing cell cycle arrest, inhibiting proliferation, and inducing apoptotic cell death (34). These properties of type I IFNs have been found to be therapeutically exploitable in the treatment of viral, malignant, and autoimmune diseases, resulting in the approval of IFN- and IFN-β for treatment of hairy cell leukemia, chronic myeloid leukemia, multiple myeloma, non-Hodgkin's lymphoma, metastasizing renal cell carcinoma, malignant melanoma, hepatitis B and C, multiple sclerosis, and others (16, 17, 36). However, IFN therapy is accompanied by undesired side effects, limiting the benefits of high-dose IFN treatment. Renal impairment was, for example, observed in 
20% of the patients treated. Symptoms ranged from subclinical to severe dysfunction such as acute renal failure requiring dialysis (15, 20). Clinical findings revealed tubular cell dysfunction and/or tubular cell death (2, 20). Analysis of urinary protein revealed pathological urinary excretion of 1-microglobulin in 20% of patients and albumin in 15% of patients (20) and the presence of urinary tubular casts (2). Renal biopsies performed on patients with IFN--induced acute renal failure showed diffuse interstitial edema of rapid onset, acute tubular injury characterized by tubular dilatation and sloughing of epithelial cells, signs of interstitial nephritis, and/or glomerulonephritis (4, 12, 31, 33). These clinical findings suggest that IFN- could interfere with the function of tubular epithelial cells. Therefore, we previously studied the effect of IFN- on proximal tubular epithelial barrier function (23, 24). LLC-PK1 monolayers were found to display a reversible decrease in transepithelial electrical resistance (TEER), implying an IFN--induced increase in paracellular permeability. This impairment of epithelial barrier function was accompanied by a displacement or missorting of the junctional proteins occludin and E-cadherin, as demonstrated by prominent staining at the basal cell pole in addition to localization at the junctional region in immunofluorescence confocal microscopy. Furthermore, the expression of occludin and E-cadherin was found to be induced by IFN-. These findings provide evidence showing that IFN- can directly affect barrier function in renal epithelial cells.
The present study investigated whether IFN- also affects renal tubular function by inducing apoptotic cell death. Since at least two apoptotic pathways, the extrinsic via death receptors involving caspase-8 (3) and the intrinsic mitochondrial pathway resulting in caspase-9 activation (13), are known to activate effector caspases, we sought to analyze the apoptotic signaling mechanisms utilized by IFN- to induce cell death in proximal tubular cells. The potential link between regulation of epithelial barrier function and apoptosis is also discussed.
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MATERIALS AND METHODS |
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treatment was started and kept in serum-free medium until the end of the experiment. The IFN- preparation used for the described experiments was recombinant human IFN-2b, either solubilized in phosphate buffer (Strathmann-Biotech, Hamburg, Germany) or as a clinical IFN-2b preparation (IntronA, Schering, Berlin, Germany) with no differences detected between the two preparations. Cyclosporine A (CsA) was the kind gift of Novartis (Basel, Switzerland). DNA fragmentation assay by flow cytometry. LLC-PK1 cells were harvested by trypsination and combined with cells floating in the supernatant medium. Cells were fixed in 70% ethanol on ice for 30 min. Alternatively, the short protocol of Nicoletti et al. (27), utilizing detergent-permeabilized nuclei, was applied. DNA was stained with propidium iodide for flow cytometric analysis. DNA fixation using these protocols is incomplete, allowing a selective loss of small DNA fragments produced by apoptotic DNA cleavage. Thus, apoptotic cells partition into the sub-G0/G1 peak. Propidium iodide fluorescence was measured at an excitation of 488 nm and an emission of 585/42 nm [fluorescence-2 (FL-2)] or 670 nm long pass (fluorescence 3) in a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany). At least 25,000 events were analyzed in the slow mode excluding debris and aggregates. The quantitative analysis of the sub-G0/G1 peak was performed on histograms showing FL-2 height on a logarithmic scale using Cellquest software (Becton Dickinson).
Microscopic detection of nuclear condensation. LLC-PK1 cells were harvested by trypsination and combined with cells floating in the supernatant medium. Cells were fixed in methanol for 10 min at –20°C and subjected to staining with 0.5 µg/ml Hoechst 33258 (Calbiochem-Merck, Darmstadt, Germany). Cells were washed twice in PBS and mounted in 3 mg/ml p-phenylene-diamine glycerol solution. Photographs were taken from an Axiophot microscope using the x63 oil-immersion objective (1.4 numerical aperture, Zeiss, Göttingen, Germany) with a cooled charge-coupled device camera (Spot, Diagnostic Instruments, Sterling Heights, MI).
Caspase enzymatic assays. Caspase activity was determined using caspase fluorescence-based enzymatic assays (Molecular Probes, Leiden, The Netherlands) according to the manufacturer's instructions.
Cellular extracts were obtained by incubating adherent cells on ice for 30 min in lysis buffer (10 mM Tris, 1 mM EDTA, and 1% Triton X-100; pH 7.4). Caspase-3 activity was measured by cleavage of Ac-DEVD-R110, caspase-8 activity by cleavage of Ac-IETD-R110, and caspase-9 activity by cleavage of Ac-LEHD-R110. These substrates are recognized mainly by the indicated caspases. DEVD may, however, also be cleaved to a lesser extent by caspase-6, -7, -8, and -10, IETD by granzyme B, and LEHD by caspase-4 and -5. Fluorescence was measured in a spectrofluorimeter (TECAN, Grödig, Austria) at 485/535-nm excitation/emission. Background fluorescence of the substrate was subtracted from relative fluorescence units. Fluorescence signals were also corrected for protein content as determined by a BCA assay (Pierce, Rockford, IL) according to the manufacturer's instructions. Caspase-3 activity was expressed as fold over control.
For caspase inhibition, the following cell-permeable irreversible inhibitors (all from Calbiochem-Merck) were used: 5 µM z-VAD-fmk (pan-caspase inhibitor), 20 µM z-DEVD-fmk (caspase-3 inhibitor), 20 µM z-IETD-fmk (caspase-8 inhibitor), and 20 µM z-LEHD-fmk (caspase-9 inhibitor); inhibitors were applied to the cells 1 h before and together with IFN-.
Lactate dehydrogenase activity in the culture medium. The cell culture supernatant was collected and centrifuged for 5 min at 2,500 rpm and 4°C to eliminate floating cells. Lactate dehydrogenase (LDH) enzymatic activity was determined with the Cytotoxicity Detection Kit LDH (Roche Diagnostics, Penzberg, Germany) according to the manufacturer's instructions and expressed as fold over control.
Analysis of the inner mitochondrial membrane potential. The probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1; Molecular Probes) was used to determine mitochondrial membrane potential. JC-1 monomers are able to selectively enter mitochondria, presenting green fluorescence at low concentration or low potential (<140 mV). At high potential, JC-1 monomers convert to J-aggregates, which emit red fluorescence. Cells were harvested by trypsination and combined with cells floating in the supernatant medium. Incubation with 3.75 µM JC-1 was performed in serum-free medium containing 0.0125% pluronics (Sigma-Aldrich) and 20 µM PSC833 (gift of Novartis, Basel, Switzerland) for 1 h at 37°C and 5% CO2. PSC833 blocks p-glycoprotein, allowing greater accumulation of JC-1 within cells. Green (FL1 channel 530/30 nm) and red fluorescence (FL2 channel 585/42 nm) were determined by flow cytometry (FACScan, Becton Dickinson). At least 10,000 events were analyzed, excluding debris identified by forward scatter/side scatter analysis.
TEER measurement.
TEER measurements were measured with fixed electrodes using an Endohm (World Precision Instruments, Sarasota) coupled with an Evom volt-ohm meter (Millipore, Vienna, Austria). TEER was calculated by multiplying raw values by the surface area of the filter after subtracting recordings obtained from blank filters. Graphs show the differences to time-matched controls (in 
·cm2).
Statistical analysis. Values are expressed as means ± SD. At least three independent experiments were performed in triplicate or more. Statistical evaluation was performed using Student's t-test or ANOVA where appropriate. A P value of <0.05 was deemed statistically significant.
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RESULTS |
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treatment induces apoptosis in renal proximal tubular LLC-PK1 cells, as shown in Fig. 1A by an increased hypodiploid cell population, a sign of apoptotic DNA fragmentation. Microscopic inspection of IFN--treated proximal tubular LLC-PK1 cells stained with the DNA dye Hoechst 33258 morphologically confirmed apoptosis by showing nuclear condensation in about the same percentage of cells (Fig. 1B).
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-treated monolayers. As shown in Fig. 1C, IFN- induced a concentration-dependent increase in caspase-3 enzymatic activity in LLC-PK1 cells. IFN--induced caspase-3 activation was also demonstrated in extracts from LLC-PK1 monolayers grown on microporous growth supports (Fig. 1D). This cell culture procedure allows culture medium supply from both the apical and basolateral sides of the monolayer and thus more closely resembles the in vivo situation of proximal tubular cells. Furthermore, it was shown that IFN- added only to the apical or basolateral medium compartment, respectively, was effective to induce caspase-3 activation, indicating the presence of IFN type I receptors on both the apical and basolateral cell surfaces of proximal tubular epithelial cells.
To analyze the apoptotic cascade upstream of caspase-3 in more detail, the potential implication of caspase-8 and -9, key initiator caspases of the death receptor or mitochondrial pathway, respectively, was investigated. Both caspase-8- and -9-like enzymatic activities were induced by IFN- treatment and showed the typical early and transient activation patterns of initiator caspase activation (Fig. 2, A and B). Maximal caspase-9 activity induced by IFN- was reduced by coincubation of the cells with IFN- and a caspase-8 inhibitor (z-IETD-fmk), in contrast to caspase-8 activity, which was not prevented by a caspase-9 inhibitor (z-LEHD-fmk; Fig. 2, A and B, insets). This result suggests that caspase-9 activation depends on active caspase-8, indicating sequential activation.
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in the presence of inhibitors of caspase-8 (z-IETD-fmk) and caspase-9 activity (z-LEHD-fmk) were also analyzed for caspase-3 activation (Fig. 3A). Both caspase-8 and -9 inhibitors were shown to prevent caspase-3 activation to a degree similar to that of a caspase-3 inhibitor (z-DEVD-fmk) or a pan-caspase inhibitor (z-VAD-fmk), indicating that both the death receptor and mitochondrial pathway are necessary for efficient caspase-3 activation by IFN- in proximal tubular LLC-PK1 cells. The inhibitors did not significantly alter basal caspase-3 activity (Fig. 3B).
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(Fig. 3C). These results implicate caspase-dependent pathways in the control of apoptotic DNA fragmentation. However, caspase-independent death program execution was also possible, although in a reduced number of cells.
To determine plasma membrane integrity, LDH release into the supernatant medium was also analyzed. After prolonged IFN- incubation (48 h), LLC-PK1 cultures showed a significant increase in LDH release into the culture medium compared with time-matched controls. A slight but statistically significant reduction in LDH release was observed after treatment of the cells with IFN- in the presence of caspase inhibitors (Fig. 3D).
Since caspase-9 is activated by the release of mitochondrial proteins into the cytoplasm, IFN--treated cell cultures were further evaluated for signs of mitochondrial involvement. Inner mitochondrial membrane potential was determined using the mitochondrial potential-sensitive dye JC-1. As shown in Fig. 4A, IFN- was able to depolarize the inner mitochondrial membrane in a significant number of cells, as shown by a decrease in red JC-1 fluorescence in 40% of the cell population (mean ± SD, 40.76 ± 5.58% vs. 15.51 ± 1.78% in IFN--treated cells vs. controls). The pan-caspase inhibitor z-VAD-fmk was able to markedly reduce the number of cells having depolarized mitochondria (26.21 ± 1.8%) compared with treatment with IFN- alone. Incubation of LLC-PK1 cultures with IFN- in the presence of CsA (1 µM), which is known to bind to and inactivate cyclophilin D, a component of the mitochondrial megapore (6), resulted in partial inhibition of mitochondrial depolarization. This was evidenced by a right shift of the peak containing affected cells, indicating that the mitochondria were not fully depolarized. However, at a higher concentration (10 µM), CsA exacerbated the mitochondrial depolarizing effect of IFN-, resulting in a complete loss of red JC-1 fluorescence in the whole cell population. As shown in Fig. 4B, CsA was able to reduce IFN--induced caspase-3 activation at low concentrations (0.1 and 1 µM). At a concentration of 10 µM, however, CsA induced caspase-3 activation itself. Cotreatment consisting of 10 µM CsA and IFN- produced a synergistic increase in caspase-3 activity compared with treatment with IFN- alone. Similar, albeit less pronounced, effects were induced by low and high concentrations of CsA on IFN--induced LDH release (Fig. 4C). These results indicate that CsA influences IFN--induced apoptosis in a concentration-dependent manner.
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also induces a decrease in TEER of proximal tubular LLC-PK1 monolayers (23), the impact of apoptosis on paracellular permeability regulation was determined in this study. Time courses of apoptotic death markers and effects of a pan-caspase inhibitor (z-VAD-fmk) were compared with TEER decrease (Fig. 5). Figure 5A shows the time course of caspase-3 activation by IFN-. DNA fragmentation, as determined by an increase in the sub-G0/G1 peak, showed the same time course with a delay of 6 h versus caspase-3 activation. After a further time delay, LDH measured in the cell culture medium was higher than in controls, indicating a loss of plasma membrane integrity, most likely by late apoptotic LLC-PK1 cells.
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(Fig. 5B). DNA fragmentation was completely prevented by z-VAD-fmk at 24 h of treatment and reduced by 50% at 48 h of IFN- treatment (Figs. 3C and 5B). Similarly, LDH release was statistically significantly reduced by the pan-caspase inhibitor (Figs. 3D and 5B).
To determine the impact of apoptosis on the IFN--induced TEER decrease, we measured TEER in the presence of IFN- and z-VAD-fmk (Fig. 5C). Inhibition of the caspase cascade, however, did not influence the TEER decrease induced by IFN-. Furthermore, when the time course of the TEER decrease was compared with caspase-3 activation, DNA fragmentation, and LDH release, the IFN--induced TEER decrease was seen to precede apoptotic death program execution.
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DISCUSSION |
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is able to induce apoptosis in renal tubular epithelial cells. This finding is a novel, previously unrecognized aspect of IFN- actions. Induction of apoptosis is one way in which IFN- is able to directly affect proximal tubular integrity. Furthermore, IFN- is able to impair epithelial barrier function, as we have previously shown (23). IFN- might, thereby, contribute to epithelial dysfunction during immune reactions and IFN- therapy.
To better understand the mode of action of IFN- in proximal tubular cells, we determined the impact of the apoptotic death receptor pathway and mitochondrial signaling on IFN--induced apoptotic cell death. IFN- induced the activation of caspase-8, a key event in death receptor signaling, and caspase-9, which is activated by mitochondrial apoptosis. Caspase-3 activation was not possible when cells were exposed to inhibitors of either caspase-8 or -9. Thus, the death receptor pathway and mitochondrial pathway both appear to be necessary for efficient executor caspase activation by IFN-.
In addition, IFN- induced a breakdown of the inner mitochondrial membrane potential, further implying mitochondrial signaling in IFN--induced apoptosis. A pan-caspase inhibitor attenuated this response, suggesting a caspase event upstream of mitochondrial involvement in IFN--induced apoptosis. One potential candidate for this effect is the caspase-8 substrate Bid, a Bcl-2 family member known to trigger mitochondrial membrane permeabilization by activating pore-forming Bax-like proteins (18, 19). A role of caspase-8 upstream of the mitochondrial pathway is also suggested by the finding that caspase-9, which is known to be activated by the cytoplasmic release of mitochondrial proteins (13), was found to depend on caspase-8 activation.
Taken together, the presented findings indicate that the death receptor pathway activated by IFN- is not sufficient to directly activate executor caspases, such as caspase-3, in renal proximal tubular cells. Progression of programmed cell death is only possible if the death receptor pathway is followed by mitochondrial activation (1, 21).
To further study the role of mitochondria in IFN--induced apoptosis, cells were treated with IFN- and CsA, which is known to bind to and inactivate cyclophilin D, thus blocking the voltage-dependent anion channel (VDAC)/adenine nucleotide translocase (ANT) mitochondrial megapore (6). Coincubation with IFN- and 1 µM CsA partially prevented mitochondrial membrane depolarization, suggesting a role of the VDAC/ANT megapore (13, 19). Moreover, 1 µM CsA partially relieved IFN--induced caspase-3 activity, thus linking megapore opening to effector caspase activation.
In contrast, at a higher concentration (10 µM), CsA did not slow down, but even exacerbated, IFN--induced apoptosis. Furthermore, 10 µM CsA per se was able to induce a slight but significant increase in caspase-3 activity and LDH release that was consistent with previous findings concerning CsA-induced apoptosis in LLC-PK1 cells (14). In this report, it was suggested that apoptosis by CsA may be linked to CsA-induced Fas expression, thus potentially facilitating the onset of a death receptor pathway. Such an increased Fas expression by CsA might facilitate IFN--induced death receptor activation and thus explain the increased apoptosis induced by 10 µM CsA and IFN- compared with IFN- alone, as observed in our study. These results reveal a pleiotropic nature of CsA acting as an antiapoptotic or a proapoptotic factor depending on the concentration.
Taken together, our results imply that IFN--induced apoptosis of renal epithelial cells involves a death receptor pathway followed by a mitochondrial mechanism. Figure 6 shows a schematic representation of the proposed signaling pathways. Caspase-8 activation was previously also implicated in IFN--induced apoptosis of melanoma and bladder carcinoma cells (11). A role of mitochondria in IFN--induced apoptosis in melanoma cells was, furthermore, demonstrated by cytochrome c release and disruption of the mitochondrial membrane potential (22). The present study indicates that IFN- mediates apoptosis in renal proximal tubular cells through similar death signaling pathways as in these malignant tumor cell lines. Furthermore, IFN- doses described to induce apoptosis in IFN--sensitive melanoma and bladder cancer cells in vitro (11, 30) were similar to those seen to be effective in LLC-PK1 cells. The physiological and pathophysiological relevance of IFN--induced effects on renal epithelial cells is also suggested by pharmacokinetic studies in humans and mice (28, 37) implying that maximal serum concentrations during high-dose IFN- therapy may reach concentrations that have been shown to cause caspase-3 activation in proximal tubular cells.
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-induced effects in renal epithelial cells could have potential clinical implications. Caspase inhibitors have been discussed as potential therapeutic agents for preventing apoptotic cell death (21). However, these inhibitors can be effective only when the life-death decision occurs downstream of caspase activation (9). To analyze whether caspase inhibition could potentially prevent IFN--induced apoptotic cell death, the impact of caspase inhibitors on late stages of apoptosis was determined. DNA fragmentation in LLC-PK1 cells was partially prevented by caspase inhibition, suggesting that IFN- was able to activate caspase-dependent DNases. However, a reduced hypodiploid population was detected at late apoptotic stages despite complete caspase-3 blockage. This result implies that caspase-independent nucleases are also involved in IFN--induced apoptosis, which would account for the delayed and reduced DNA fragmentation under conditions of caspase inhibition. Thus, the "point of no return" in the life-death decision seems to lie upstream of the caspase cascade.
Similar results were obtained by analyzing loss of plasma membrane integrity as quantified by the release of LDH into the cell culture medium. A statistically significant increase in LDH activity in the medium was induced by long-term IFN- treatment and was reduced by caspase inhibition. LDH release may, thus, result from "secondary necrosis," a process caused by swelling and lysing of apoptotic bodies in the absence of phagocytosis in vitro (29).
Since we have previously reported an IFN--dependent decrease in TEER in LLC-PK1 monolayers (23), the present study also analyzed the link between barrier dysfunction and apoptosis. It was demonstrated that caspase inhibition was not able to influence the IFN--induced TEER decrease while significantly attenuating and delaying effector caspase activity, DNA fragmentation, and LDH release. This finding demonstrates that barrier destabilization is not the consequence of apoptotic cell death. This was a surprising outcome since TEER as an indicator of epithelial barrier integrity would be expected to be compromised by an apoptotic cell death rate involving 40% of the cell population. However, caspase inhibition had no effect on TEER.
Recently, other studies have revealed an unexpected stability of epithelial barriers in the presence of high-rate apoptosis with no effects on TEER. High-resolution epithelial surface analysis demonstrated that a continuous epithelial cell layer is maintained during apoptotic cell extrusion (39). An actin ring was shown to form between the apoptotic cell and its neighbors, allowing the gap in the monolayer caused by the extruding cell to immediately seal (32). Electronmicroscopic observations of cell extrusions at the villus tip of intestinal epithelium have, furthermore, revealed de novo formation of tight junctional strands between the new neighbor cells while the extrusion process was not yet completed (25). These reports suggest that apoptotic cell extrusions might be possible without a significant loss of epithelial barrier integrity. Thus, apoptotic cell death is not necessarily accompanied by a TEER decrease, and barrier destabilization might be mediated by apoptosis-independent mechanisms. Apoptosis-independent mechanisms have been, for example, previously implicated in intestinal barrier destabilization by TNF-, whereas TNF- also induced apoptosis (10), similarly to what we found for IFN- in the renal proximal tubular epithelium.
Apoptosis-independent barrier destabilization may be mediated by IFN- by junctional complex restructuration, since we have previously found IFN- to induce an intracellular redistribution of E-cadherin and occludin (23). While IFN--induced reorganization of junctional complexes could result in apoptosis-independent epithelial barrier destabilization, these events may well be the trigger for proapoptotic signaling. Tight and adherens junctions have been recognized as signaling platforms integrating extra- and intracellular signal inputs and regulating numerous and diverse cellular functions (5, 35). E-cadherin in the adherens junctional complex has been, for example, shown to be crucial for survival signaling by growth factor receptors (7). Occludin function has been recently suggested to be involved in apoptotic mechanisms (26, 38). Thus, IFN--induced relocation of E-cadherin and occludin might induce proapoptotic signals in renal proximal tubular cells in addition to increasing paracellular permeability.
In conclusion, we show that IFN- is able to directly induce apoptosis and barrier dysfunction in the renal tubular epithelium. The apoptotic signaling pathways activated in proximal tubular cells resemble the pathways induced by IFN- in melanoma and bladder carcinoma cells. Epithelial permeability was increased by IFN- independently of concomitant apoptotic cell death, since caspase inhibition did not influence permeability regulation while significantly attenuating and delaying cell death. These findings reveal a novel aspect of IFN- actions with implications for renal epithelial function during immune reactions and IFN- therapy.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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). z-VAD-fmk or CsA alone did not produce a significantly different fluorescence pattern than seen in the control (not shown). B: IFN-
