(Received for publication, October 5, 1994; and in revised form, December 14, 1994)
From the
crmA is a cowpox virus gene that encodes a protease
inhibitor of the serpin family. The only reported target for the CrmA
protein is the cysteine protease interleukin-1 converting enzyme
(ICE). ICE, by virtue of its homology to the Caenorhabditis elegans cell death protein Ced-3, has been suggested to play a
fundamentally important role in mammalian apoptosis. We hypothesized
that a function of crmA may be to inhibit cell death, since a
major mechanism of viral clearance is the immune system-mediated
induction of apoptosis of infected cells. The tumor necrosis factor
receptor and the Fas antigen are two cytokine receptors which, by
engaging and activating the death pathway, can eliminate virus-infected
cells. Remarkably, crmA was found to be an exceptionally
potent inhibitor of apoptosis induced by both these receptors, capable
of blocking the cell death program even at pharmacological doses of the
death stimulus. Therefore, an important new function for crmA is the inhibition of cytokine-induced apoptosis. Further, the data
suggest that a protease, either ICE or a related crmA-inhibitable protein, is a component of the Fas- and tumor
necrosis factor-induced cell death pathways.
Apoptosis, or programmed cell death (PCD), ()is
critical to many biological processes, including embryogenesis,
development of the immune system, elimination of virus-infected cells,
and the maintenance of tissue homeostasis (reviewed in (1) and (2) ). Although the molecular components comprising the death
pathway in mammalian cells await definition, recent work suggests that
in the worm Caenorhabditis elegans, the ced-3 gene
encodes a component of the death pathway(3) . ced-3 was found to be obligatory for apoptosis (4) and sequence
analysis revealed it to be similar to the mammalian protease
interleukin-1
converting enzyme (ICE)(3) . ICE is a
cysteine protease that catalyzes the proteolytic processing of the
pro-inflammatory cytokine interleukin-1
from an inactive precursor
form to the active mature form(5, 6) . Sequence
similarity between the predicted Ced-3 and ICE proteins was greatest in
the active site region(3) , and the recent description of the
crystal structure of ICE revealed that structurally important residues
are conserved between the two proteins(7, 8) . Taken
together, these data suggest that ced-3 encodes a
cysteine protease and that a homologue of Ced-3,
either ICE or a related protease, may be a component of the death
pathway in mammalian cells.
Apoptosis is of particular relevance to
viral pathogenesis, as a major mechanism for viral clearance by the
mammalian immune system is the induction of apoptosis of infected
cells(9) . It is becoming increasingly apparent that many
viruses have evolved proteins that are capable of attenuating
apoptosis(10) . The cowpox virus encodes a protein designated
CrmA, which is an ICE inhibitor(11) . CrmA was originally
described as a modulator of the host inflammatory response, presumably
functioning by decreasing interleukin-1 production through
inactivation of ICE(11) . However, now given that ICE might be
a component of the death pathway in mammalian cells, we hypothesized
that crmA may additionally function to inhibit apoptosis. This
line of thinking was substantiated by a recent report in which
microinjection of crmA cDNA into chicken neuronal cells
afforded partial protection from growth factor withdrawal-induced
apoptosis(12) . There are multiple triggers for PCD, however,
and it is not clear whether cells undergoing apoptosis triggered by
different stimuli traverse biochemically identical or distinct
pathways. Thus, it is not possible to extrapolate the finding in
neuronal cells to cell death induced by other stimuli. In addition, the
relevance of growth factor withdrawal-induced neuronal apoptosis to the
physiologic function of crmA in the context of viral infection
is not clear.
For these reasons, we chose to address the hypothesis in the context of apoptosis driven by two cell-surface cytokine receptors: the receptor for tumor necrosis factor (TNF) and the Fas antigen. These have been shown to induce cell death upon activation by either their respective ligands or by cross-linking with agonist antibody(13, 14, 15, 16) . PCD triggered by these receptors is especially relevant to the viral life cycle since both receptors have been implicated in viral clearance. TNF itself is a potent antiviral agent (17) and cytotoxic T-cells, which comprise a major antiviral defense, utilize the Fas antigen to induce apoptosis of target cells(18, 19, 20, 21) . To investigate whether crmA could modulate cytokine-induced apoptosis, we expressed the gene for crmA in cell lines susceptible to cell death mediated through either the TNF receptor or Fas. crmA acted as a powerful inhibitor of apoptosis, capable of completely abrogating cell death induced by activation of either of these receptors. This finding has implications for understanding the physiologic function of crmA as well as for identifying the components that comprise the cytokine-driven death pathway.
For electron microscopy, cells were fixed and processed as per standard electron microscopy procedures.
BJAB
cells were grown at 3 10
cells/ml and treated with
anti-Fas antibody at a concentration of 250 ng/ml (unless indicated
otherwise) for 18 h after which an aliquot was stained with acridine
orange as described above. Apoptotic cells and non-apoptotic cells were
quantitated and normalized to untreated samples. Assays were done at
least in duplicate.
Secondary assays of cell death used were the MTT conversion assay and crystal violet staining and were done as described previously(22, 23) .
Cell death can occur by two biochemically and morphologically distinct processes: apoptosis and necrosis(26) . We first confirmed that death seen in response to TNF or anti-Fas occurred by apoptosis. Although various markers of apoptosis have been reported, the phenomenon is ultimately defined at the morphological level and is characterized by chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing without disintegration of the cellular membrane(26) . Necrosis, conversely, is defined by cytoplasmic swelling and lysis of the cell membrane and, importantly, does not exhibit the chromatin margination characteristic of apoptosis(26) . DNA laddering, representative of cleavage at internucleosomal intervals, is seen in some but not all forms of apoptosis, further emphasizing the importance of morphological criteria in defining apoptosis(27) . Nuclear morphology of cells dying in response to TNF or anti-Fas antibody was examined following staining with the DNA-binding dyes propidium iodide (MCF7 cells) and acridine orange (BJAB cells). Fluorescence microscopy and laser-scanning confocal microscopy demonstrated marked changes in nuclear morphology in the MCF7 cells in response to either TNF or anti-Fas+CHX and in the BJAB cells in response to anti-Fas. Chromatin condensation was clearly visible by immunofluorescence microscopy in both cell lines and formed the basis for our later assays of apoptosis in transfected cell lines (Fig. 1B for BJAB and data not shown for MCF7). Confocal microscopy confirmed margination along the inner nuclear membrane (Fig. 1, A and B (inset)). These morphological criteria of apoptotic cell death were further confirmed by transmission electron microscopy. The MCF7 cells clearly demonstrated chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and increased membrane blebbing in response to either TNF or anti-Fas+CHX (Fig. 1A). BJAB cells treated with anti-Fas antibody demonstrated chromatin margination and cellular shrinkage typical of apoptosis in lymphoid cells (data not shown). Thus, both TNF and Fas triggered genuine apoptotic death in these cell lines.
Figure 1: A, TNF and anti-Fas+CHX induce apoptosis in MCF7 cells. Treatment with TNF or anti-Fas+CHX was carried out as described under ``Materials and Methods.'' Upperrow, nuclei of untreated, TNF-treated, or anti-Fas+CHX-treated MCF7 cells stained with propidium iodide and visualized by laser-scanning confocal microscopy. Arrows indicate examples of apoptotic nuclei. Lowerrow, transmission electron microscopy of untreated, TNF-treated or anti-Fas+CHX-treated MCF7 cells. B, anti-Fas antibody induces apoptosis in BJAB cells. Anti-Fas treatment was carried out as described under ``Materials and Methods.'' Fluorescence microscopy of untreated or anti-Fas-treated BJAB cells stained with acridine orange. Arrows indicate examples of apoptotic nuclei. Inset, laser scanning confocal microscopy of untreated or anti-Fas-treated BJAB cells stained with acridine orange.
Figure 2: Inhibition of apoptosis by crmA in pooled populations of transfectants. Pooled populations of the vector- or crmA-transfected cells indicated were analyzed for sensitivity to TNF- or Fas-mediated apoptosis.
In addition to the pools, clonal lines were derived from both MCF7 and BJAB transfectants and challenged by activation of the TNF and Fas death pathways. In the MCF-7 cell line, vector clones were uniformly sensitive to apoptosis induced by either TNF or anti-Fas+CHX, whereas among the transfected clones, those that expressed detectable crmA transcript were markedly resistant (Fig. 3A). Indeed, lines expressing the highest levels of crmA were totally resistant to apoptosis (Fig. 3A) and showed no morphologic cytopathic effects (Fig. 3B), demonstrating that crmA can completely block the TNF- and Fas-mediated death pathways. Similarly, among BJAB-transfected clones, the crmA-expressing lines were markedly resistant to anti-Fas-induced apoptosis, whereas the vector clones were universally sensitive (Fig. 4A). Importantly, in both MCF7 and BJAB transfectants, those clones expressing the highest levels of crmA were the most resistant, while those clones expressing little or undetectable levels were the most sensitive (Fig. 3A and Fig. 4A). Although direct visual quantitation of apoptotic nuclei is the most accurate measure of apoptosis, comparable results were obtained when either an MTT conversion-based death assay (Fig. 3A, inset, and 4A, inset) or crystal violet staining (Fig. 3C) was employed to assess cell survival.
Figure 3:
A, inhibition of TNF- and
anti-Fas+CHX-induced apoptosis of MCF7 cells by crmA. Top, sensitivity of MCF7 vector-transfected clones (V1-V5) or crmA-transfected clones (crmA1-crmA5) to TNF- and
anti-Fas+CHX-induced cell death assessed by propidium iodide
apoptosis assay. Inset, sensitivity of selected clones to TNF-
and anti-Fas+CHX-induced cell death assessed by MTT conversion
assay. Middle, Northern analysis of corresponding cell lines
to detect crmA transcript. Bottom, Northern analysis
to detect -actin transcript to assess loading of RNA. B, crmA inhibits TNF- and anti-Fas+CHX-induced cytopathic
effects on MCF7 cells. Phase contrast photomicrographs of cultures of a
vector-transfected clone (MCF7V4) or a
crmA-expressing clone (MCF7crmA3) treated with TNF,
anti-Fas+CHX, or no treatment (UnRx) for 36 h. C, crmA inhibits TNF and anti-Fas+CHX-induced
death of MCF7 cells when assessed by crystal violet staining. A
vector-transfected clone (MCF7 V4) and a crmA-expressing clone (MCF7crmA3) were
grown in a six-well culture dish and treated with TNF,
anti-Fas+CHX, or no treatment (UnRx) for 48 h. Surviving
cells were stained with crystal violet as described under
``Materials and Methods.''
Figure 4:
A, inhibition of anti-Fas-induced
apoptosis in BJAB cells by crmA. Top, analysis of
sensitivity of BJAB vector-transfected clones (V1-V6) or crmA-transfected clones (crmA1-crmA10)
to anti-Fas-induced cell death assessed by acridine orange apoptosis
assay. Inset, sensitivity of selected clones to
anti-Fas-induced cell death assessed by MTT conversion assay. Middle, Northern analysis of corresponding cell lines for
detection of expression of crmA transcript. Bottom,
Northern analysis to detect -actin transcript to assess loading of
RNA. B, crmA inhibits apoptosis induced by increasing doses of
anti-Fas antibody. The acridine orange apoptosis assay was used to
analyze the sensitivity of vector-transfected (BJAB V1) or crmA-transfected (BJABcrmA2, BJABcrmA3) clones to apoptosis induced by the indicated doses
of anti-Fas antibody.
We proceeded to determine if protection conferred by crmA from cytokine-induced apoptosis could be attenuated by increasing the dose of the death stimulus. Remarkably, crmA afforded comparably high levels of protection from anti-Fas-induced apoptosis in response to doses of antibody 250 times greater than those needed to kill greater than 95% of the vector-transfected cells (Fig. 4B). Similar results were obtained when the dose of TNF was varied for the MCF7 transfectants (data not shown), implying that crmA is functioning as an exceptionally potent inhibitor of cell death at a presumably critical step in the death pathway.
Our findings describe an important new function for crmA: the blockade of TNF- and Fas-mediated apoptosis. Given
the importance of both TNF and Fas in the host anti-viral response, it
is likely that this function of crmA is important for
productive viral infection in vivo. crmA represents
yet another example of viral economy in which two important functions,
namely the inhibition of interleukin-1 production and the
prevention of apoptosis, are embedded in one protein.
An important implication of our data is that a protease is a component of both the TNF- and Fas-induced cell death pathways. Earlier experiments with small molecule protease inhibitors and with overexpression of plasminogen activator inhibitor-2 (28, 29, 30) provided evidence for protease activation in TNF-induced cytotoxicity, although protection was partial and the physiologic relevance of the studies unclear. crmA is a powerful inhibitor capable of conferring complete protection and, by virtue of its known inhibition of ICE, has particular relevance to apoptosis. Although the only reported target for CrmA is ICE, it is becoming increasingly clear that ced-3 and ICE are members of a gene family, of which at least one other member, the murine nedd2 gene, has been reported(31) . Hence, it is possible that the protease involved in TNF- and Fas-mediated apoptosis may not be ICE itself but instead one of its relatives. Furthermore, a recent report suggests that CrmA functions as a ``cross-class inhibitor'' and may be capable of inhibiting serine proteases as well as ICE(32) . Thus, the possibility that the relevant CrmA target is a serine protease cannot be excluded.
Additionally, the findings have implications for the unification of death pathways in general. First, the fact that crmA blocks both TNF- and Fas-mediated apoptosis, especially in the MCF7 cells which possess both receptors, suggests that the two receptors signal death through a biochemically common pathway. This hypothesis is supported by the finding that the cytoplasmic regions of both these receptors encompass a region of homology which has been defined by mutational analysis as a ``death domain'' and which presumably interacts with a common set of signal transduction molecules(23, 33) . Furthermore, it is now apparent that crmA blocks cell death triggered by two very different stimuli: growth factor withdrawal in neuronal cultures (12) and, from our studies, activation of cytokine receptors. It is of note that apoptosis in these two systems has been suggested to occur through biochemically distinct pathways, in that apoptosis in the former system is dependent on new protein synthesis and death is blocked by cycloheximide(34) , whereas in contrast TNF- and Fas-mediated cytotoxicity is independent of new protein synthesis and is, in fact, enhanced by cycloheximide(13) . Our data would suggest that at some point, the death pathway in both systems converges upon a crmA-inhibitable step, likely the activation of a protease. Indeed, given that in C. elegans the ced-3 gene is required for all apoptosis in the organism during development(4) , it is possible that the functional homologue of ced-3 in mammalian cells might well play a similar universal role. Therefore, the identification of the apoptosis-relevant CrmA target, be it ICE or another protein, may define the first component of a general mammalian cell death pathway.