AN ABSTRACT OF THE THESIS OF
Cathleen M. Moscibrocki for the degree of Master of Science in Microbiology
presented on June 6. 2001. Iffle: LLncovering_th Mechanism of IL-4-mediated T
Cell Survival.
Abstract approved:
Redacted for Privacy
Anthony T. Vella
Although IL-4 is a well-characterized multi-functional cytokine, its role as a
survival factor in T cells is not completely understood. In an attempt to uncover
IL-4-mediated survival, we studied caspase activation in primary mouse T
lymphocytes undergoing death by neglect, activation-induced or steroid-induced
cell death. Here, we identify the executioner caspases-3, and -6, and, to a lesser
extent, caspase-4 to be involved in the apoptotic process of T cells in our model, as
demonstrated by a peptide inhibition assay. Furthermore, we show that IL-4 blocks
caspase-3 activation in T cells undergoing all three forms of apoptosis. Western
blot and flow cytometric analysis showed a substantial decrease in dexamethasoneinduced and death by neglect-induced caspase-3 activation upon IL-4 treatment,
indicating IL-4 to be an important modulator of caspase-3 activity. Since a major
pathway leading to caspase activation involves mitochondrial disruption and/or the
release of pro-apoptotic proteins, we analyzed mitochondrial membrane potential
(''m)
in T cells undergoing various forms of apoptosis in an attempt to define the
mechanism(s) by which IL-4 blocks caspase-3 activation. IL-4 treatment resulted
in a reduction of mitochondrial disruption, as defined by iWm. Moreover, in T
cells derived from B ax-deficient mice, it appeared that IL-4-mediated survival
involved a mechanism that does not include the modulation of the pro-apototic Bax
protein. These data suggest that IL-4-mediated T cell survival involves the
blockage of caspase-3 activation through a mechanism that relies on the
maintenance of LWm.
Uncovering the Mechanism of IL-4-mediated T Cell Survival
by
Cathleen M. Moscibrocki
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented June 6, 2001
Commencement June 2002
Master of Science thesis of Cathleen M. Moscibrocki presented on June 6. 2001.
APPROVED:
Redacted for Privacy
Major Professor, representing Miio1ogy
Redacted for Privacy
Department of Microbiology
Redacted for Privacy
Dean of theraduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to
any reader upon request.
Redacted for Privacy
Cathleen M. Moscibrocki, Author
CONTRIBUTION OF AUTHORS
Dr. Anthony T. Vella was involved in the design, analysis, and writing of
this manuscript. Lisa Finkeistein performed the IL-4Ra immunoprecipitation and
Western blot, and assisted in the caspase inhibition assay. Chen-Yao Chen assisted
in the P1-3 K inhibition assay, as well as the interpretation of the corresponding
data. Rhonda Kaetzel kindly assisted in the mitochondrial staining and flow
cytometric analysis of mitochondrial membrane potential.
TABLE OF CONTENTS
INTRODUCTION ............................................................................. 1
The Immune System .................................................................. 1
Apoptosis .............................................................................. 7
Caspases .............................................................................. 10
Mitochondria and Apoptosis ...................................................... 20
Interleukin-4/Interleukin-4 Receptor ............................................. 24
Preliminary Data and Research Goals ............................................ 31
References ........................................................................... 33
UNDERSTANDING HOW IL-4 MEDIATES T CELL SURVIVAL ............... 48
Introduction .......................................................................... 49
Materials and Methods ............................................................. 51
Results ................................................................................ 56
Discussion ........................................................................... 76
References ........................................................................... 83
EXPLORING IL-4-MEDIATED MECHANISMS IN MODULATING CASPASE
ACTIVITY .................................................................................... 86
Introduction .......................................................................... 87
Materials and Methods ............................................................. 89
Results ................................................................................ 94
Discussion .......................................................................... 113
References .......................................................................... 120
TABLE OF CONTENTS (Continued)
CLOSING THOUGHTS AND CONCLUSIONS ..................................... 122
BIBLIOGRAPHY ........................................................................... 130
LIST OF FIGURES
Figure
1.1
Proteolytic activation of caspases
11
1.2
Caspase activation by cell surface death receptors
13
1.3
Model of caspase-3 activation
15
1.4
A schematic representation of the components spanning the
inner and outer mitochondrial membranes
22
1.5
The IL-4 Receptor complex
26
1.6
The P1-3 K pathway
29
1.7
The Jak-Stat pathway
31
2.1
IL-4 rescues resting T cells from death by neglect and
dexamethasone-induced death in a dose-dependent manner
58
2.2
IL-4 treatment leads to increased tyrosine phosphorylation of
the IL-4Ra chain on activated T cells
60
2.3a
IL-4 inhibits the degradation of poly (ADP-ribose) polymerase
(PARP) in resting T cells
62
2.3b
IL-4 inhibits the degradation of PARP in activated T cells
64
2.4
Caspase-4 and -6 are involved in AICD and death by neglect
of T cells
66
2.5
Caspase-3 is involved in AICD and death by neglect
of T cells
68
LIST OF FIGURES (Continued)
Figure
Page
2.6a
IL-4 blocks caspase-3 activation in resting T cells undergoing
death by neglect or dexamethasone-induced death
70
2.6b
IL-4 blocks caspase activation during death by neglect
or dexamethasone-induced death, as detected in intact T cells
72
2.7a
IL-4 delays the dissipation of mitochondrial membrane
potential in resting T cells
75
2.7b
IL-4 maintains mitochondrial membrane potential in resting
T cells
76
3.1
Recombinant active caspase-3 cleaves a caspase-3-specific
substrate
95
3.2
The activity of recombinant caspase-3 is not blocked by
T cell lysate
97
3.3
Titration of cell lysate and caspase-3 substrate
100
3.4
Titration of caspase-3, -6, -8, and -9 inhibitors
101
3.5
IL-4 rescues T cells from death by neglect and
dexamethasone-induced death when P1-3 K is inhibited
103
3.6
IL-4 rescues T cells from death by neglect death in the absence
of active P1-3 K
104
3.7
IL-4 rescues I cells from death by neglect and
dexamethasone-induced death in the presence or absence of Bax
106
3.8
IL-4 prevents mitochondrial membrane dissipation in the
presence or absence of Bax
108
3.9
IL-4 blocks caspase-3 activity in the presence or absence of Bax
110
LIST OF FIGURES (Continued)
Figure
3.10
Tracking and translocation of cytochrome c in T cells
treated with or without IL-4
112
UNCOVERING THE MECHANISM OF IL-4-MEDIATED T CELL SURVIVAL
INTRODUCTION
THE IMMUNE SYSTEM
In order for living creatures to survive the constant onslaught of invading
organisms it is imperative that an effective and robust defense mechanism is
activated when encountering such microbes. This defense mechanism is
orchestrated by the immune system that is comprised of numerous cells and organs,
and each of these components possesses a specific function. In response to an
infection, cells of the adaptive arm of the immune system differentiate and
proliferate, and eventually undergo programmed cell death. The successful
elimination of a pathogen involves a quick removal of circulating invaders and
infected host cells while recognizing and avoiding damage to healthy host cells.
The ability of a host to recognize foreign material, otherwise known as a foreign
antigen, while simultaneously tolerating self-antigens is achieved by a complicated
process. This includes the necessary deletion of once-activated immune cells with
a simultaneous preservation of a subset of cells destined to become long-lived.
This 'memory" subset is crucial in providing lost-lasting immunity against a
pathogen a host has already encountered.
There exists more than one line of defense against infection within a host.
The cells of the first line of defense, known as the innate immune system, are
mainly phagocytic cells that have evolved to recognize, engulf, and destroy
2
potentially pathogenic organisms (1). These phagocytic cells known as
macrophages and neutrophils, as well as non-phagocytic eosinophils, are derived
from myeloid progenitor cells within the bone marrow (1). Macrophages,
neutrophils, and eosinophils can neutralize almost any invading pathogen
immediately and non-specifically, and they do not require pre-activation or
"priming" to carry out their functions (1). These cells, like others of the immune
system, are able to secrete cytokines, chemical messengers that affect the
physiology of cells within the immune and other systems (2). Importantly, among
a myriad of functions, cytokines mediate inflammation in response to phagocytized
pathogens (1). Inflammation is a mechanism used by the host to isolate the
pathogen at the site of invasion and attract and focus immune cells that serve to
recognize and eliminate the pathogen (3).
In contrast, the adaptive immune system is less immediate but more
powerful and, most importantly, antigen-specific. The adaptive arm of the immune
system contains a diversity of cells that can each recognize only one type of
antigen, such as a viral proteinor a component of a bacterium. These antigenspecific cells are called B lymphocytes (B cells) or T lymphocytes (T cells), and are
derived from common lymphoid progenitor cells within the bone marrow (4). B
cells and T cells can be activated, or "primed," upon recognizing a portion of an
antigen that is displayed on the surface of antigen-presenting cells (APCs) (5, 6). T
cells "see" displayed antigen in the context of major histocompatability complex
(MHC) I or 11(5). In addition to antigen recognition in the context of MHC, a T
3
cell must also receive a second costimulatory signal to become activated (7). Once
activated, lymphocytes proliferate and subsequently differentiation into specialized
effector cells capable of recognizing and destroying the antigen.
The fundamental principle of immunology is that a host distinguishes "self'
from "non-self." In other words, a healthy organism has developed tolerance for its
own tissue, or antigens, while it recognizes and destroys foreign antigen. In the
context of B cell maturation, which occurs in the bone marrow, B cells that are
reactive to self-antigen are prevented from maturing (8). Since self-reactive
lymphocytes mediate autoimmunity, inhibiting the survival of self-reactive B cells
is one way in which the host can eliminate the development of autoimmune
diseases (9). Still, self-reactive B cells can escape tolerance induction within the
bone marrow and thus must be tolerized after they enter the periphery (10). Selftolerized B cells do not recognize self-antigen and are allowed to enter and
circulate throughout the periphery as mature naïve B cells (8). When a naïve B cell
encounters an antigen that it specifically recognizes, activation can occur with the
help of a subset of T cells known as CD4 T helper cells (11). An activated B cell
differentiates into a plasma cell that secretes large quantities of an antibody (6). A
prototypical antibody is a Y-shaped protein structure that has a number of
functions, including the ability to bind an antigen, thus neutralizing it and
preventing it from gaining access to and infecting a host cell (12). Lastly antibody
can coat an antigen, called opsonization, which allows the antigen to be
4
phagocytized by a macrophage (13). Antibody can also activate the complement
cascade, facilitating the destruction of an opsonized antigen (14).
Like B cells, T cells originate in the bone marrow (4). However, precursor
T cells migrate from the bone marrow to the thymus where they undergo
maturation (4). T cell precursors undergo a selection process that is eliminates selfreactive T cells (15). Following selection and release into the periphery, naïve T
cells must receive two signals in order to become activated (7). The first signal
constitutes ligation of the T cell receptor (TCR) by MHC molecules presenting
specific peptides (7). The second signal involves a costimulatory signal whereby
the APC ligates a T cell constimulatory molecule (7). When a mature naïve T cell
encounters an antigen it is specific for, and it receives the proper costimulation by
other immune system cells such as B cells or dendritic cells, it begins to proliferate
and produce interleukin-2 (IL-2) (16). IL-2 further drives proliferation and
differentiation into effector T cells (17). Effector T cells become either cytotoxic
CD8+ T cells or helper CD4+ T cells, depending on whether they express CD4 or
CD8 on their surface. Furthermore, CD4+ T cells can be divided into two classes,
existing as either Thi (pro-inflammatory) T cells or Th2 (anti-inflammatory) T
cells (18).
Collectively, T cells work to eliminate antigen-bearing pathogens via a
variety of mechanisms. Cytotoxic T cells contain stores of cytotoxic proteins and
proteases that can puncture or degrade infected cells. Cytotoxic T cells can also
target and kill infected host cells by releasing pro-inflammatory cytokines like
TNFa and INFy which can activate macrophages and inhibit viral replication.
Lastly, cytotoxic I cells can also destroy infected host cells by ligating the host cell
surface receptor Fas with Fas-ligand, which results in the programmed cell death
of, or apoptosis of, the infected cell (19). On the other hand, the Thi subset of
CD4+ T cells can produce cytokines that enhance the inflammatory response by
activating macrophages (20). The Th2 subset of CD4+ I cells provide the
necessary costimulatory signal a B cell requires in the presence of antigen to
proliferate and differentiate into an antibody-secreting plasma cell (6).
At the end of an immune response, the majority of antigen-specific B cells
and T cells undergo apoptosis, yet it is vital that not all of these B cells and T cells
become deleted. Those that survive recognize an already-encountered pathogen.
This recognition is what defines adaptive immunity. A host that has "seen" an
antigen previously can mount an already-primed immune response, thus offering
effective protection from that antigen. Elimination of the previously encountered
pathogen is markedly swifter and more efficient compared to the initial exposure.
The survival of
lymphoctyes is critical not only in the context of infection, but also
in the ability of a host to combat dangerous tumor cells (21) and toxic substances
(22).
The mechanisms by which lymphocytes escape death are still unclear, and
our primary interest lies in deciphering ways in which T cells avoid deletion and
become long-lived. Although this can be an obvious benefit to an organism in
terms of acquiring immunity, T cells that avoid deletion can also be detrimental if
they attack the host. Nonetheless, it has become increasingly apparent that
environmental factors can influence the fate of T cells.
During the early stages of cell activation by antigen T cells exposed to
inflammatory agents, like bacterial lipopolysaccharide (LPS), can supply an
autocrine signal that allows them to avoid activation-induced cell death (AICD)
(23). Ligation of the T cell co-stimulatory molecule 0X40, in conjunction with
LPS during a T cell response, enhances survival more than LPS alone, signifying
that more than an inflammatory environment is required for optimum T cell
survival (24). Cytokines belonging to the IL-2 superfamily play a role in T cell
survival and are capable of protecting T cells from death. In particular, IL-4 has
been shown to block T cell death in vitro and in vivo, although the mechanism is
still not understood (25).
The importance of understanding T cell survival is crucial in terms of
uncovering the mysteries of T cell behavior. As mentioned previously, most of the
cells involved in an immune response must subsequently be deleted, while a select
few are preserved to become memory cells. However, T cells which are naïve or
activated, and which avoid deletion, are permitted to survive, providing the
opportunity for these T cells to become autoreactive. Understanding the
mechanism(s) of autoreactive T cell survival is key in improving therapies, such as
glucocorticoids, aimed at eliminating autoimmune disease. Glucocorticoids, via
their immunosuppressive activity, are frequently used to treat inflammatory
diseases such as asthma, rheumatoid arthritis, and autoimmune diseases. Thus, by
deciphering exactly how T cells could escape glucocorticoid-induced death,
improvement of existing therapies becomes a possibility. Lastly, investigating T
cell survival provides information of the basic requirements a T cell must fulfill in
order to avoid death.
APOPTOS IS
Apoptosis, or the process known as programmed cell death, is widely
observed throughout Nature. More specifically, apoptosis is a coordinated series of
events leading to genetically controlled cell demise in developing organisms (26).
Although Currie et al. coined the term apoptosis in 1972, the exact mechanisms of
apoptosis and key players involved have only recently begun be described.
Apoptosis involves the ordered and programmed breakdown of the cell, involving
drastic biochemical, physiological, and morphological changes that lead to death.
These events include the proteolysis of targeted substrates, degradation of
chromosomal DNA into distinct oligonucleosomal fragments and radical cell
membrane alterations, including membrane blebbing and externalization of
phosphatidylserine (27). Programmed cell death is essential for the orderly
development and maintenance of an organism, specifically eliminating
unnecessary, unneeded, or dangerous cells.
Apoptosis occurs constantly throughout the immune system. It is vital
during the selection and maturation of lymphocytes, as well as during the
elimination of effector cells that are no longer needed. As T cells mature in the
thymus, they are negatively or positively selected, and those that are negatively
selected die via apoptosis (15). In the periphery, T cells that have become activated
need constant exposure to antigen in order to avoid death. As soon as the antigen is
no longer available, the antigen-specific T cells are eliminated. Moreover, during T
cell activation, the surface receptor Fas is upregulated (19). Fas, when engaged
with Fas-ligand, triggers AICD, promoting tolerance and eliminating the circulation
of activated T cells that are no longer needed (19).
Conversely, circulating naïve T cells need stimuli that promote survival. If
these requirements are not met, the T cell undergoes spontaneous cell death. This
phenomenon is frequently observed when resting naïve T cells are neglected such
as when cultured in vitro without survival factors. These otherwise healthy cells
consequently die by apoptosis unless survival factors are provided. Nonetheless,
the exact environmental factors and conditions necessary for basic T cell survival
remain unclear.
T cells, like all cells, can be exposed to an array of harmful chemicals and
toxins that trigger apoptosis. Agents that have been identified as successful in
triggering apoptosis of T cells, such as the glucocorticoids, have been used
therapeutically against a wide variety of diseases, particularly diseases that involve
inflammation. Glucocorticoids, including the synthetic glucocorticoid
dexamethasone, are recognized as immunosuppressive and inhibit the production of
a large number of cytokines, including the pro-inflammatory IL-2, TNFa and
IFNy (28). Indeed, glucocorticoids will trigger apoptosis in CD4 CD8
thymocytes, also known as double positive thymocytes, during the selection
process within the thymus. We have shown that glucocorticoid-induced apoptosis
also occurs in activated and resting murine T cells in vitro (Chapter 2).
Glucocorticoid hormone (GCH)-induced apoptosis involves the binding of
GCH to the glucocorticoid hormone receptor (GR) (29). However, the exact
molecular mechanisms of GCH-induced death are not fully understood.
Dexamethasone has been shown to induce sphingomyelinase (Smase) activity in a
number of cell types (30, 31). Signaling through the sphingomyelin (SM) pathway
is mediated via generation of ceramide, which behaves as a second messenger
molecule in the SM cycle (32, 33), apoptosis (34), and cellular differentiation in a
number of cell types (34). Cifone et al. (35) showed that the dexamethasone/GR
interaction in mouse thymocytes activated a signaling pathway required for death.
Specifically, early generation of ceramide through sequential activation of
phosphatidylinositol-specific phospholipase C (P1-PLC) and acidic Smase
(aSMase) was required for dexamethasone-induced caspase activation and
apoptosis.
With respect to thymocyte apoptosis, it is known that, in response to
glucocorticoid treatment, these cells undergo what is known as a mitochondrial
death pathway. The anti-apoptotic proteins Bcl-2 and Bcl-XL interact with
components of the mitochondria and inhibit this process. Also, the proteins Apaf- 1
and caspase-9 are required for mitochondrial death by this pathway (36, 37).
Caspase-9 activation involving Apaf- 1 requires the release of the mitochondrial
10
protein cytochrome c, which occurs in response to death stimuli andlor
mitochondrial disruption (38). Still, the exact mechanisms by which
glucocorticoids mediate apoptosis have yet to be elucidated, especially in relation
to normal mature T cells.
CASPASES
Caspases have recently become recognized as the central executioners of
apoptosis. They are responsible for mediating the extensive morphological and
biochemical changes that occur during programmed cell death. The term "caspase"
comes from cysteinyl aspartate-specific proteinase, which defines a family of
proteases that specifically cleaves substrates on the carboxyl side of an aspartate
residue (39). Over the last ten years, much information has become available
concerning the structure, activation, and function of caspases. These enzymes are
divided into two major phylogenetic subfamilies based on proteolytic specificities,
including the ICE subfamily and the CED-3 subfamily (40). The ICE subfamily
includes caspases that are not involved in cell death, but instead mediate cytokine
maturation (40). The CED-3 subfamily, however, includes cell death-mediating
caspases, both upstream initiators and downstream effectors (40).
These highly specific cysteine proteases are synthesized as inactive
zymogens, or proenzymes, consisting of a prototypic N-terminal prodomain
followed by a large subunit alongside a small subunit (41) (Fig. 1.1).
11
N-peptide
large subunit
small subunit
IPVETEEQPYLEM11.S
Proteolytic
297
activation
Figure 1.1 Proteolytic activation of caspases. Salvesen, G.S., and V.M. Dixit.
1999. Caspase activation: The induced-proximity model. Proc. Nail. Acad. Sci.
USA 96:10964
Caspases that are upstream in a death-signaling pathway are considered initiator
caspases and they typically have longer prodomains (42). These prodomains
contain motifs that assist association with the cytoplasmic regions of cell surface
receptors. Motifs within the prodomains of initiator caspases -8 and -10 include
tandem repeats of the death effector domain, or DED, that can interact with
receptor-associated adapter proteins such as TNF receptor-associated death domain
(TRADD) and Fas-associated death domain (FADD) (43). Furthermore, caspases
1, -2, -4 and the initiator caspase-9 contain a caspase-recruitment domain, or
CARD, within the prodomain that assists in the interaction of these caspases with
adaptor molecules, as well, or regulatory proteins (44). Conversely, downstream or
12
effector caspases possess short prodomains and depend on initiator caspases for
their activation (45).
Formation of an active caspase requires distinct processing of the precursor
enzyme followed by the association of cleavage products into an active
conformation. The caspase precursor requires cleavage carboxy-terminal to an
aspartate residue at two sites (46), and the sites cleaved appear to occur in a specific
order (47). The first cleavage separates the large subunit from the small subunit,
and the second cleavage separates the prodomain from the large subunit (41). The
following arrangement of two large subunits with two small subunits comprises the
active enzyme, which contains two catalytic regions.
There are a few fundamental ways in which a zymogen is processed into an
active molecule. These include recruitment-activation involving cell surface death
receptors such as Fas or intracellular regulatory proteins such as Apaf- 1, as well as
cleavage of downstream caspases by upstream active caspases (41). Also,
zymogen processing may occur as a result of autoactivation (41). Lastly, caspases
may be activated via cytotoxic lymphocyte activity (41).
The activation of upstream initiator caspases by recruitment-activation
involves the organization of numerous proenzymes within close proximity to each
other. Intracellular death domains of death receptors, such as CD95/Fas/APO- 1,
TNFR1, DR-3/Apo-3IWSL-1/TRAMP, and the TRAIL receptors DR4/TRAIL-R1
and DR5/TRAIL-R2/TRICK2/KILLER (48), can recruit adapter proteins, which, in
turn, recruit procaspases to the receptor complex (49) (Fig. 1.2). Adapter proteins,
13
such as TRADD (TNF-receptor associated death domain), RIP (receptor interacting
protein), RAIDD, and MADD have been found to contain homologous death
domains (46). In the case of the receptor Fas, the cytosolic adapter protein FADD
(Fas-associated death domain) intracellularly associates with Fas via its own Cterminal death domains (DD)
TNFa.
APO3L
Caspase Recruitment
and Activation
Caspase 3,6,7
-
TRAIL
FasL
Mitc'cc nd rial
>BDCeava9e
Li
lAP's
AmpIcation
Cyt. c
Caspase 9
AivatiOn
/
release
Figure 1.2 Caspase activation by cell surface death receptors. Budihardjo, et al.
1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev.
Cell Dev. 15:269
14
with the receptor's clustered cytosolic DD (43). These DD-DD interactions anchor
FADD and allow its N-terminal death effector domain (DED) to associate with
recruited procaspase-8 molecules (50). The procaspase-8 protein, as mentioned
previously, contains tandem DED within its prodomain which facilitates its
association with FADD through DED-DED interactions (51). The
FADD/procaspase-8 complex, known as the DISC, or death-inducing signaling
complex, recruits more procaspase-8 molecules to the site (40). As procaspase-8
molecules cluster together, the low level of endogenous catalytic activity that each
proenzyme harbors allows trans-catalysis to generate active caspase-8 (49).
Another example of recruitment-activation occurs with the interaction of
procaspase-9 with Apaf- 1. Apaf- 1 is a 130 kD cytoplasmic protein that shares
limited homology with the nematode C.
elegans ced-4
apoptosis regulator (52).
When a cell receives apoptosis-triggering stimuli that do not involve death
receptors, the mitochondria release cytochrome c into the cytosol (53). Apaf-1 can
bind the liberated cytochrome c in a dATP-dependent fashion (47), allowing
procaspase-9 to associate with Apaf-1 via CARD-CARD interactions (47).
Furthermore, the oligomerization of Apaf-1 allows the recruitment of multiple
procaspase-9 molecules, which, similar to procaspase-8 activation, allows
procaspase-9 to become activated via trans-catalysis (47).
The activation of caspases by other caspases is an intensively studied area
(44). Generally, initiator caspases can act as potent activators of effector caspases,
and evidence from a number of studies highly suggest that this method of activation
15
is through a cascade. A study using a cell-free system demonstrates that caspase
activation can be achieved through a hierarchic activation network in which key
caspases, including caspase-8, are able to activate other caspases (54). Similarly, as
caspase-9 becomes activated via association with Apaf-1, it can, in turn, activate
downstream caspases, apparently simultaneously activating caspase-3 and caspase-
7 (55) (Fig. 1.3). Importantly, the removal of any components of the caspase
cascade, as described in the study above, essentially blocks downstream events,
further supporting the idea that activation is indeed through a true cascade.
Cytochronc c
(Apaf-2)
dATP
Appwiic Sn,uI
$
A pat- I
Caspasc-9 Precursor
DQ4DA
IATP
Activated Caspasc-9
Caspasc-3 Precursor
Actived Caspase3
Figure 1.3 Model of caspase-3 activation. Li, et al. 1997. Cytochrome c and dATPdependent formation of Apaf- 1/Caspase-9 complex initiates an apoptotic protease
cascade. Cell 9 1:479
16
The significance of the identification of the caspase activation cascade stems from
the fact that a myriad of apoptotic stimuli can eventually activate the same set of
effector caspases. Thus, regardless of the death trigger, whether it's through a
death receptor or exposure to drugs, the cell is able to undergo apoptosis by
utilizing its defined set of proteolytic machines that have specifically evolved to
carry out cell degradation.
Autoactivation is a third mechanism by which caspases may be activated.
Nevertheless definitive evidence proving this theory has yet to be demonstrated.
Although a non-recruitment type of autoactivation has not yet been established,
there are indications that suggest such a mechanism may exist (56). Moreover, the
fact that downstream capsases cleave and activate upstream caspases suggests a
more complex system of caspase activation that involves more than an activation
cascade simply proceeding from top to bottom. Instead, a mechanism similar to a
positive feedback ioop may occur, to further commit the cell to apoptosis.
Cytotoxic CD8 T cells as well as NK cells possess granules that contain
granzyme B and perform. Granzyme B is a serine protease that cleaves proteins
carboxy-terminal to aspartic acid residues, and perform forms pores by which
granzyme B can enter the target cell. Granzyme B cleaves and activates many
caspases in vitro (57). It appears that the preferred target of granzyme B is caspase3, which, once activated, can activate caspases-7, -8, and -9 (58). Caspase
activation is an event that defines the death checkpoints in the common
17
programmed cell death pathway (59); once caspases are activated a cell is past the
point of no return and is irreversibly committed to death.
Caspases are very selective proteases that cleave specific target proteins
carboxy-terminal to aspartic acid residues. Functioning as a network of machinery
to achieve cellular breakdown during apoptosis, caspases target and degrade
cytoskeletal proteins, DNA repair proteins, as well as a number of proteins
involved in survival signaling pathways (40). Approximately 70 polypeptides have
been identified as targets for caspase-mediated degradation (60). Generally,
caspase-mediated processing of most of the 70 different substrates results in events
conducive to apoptosis, including the arrest of events that protect the cell from
death as well as the initiation of cellular degradation. A clear sign of apoptosis is
the fragmentation of DNA, and caspases aid in this event via inactivating DNA
repair enzymes such as poly-ADP ribose polymerase (PARP) and DNA-dependent
protein kinase (DNA-PK) as well as activating caspase-activated DNAse (CAD), a
pro-apoptotic endonuclease (41). Furthermore, caspases contribute to apoptosis via
direct disassembly of the cell, cleaving proteins such as lamins, the nuclear mitotic
apparatus protein (NuMA), as well as RNA-binding and ribonuceloproteinassociated proteins (41).
Substrate processing by caspases is carried out one of four ways. Caspases
can inactivate the substrates, thus preventing these proteins from carrying out their
normal functions (39). Conversely, caspases can also activate substrates by
removing regulatory domains (40). Caspases can alter or invert substrates, turning
once-protective proteins into pro-apoptotic molecules. Lastly, caspases can
disassemble substrates, ultimately destroying cell structure (61).
Since caspase precursors are constitutively expressed in cells and apoptosis
can be rapidly induced, these observations suggest that cell survival requires a high
level of regulation of caspase activation (39). The presence of abundant effector
caspases that remain inactive until the appropriate apoptotic signal has been
received demonstrates that there must be tight regulation. FADD-like ICE
inhibitory proteins (FLIPs) are similar in sequence to procaspase-8 and may act as
competitors for the adapter molecule FADD, thus blocking procaspase-8 activation
(62-64). Furthermore, relatively recently discovered endogenous cellular proteins
have been found to influence caspase activity, illustrating yet another level of tight
regulation of the protease-driven breakdown of the cell.
lAPs, or inhibitors of apoptosis proteins, are found in baculovirus,
Drosophila, and humans (50). lAPs possess up to three homologous baculovirus
TAP repeats (BIR) (65). BIR motifs are apparently required for the anti-apoptotic
functions of lAPs (66). Along with BIR motifs, two human lAPs possess CARD,
suggesting that lAPs may be able to directly interfere with procaspase-9 activation
via Apaf- 1/cytochrome c (50). Although the exact targets of lAPs have yet to be
defined, studies have suggested that TAPs are able to directly interfere with caspase
activity or the processing of caspases. lAPs do not interact with caspases-1, -6, -8,
or -10 (67). As lAPs do not bind and inhibit the upstream caspases-8 or -10, they
19
do inhibit the caspase-8-activated effector caspase-3 as well as the effector caspase7 (50), thus arresting the caspase activation cascade initiated by death receptors.
Moreover, lAPs have also been shown to interrupt procaspase-9 activation
three ways. First, lAPs may directly bind to inactive caspase-9 molecules (68, 69).
lAPs may also compete with procaspase-9 for Apaf-1 via their CARDs. Lastly, as
TAPs inhibit active caspase-3, including the active caspase-3 produced as a result of
caspase-9 activation, lAPs could attenuate an amplification loop involving
subsequent caspase-3-mediated processing of procaspase-9 (47).
The Bcl-2 family of proteins are intracellular proteins that share homology
within conserved regions and have the ability to dimerize and function as regulators
of apoptosis (59). Certain members of this family may regulate mitochondriamediated caspase activation. Specifically, Bcl-2 proteins may regulate the
permeability transition pore (PTP) of mitochondria (70). The PTP itself regulates
the inner mitochonrial membrane potential and is comprised of a number of
proteins, including cyclophilin D, adenine nucleotide transporter (ANT), and the
voltage-dependent anion channel (VDAC) (71). Exactly how the PTP initiates loss
of mitochondrial outer membrane potential is still unknown, but disruption of the
membrane potential may lead to osmolarity changes that lead to mitochondrial
swelling and subsequent release of intermembrane proteins (72). A recent study
conducted by Wei et al. (2001) demonstrated that cells lacking both pro-apoptotic
Bcl-2 members Bak and Bax were resistant to mitochondrial disruption.
Furthermore, these cells were completely resistant to mitochondrial cytochrome c
20
release by another pro-apoptotic protein called tBID (73). It appears that antiapoptotic Bcl-2 family members, specifically Bcl-2, maintain mitochonclrjal
integrity (74), thus potentially preventing the release of cytochrome c into the
cytosol and the activation of caspase-9.
MITOCHONDRIA AND APOPTOSIS
Recently, mitochondria have been examined for the possibility that these
organelles are involved in cell death. The intriguing observation linking
mitochondria to cell death involves the changes that take place within the
mitochondria prior to death. Pre-death alterations of the mitochondria generally
include mitochondrial membrane permeabilization (which can be caused by a
number of proteins and agents) followed by the liberation of protease and nuclease
activating proteins. Observations from a number of studies outline a three-step
model for mitochondrial-induced apoptosis: an initiation phase involving activation
of apoptosis-triggering pathways, an effector phase involving mitochondrial
membrane permeabilization, and a degradation phase involving the release of proapoptotic proteins from within the mitochondria (75, 76).
An important point discussed by Leoffler and Kroemer (2000) is that
mitochondria may not always be involved in cell death. They describe "type-i"
cells that, upon ligation of Fas, activate the initiator caspase-8, which in turn
activates the effector caspase-3. In this case, mitochondrial membrane permeability
in response to receptor-mediated apoptosis may be dispensable (77). However,
21
in"type-2" cells, caspase-8-mediated cleavage of the proapoptotic Bid protein is
required for apoptosis (77). Here, cleaved Bid can translocate to the mitochondrial
membranes and cause permeabilization (78).
Whether the mitochondria are required or not in cell death it is clear that a
number of proteins and agents regulate permeabilization of mitochondrial
membranes (79, 80), which in turn leads to disruption of
/X'I'm.
Furthermore, it
appears apoptosis is accompanied by liberation of cytochrome c into the cytosol
(77). Studies involving cell-free systems demonstrate that the recombinant
proapoptotic Bcl-2 family proteins Bax and Bak, when added to purified
mitochondria, permeabilize the mitochondrial membrane (81, 82). Conversely,
anti-apoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-XL protect
mitochondrial membranes from permeabilization (72).
Mechanisms by which mitochondrial permeabilization is induced or
prevented are controversial, and there are opposing observations that exist in terms
of how permeabilization may be achieved. Close examination of a complex called
the "permeability transition pore complex" (PTPC) has been conducted to elucidate
the possible mechanisms of regulation. Kroemer and colleagues find that the PTPC
is constituted of a number of proteins spanning the inner and outer mitochondrial
membranes. The components include matrix-bound cyclophilin D, ANT, VDAC,
and hexokinase-1 (70) (Fig. 1.4).
23
not exert antiapoptotic effects when added to intact cells, or when effects are
observed several hours or days later, suggesting that the PTPC is not rate-limiting
(77).
An effective model explored by Gross and Korsmeyer (1999) involves the
anti-apoptotic proteins "guarding the mitochondrial gate" from the pro-apoptotic
members that "gain access" after a death signal. The pro-apoptotic and antiapoptotic Bcl-2 family members reside in different locations prior to a death signal.
Anti-apoptotic members such as Bcl-XL and Bcl-2 are integral membrane proteins
localized to the mitochondria, endoplasmic reticulum, or nuclear membrane (71,
85). Conversely, the pro-apoptotic Bcl-2 family members are located within the
cytosol prior to a death trigger (86), and only when a death signal has been received
do these proteins undergo conformational changes that allow them to integrate into
the mitochondrial membrane (71).
Bcl-2, when localized to the mitochondrial membrane, can prevent the
events that normally follow PTPC opening, including
dissipation (87), the
release of pro-apoptotic proteins (53), as well as the overproduction of reactive
oxygen species (ROS) (88). Moreover, Bcl-2 retains ceramide, a death-triggering
molecule, within the cytosol of ceramide-treated cells (89). Because the NMR
structure of Bcl-XL is similar to pore-forming toxins such as the diptheria toxin, it
was suggested that this anti-apoptotic protein, and potentially other anti-apoptotic
Bcl-2 family proteins, could themselves form pores within mitochondrial
membranes. Indeed, studies demonstrate that Bcl-XL, as well as Bcl-2 and pro-
24
apoptotic Bax, form ion channels in artificial membranes (90, 91). Dimerization or
induced expression of Bax, or another pro-apoptotic protein Bak, causes
I.'I'm
dissipation, ROS production, and cytochrome c release, the latter depending on the
setting (92). In most cases, overexpression of Bcl-2 or Bcl-XL will block these
deleterious effects. Agents such as atractyloside that induce opening of the PTPC
mimic the effects of recombinant Bax or Bak on purified mitochondria (71). It is
proposed that Bax may directly associate with components of the PTPC thus
causing its opening (82, 93). On the other hand, Bcl-2 or
Bcl-XL
may interact with
the components of the PTPC that govern ion transport (94). Relative ratios of proapoptotic and anti-apoptotic Bcl-2 family members may influence ion flow and
volume regulation within the mitochondria, though the exact mechanisms are still
under investigation.
INTERLEUKIN-4IINTERLEUKIN-4 RECEPTOR
Interleukin-4 (IL-4) is a multifunctional cytokine that is produced by a
number of cells, including the Th2 subset of CD4+ T cells, polymorphonuclear
basophils and mast cells, in response to receptor-mediated activation events (95).
Other cells, such as specialized T cells that express NK1.1 (96), as well as
eosinophils, also produce IL-4 (97). IL-4 plays a key role in regulating the
differentiation of naïve T cells that have been stimulated with antigen (98). It
causes these stimulated cells to behave as Th2 cells, secreting IL-4, as well as other
"anti-inflammatory" cytokines such as IL-5, -10, and -13 (99, 100). Isotype
switching is dictated by the cytokines that a B cell encounters, and IL-4 drives
isotype switching to IgE (101). IgE production is important in combating parasitic
infections, and is a key player in Type I hypersensitivity (102). Thus, IL-4 plays a
crucial role in regulating immediate hypersensitivity and a role in developing
immune responses against parasites. Other functions of IL-4 include inducing
increased expression of MHC II molecules in B cells (103), enhancing expression
of CD23 (104), and acting as a B cell growth factor (105). Furthermore, IL-4 is
involved in adhesion and inflammation (98). IL-4 contributes to the induced
expression of vascular cell adhesion molecule-i (VCAM-1) on vascular endothelial
cells (106), and downregulates expression of E-selectin (107), favoring the
recruitment of T cells and eosinophils to a site of infection (98). Finally, IL-4
substantially prolongs the life of T cells and B cells in vitro (108), an observation
that is the main focus of this thesis.
The IL-4 receptors (IL-4R) are present on many different cells, including
hematopoeitic, endothelial, epithelial, muscle, fibroblast, hepatocyte, and brain
tissue (109, 110). The IL-4R consists of a l4OkD a chain that is able to
heterodimerize with a second chain, namely the common gamma chain (yc), which
allows the generation of signals within the cell (98). Although the IL-4Ra chain
can homodimerize (111, 112) or heterodimerize with a component of the IL-13R
(113), the yc appears to be the major component in IL-4-mediated signal
transduction in hematopoeitic cells (98). The IL-4Ra is a member of the
hematopoeitin receptor superfamily (98). These family members share common
26
motifs within the extracellular and intracellular regions of the a chain, and these
conserved motifs serve as functionally significant regions important for IL-4mediated signaling (Fig. 1.5).
IL-4 Receptor
IL-4
IL-4Ra
Motif:
14R
(Jak1
Y497 [Yl]
Y575{Y2]
Stat-6
<
ITIM
proliferation!
P1-3-K activation
Y603 {Y3]
IL-4-inducccl genes
Y713 [Y5]
negative regulation
Y631[Y4]
Figure 1.5 The IL-4 Receptor complex. Adapted from Neims, et al. 1999. The IL-4
Receptor: Signaling mechanisms and biologic functions. Annu. Rev. Immunol.
17 :70 1
The extracellular, membrane proximal WSXWS motif has been proposed to be
required for the maintenance of productive cytokine binding (114). The
intracellular, membrane proximal "box 1 motif" of the a chain, found in many
hematopoeitin receptor family members, appears to be important for the function of
the receptor (98).
IL-4 binding to IL-4Ra causes this chain to heterodimerize with the yc,
which leads to activation of IL-4-induced signaling pathways (115). Signal
initiation requires the subsequent activation of receptor-associated kinases, namely
the Janus-family tyrosine kinases (Jaks), since neither the a chain nor the yc
possess endogenous kinase activity (116). Jak-1 has been proposed to be
associated to the a chain while Jak-3 is proposed to associate with the yc (117).
Upon ligation of IL-4, Jak- 1 and Jak-3 become phosphorylated at tyrosine residues
(98), and shortly thereafter, the IL-4Ra becomes phosphorylated at any of five
conserved tyrosine residues within the cytoplasmic region (Fig. 1.5). The
importance of phosphorylation at these tyrosine residues is due to the fact that these
sites serve as places of interaction with downstream signaling molecules, either
through Src-homology 2 (SH2) or phosphotyrosine-binding (PTB) domains (98).
Through a series of truncation and deletion experiments, IL-4Ra has been
divided into three functionally distinct areas. The region containing one conserved
tyrosine residue (Y497) is necessary for IL-4-mediated proliferation (118, 119).
Within the region beyond residue 557, the three conserved tyrosine residues (Y575,
Y603, and Y631) are crucial for IL-4-mediated gene activation (112, 120). Lastly,
!11
the conserved tyrosine residue Y713 is located within an immunoregulatory
domain (121). Collectively, these conserved tyrosine residues, along with the
membrane-proximal region necessary for Jak interaction, comprise IL-4Ra which,
when ligated with IL-4 and the yc, initiates signaling cascades required for the cell
to respond to its environment.
Insulin receptor substrates-i or -2 (IRS-i or IRS-2) links the IL-4Ra chain
to signaling pathways involved in proliferation (98). IRS-i/2 is able to bind to the
insulin IL-4 receptor (14R) motif of the a chain (119). Upon binding to the a chain
through a PTB, IRS- 1/2 becomes phosphorylated itself, presumably by receptor-.
associated kinases (98). IRS-i and -2 each possess approximately 20 tyrosine
residues that serve as potential sites for phosphorylation (122, 123), and a number
of these residues are bound by SH2 domains. This is an indication that IRS-i/2
may serve as a docking protein for downstream signaling molecules, providing a
means of connecting the IL-4Ra to these molecules (122, 123).
One of the pathways activated by IL-4 is the phosphatidylinositide-3 kinase
(P1-3 K) pathway. The P1-3
K complex activated by IL-4 is comprised of two
subunits, an 85 kD regulatory and a 110 kD catalytic subunit (98). As the p85
subunit interacts with phosphorylated IRS-1/2 molecules bound to the a chain, a
conformational change takes place that leads to the activation of the p110 subunit
(124). The activated subunit can then phosphorylate membrane lipids and Ser/Thr
residues on other proteins (124). Specifically, active P1-3 K transfers a phosphate
29
from ATP to the D-3 position of the inositol ring of phophoinositides, generating
3' -phophorylated phosphoinositides (125).
When survival signals are transduced, certain responsive isoforms of P1-3 K
mainly generate phosphatidylinositol 3,4 bisphosphate (P13 ,4P) and
phosphatidylinositol 3,4,5 triphosphate (PI3,4,5P) (125). These molecules act as
second messenger molecules during cell signaling, including IL-4-mediated
signaling (98).
I L-4
lns (4,5)PO4..-ns
iRS1}f
I
ill
Grb2/SOS
SHP-2
(3,4,5)PO4
Ins
Ins (4)PO
(3,4)PO4
'N__
\®[PKcJ
5
Cell growth
and survival
Figure 1.6 The P1-3 K pathway. Neims, et al. 1999. The IL-4 Receptor: Signaling
mechanisms and biologic functions. Annu. Rev. Immunol. 17:70 1
30
Phosphoinositides activate downstream survival kinases, namely protein kinase B
(PKB), also known as Akt kinase (126). Akt is activated when it localizes to the
cellular membrane where it binds to phosphoinositides via N-terminal PH domains
(126). It is then phosphorylated on two C-terminal sites, a Ser and a Thr (127). A
number of factors have been proposed as targets for Akt, including the proapoptotic
Bcl-2 family member Bad, caspase-9, as well as the Forkhead family of
transcription factors and the NF-KB regulator IKK (125).
Signal transducers and activators of transcription, or STATs, are molecules
that are activated by members of the common gamma chain (yc) receptor family as
well as IFN receptors (128) (Fig. 1.7). Stat-6 is primarily activated in response to
IL-4 ligation and is responsible for the activation or enhanced expression of a
number of genes (98). Following IL-4 binding to the IL-4Ra and activation of Jak
1 and Jak 3, specific tyrosines on the IL-4Ra become phosphorylated, thus
allowing Stat-6 to bind to the receptor through a conserved 5H2 domain (98). Stat6 then becomes phosphorylated by activated kinases (128, 129), causing it to
detach from the receptor and homodimerize with another Stat-6 molecule (98). The
homodimers translocate to the nucleus and bind to specific DNA motifs within the
promoters of IL-4-responsive genes (98).
32
data, (131)). Although the many cytokines tested varied slightly in their rescue
effects, IL-4 was most effective at rescueing resting and activated T cells in vitro,
presumably through the ligation of a high affinity receptor (25, 130). Moreover,
IL-4 was recognized as a true survival factor since it did not stimulate proliferation
but instead prevented T cell death (25, 130). Also, Stat-6 did not appear to be
required for the IL-4-mediated rescue of resting T cells in vitro (130).
Interestingly, IL-4-mediated rescue of resting and activated T cells in vitro was
associated with the maintenance of the prosurvival proteins Bcl-2 and Bcl-XL(130).
Our initial approach toward uncovering the mechanism of IL-4-mediated
survival involved analyzing the physiological events associated with T cell death
and determining which events, if any, are modulated by IL-4, with the intention of
decoding the method by which IL-4 blocks various forms of apoptosis.
Specifically, our opening studies involved identifying the characteristics and events
associated with apoptosis of resting, activated, and dexamethasone-treated T cells,
and we subsequently sought to determine in what way the characteristics and
events were altered in response to IL-4 treatment.
34
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UNDERSTANDING HOW IL-4 MEDIATES T CELL SURVIVAL
Cathleen M. Moscibrocki, Lisa Finkeistein, and Anthony T. Vella
Department of Microbiology
Oregon State University, Corvallis, OR 9733 1-3804 U.S.A.
INTRODUCTION
Although it is well-established that memory T cells and B cells are
generated during the course of a normal immune response, the exact mechanisms
that promote their survival and permit their escape from death remains unclear.
The generation of long-lived memory T cells and B cells is key for survival in the
face of a constant onslaught of potential pathogens, yet the precise environmental
requirements a T cell must encounter in order to become long-lived have yet to be
fully characterized. Furthermore, the "unintended" survival of harmful T cells can
lead to the development of serious imniunopathological conditions, such as
allergies or autoimmune disease.
A number of agents and molecules have been identified as important in
rescuing T cells from apoptosis, including members of the IL-2 family of cytokines
such as IL-2, IL-4, IL-7, and IL-15 (1). Such cytokines are capable of promoting T
cell survival in vivo and in vitro. They share use of a receptor subunit known as
the common y chain (yc), and we have previously demonstrated that IL-4 requires
the yc to function in rescuing resting or activated T cells from apoptosis
(unpublished observation). Furthermore, IL-4 has been shown to maintain the
levels of the anti-apoptotic Bcl-2 and Bcl-XL proteins in resting and activated T
cells (1). Despite the well-established observation that IL-4 can prevent resting and
activated T cells from death induction, the precise mechanism(s) utilized by IL-4
are still unclear.
50
Relatively recently, the family of cysteine proteases called caspases have
been given a considerable amount of attention concerning their roles in apoptosis.
Caspases are recognized as the central executioners of apoptosis, and they are
responsible for mediating a wide variety of biochemical and physiological changes
within the cell during cell death. Caspase activation results in nuclear and
mitochondrial alterations leading to the demise of the cell (2). Depending on the
death stimulus, caspases can become activated by death receptor-mediated
signaling as well as by mitochondrial disruption.
Considering the information gathered thus far, experiments were done to
determine whether IL-4 might modulate caspase activation in T cells undergoing
spontaneous apoptosis, activation-induced cell death (AICD), or glucocorticoid-
induced apoptosis. By conducting caspase inhibition studies utilizing substrate
specific inhibitors, caspase-3, -6, and to a lesser extent, caspase-4, appeared to be
involved in spontaneous death and AICD of T cells. Caspase activity in both
resting and activated T cells was detected via analysis of poly (ADP-ribose)
polymerase (PARP) degradation in response to various treatments in vitro.
Moreover, the presence of active caspase-3 enzyme was detected via Western blot
in resting and activated T cells undergoing various treatments in vitro, and IL-4
was shown to block caspase-3 activation in T cells undergoing all three pathways
of death.
Since caspase-3 is recognized to have a major caspase in apoptosis (3), and,
once activated, commits a cell to death past the point of no return, we analyzed the
51
activity of caspase-3 in intact T cells using a caspase-3-specific fluorometric
substrate whose cleavage could be analyzed by flow cytometry. This technique
showed that IL-4 prevented caspase-3 activation in T cells incubated with
dexamethasone or nothing (death by neglect). Since various studies have shown
that mitochondrial membrane disruption most often leads to subsequent caspase
activation, we used the cationic dyes JC-1 and DiOC6 to analyze the mitochondrial
membrane potential
(Wm)
in cultured T cells responding to various treatments.
This study demonstrated that IL-4 preserved the
Wm
in T cells dying by neglect or
exposed to dexamethasone.
MATERIALS AND METHODS
Mice.
Female B1O.A and Balb/c mice were purchased from the National Cancer
Institute (Frederick, MD), and the Jackson Laboratory (Bar Harbor, ME). DO11.1O
TCR transgenic mice were bred at the Oregon State University Lab Animal
Resources and all mice were kept under specific pathogen-free conditions. In all
experiments, mice between the ages of 6 and 12 weeks were used.
Preparation and Culture
of T
cells
The axillary, bronchial, mesenteric, aortic, and popliteal lymph nodes and
spleens of mice were teased through a cell screen (Falcon). T cells were isolated by
nylon wool purification as previously described (4) and resuspended in MEM
52
(Gibco-BRL, Grand Island, NY) supplemented with 10% FCS (Gibco-BRL),
essential and nonessential amino acids (Gibco-BRL), 5 x iO M 2mercaptoethanol, sodium pyruvate at 1.16 mg/mi, 2 x iO M glutamine, and
antibiotics (Gentamycin: 47.7 mg/i; Penicillin G: 56.7 mg/i; Streptomycin Sulfate:
99 mg/i). For inhibition studies, cultures were set up in 96-well plates with
200,000 cells/well in a 200 ti volume. Caspase inhibitors (Calbiochem, La Jolla,
CA) at a stock concentration of 10mg/mi were added to the appropriate wells at the
beginning of the culture period. The final concentration for caspase-1, -3, -4, and -
6 inhibitors were 50.2 tM, 181.4 jtM, 199 pM, and 153 jtM, respectively. For the
assay analyzing caspase-3 inhibitors I, II, and III, the final concentrations were 99.9
tM, 298.4 jiM, and 362 tM, respectiveiy. For cell lysate preparation, cultures
were set up in 24-well plates with 1 x
106
or 6 x 106 cells/well in a 1 ml volume.
One or 0.005 p.g/ml IL-4 (Intergen, Purchase, NY), and/or 106 M dexamethasone
were added to the appropriate wells at the beginning of the culture period. For flow
cytometric analysis of caspase-3 activity, or staining with JC- 1 or DiOC6, cells
were set up in 96-well piates with 1 x
i05
cells/well in a 200 t1 volume. For
Annexin V staining and Trypan Blue exclusion analyses, cells were set up in 96well plates with 3 x
io
cells/well in a 200 j.tl volume.
Cell Death Analysis
T cells were isolated from DO 11.10 mice that either had or had not been
injected 48 hours prior with 20 .tg SEB (Sigma, St. Louis, MO) and placed in
53
culture for 24 or 48 hours. Samples of the cells were stained to measure DNA
content as previously described (5). Briefly, the cells were spun and the
supernatant was removed from the wells. A DNA stain, composed of 5 jtg/ml
propidium iodide (P1), 0.3% saponin, 5 mM EDTA, and 50 tg/ml RNAase, was
added to each well. The plates were incubated at room temperature for 30 minutes
in the dark and then kept on ice. The intensity of P1 staining was assayed using
either a Coulter XL flow cytometer (Miami, FL) or a FACSCalibur flow cytometer
(Becton-Dickinson, San Jose, CA). For Annexin V staining, the cells were washed,
spun, and the supernatant was removed from the wells. Five jt1 of Annexin V (BD
Pharmingen, San Diego, CA) was added to each well. The plate was incubated on
ice for 30 minutes in the dark. Approximately 300 pJ of wash buffer was added to
each well and the intensity of the Annexin V staining was assayed using a Coulter
XL flow cytometer (Miami, FL).
Cell Viability Analysis
To assess cell viability for the titration of IL-4 with dexamethasone-treated
cells, counts were performed by incubating an aliquot of each sample with an equal
amount of 0.4% trypan blue stain in PBS followed by counting the non-stained live
cells using a hemacytometer.
54
SDS-PAGE and Western Blot
T cells were isolated and cultured as described above. At designated times,
the cells were lysed in lysis buffer (0.5% NP-40, 20 mM Hepes, pH 7.2, 100 mM
NaC1, 1 mM EDTA) containing 1 mM PMSF, 1 tg/ml aprotinin, and 1 mM sodium
orthovanadate. The nuclei were pelleted for 20 minutes at 14,000rpm (16,000 g) at
4°C. For the anti-PARP and anti-active Caspase-3 Western blots, 10 tl of lysate
was transferred to a new tube. Then, 10 il of 2X sample buffer was added, and the
samples were boiled for 5 minutes. For PARP degradation analysis, lysates and
control lysates (thymocytes cultured with 1 x 106M dexamethasone [SigmaAldrich] only, or dexamethasone plus 0.1 mg/ml of a pan caspase inhibitor
[BaChem]) were separated by SDS-PAGE using a 7.5% mini-gel and blotted onto
an Immobilon PVFD membrane (Millipore, Bedford, MA) using a Trans-blot
apparatus (BioRad, Hercules, CA) for 30 minutes at 20V. For active caspase-3
analysis, lysates and 0.1 tg recombinant active caspase-3 (Calbiochem) were
separated by SDS-PAGE using a 15% mini-gel and blotted onto a PVDF
membrane (Millipore) using a Trans-blot apparatus (BioRad) for 30 minutes at
20V. For the immunoprecipitation study, lysate was transferred to a new tube and
incubated with 0.5 tg anti-phosphotyrosine mAb (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) for 1 hour on ice. Following incubation,
15
p.1 Protein A agarose
(Santa Cruz Biotechnology, Inc.) was added to the samples, then rotated overnight
in a 4°C cold room. Samples were washed 3 times with lysis buffer (supplemented
with fresh protease inhibitors), 10 p.1 of 1X sample buffer was added, and the
55
samples were boiled for 5 minutes. Immunoprecipitated samples or whole cell
lysates were separated by SDS-PAGE using a 7.5% mini-gel and blotted onto an
Immobilon PVDF membrane (Millipore) using a Trans-Blot apparatus (BioRad) for
30 minutes at 20V. Blots were blocked overnight with 1X TBS/5% non-fat dry
milk rotating in a 4°C cold room. The membrane was blotted with either anti-IL-
4Ra (Santa Cruz Biotechnology, Inc.; 0.5 tg/ml in 1X TBS/5% non-fat dry milk),
anti-PARP (Biomol, Plymouth, PA; rabbit polyclonal serum diluted to 1:1000 [or
1:5000] in 1X TBS/5% non-fat dry milk), or anti- Caspase-3 (Calbiochem; rabbit
polyclonal serum diluted to 1:1000 in 1X TBS/5% non-fat dry milk) for 2 hours at
room temperature. The membrane was then washed 3 times for 10 minutes with
lx TBS/0.1% Tween-20 and 2 times for 10 minutes with 1X TBS. The secondary
antibody, goat anti-rabbit Ig conjugated to horseradish peroxidase (BioRad; goat
polyclonal serum diluted to 1:7500 [or 1:2000] in 1X TBS/5% non-fat dry milk),
was added to the blot and incubated for 1.5 hours at room temperature, rotating.
Washes were performed as described above and the bands were detected by
chemiluminescence using the Supersignal kit from Pierce (Rockford, IL).
Analysis
of Caspase-3
activity via flow cytometry
T cells were isolated and cultured as described above. At the appropriate
times, the cells were spun down for 3 minutes and the supernatant was removed
from the wells. Then 50 tl of 10 tM caspase-3 substrate (PhiPhiLux,
Oncolmmunin, Gaithersburg, MD) in RPMI 1640 medium was added to the
56
samples followed by 5 p1 of FCS (Gibco-BRL). The samples were incubated at
37°C with 5% CO2 for 60 minutes with the plate cover removed. The intensity of
the caspase-3 substrate staining was assayed using a FACSCalibur flow cytometer
(Becton-Dickinson).
Analysis of mitochondrial membrane integrity
T cells were isolated and cultured as described above. At appropriate
times, the cells were spun down for 3 minutes and the supernatant was removed
from the wells. Then the cells were stained with either JC- 1 (Molecular Probes,
Eugene, OR; 0.5 tM in DMSO) or DiOC6 (Molecular Probes; 40 nM in DMSO)
by incubating at 37°C with 5% CO2 for 15 minutes. The cells were then washed 3
times with ice cold PBS, brought up to a 400 p1 final volume, and analyzed using a
FACSCalibur flow cytometer (Becton-Dickenson).
RESULTS
Titration of IL-4 with T cells undergoing spontaneous and dexamethasone-induced
apopotosts
Previous studies have demonstrated that IL-4 rescues murine T cells from
spontaneous and activation-induced cell death (1). Based on these findings we
examined the lowest effective dose of IL-4 on T cells undergoing death by neglect
or, dexamethasone-induced T cell death. For the analysis of death by neglect, T
cells from a B10.A mouse were isolated and cultured without or with various
57
concentrations of IL-4 for 21 hours, and the percentage of apoptotic cells was
assessed by propidium iodide staining. For the analysis of dexamethasone-induced
death, T cells from Balb/c mice were isolated and cultured with a physiological
dose of dexamethasone (1 x 1 06 M) in addition to various concentrations of IL-4
overnight. The number of viable cells in each sample was determined by Trypan
Blue exclusion. An Annexin V stain was used concurrently to analyze the percent
of dead cells in each sample.
These analyses revealed that doses of IL-4
0.0 156 .tg/ml maximally
rescued T cells from death by neglect (Fig. 2.la) and doses of IL-4
5 x iO ig/ml
maximally rescued T cells from dexamethasone-induced death (Fig. 2. ib). In the
case of death by neglect, T cells cultured with the above mentioned doses of IL-4
had significantly lower levels of apoptosis compared to untreated cells, where the
highest doses of IL-4 reduced apoptosis from approximately 45% down to
approximately 25%. Approximately 3 to 3.5 million of dexamethasone-treated T
cells treated with doses of IL-4 at or above 5 x iO pg/ml were viable as
determined by Trypan Blue exclusion (Fig. 2.lb). Approximately 1.5 million T
cells were determined viable in response to a lower dose of IL-4 (5 x i05 j.tg/ml).
Further reduction in IL-4 dosage decreased the number of viable T cells down to
approximately 250,000 cells at the lowest doses of IL-4. In analyzing non-viable T
cells, approximately 93% of the T cells treated with doses of IL-4 at or below 5 x
106 tg/ml died as determined by Annexin V staining (Fig. 2. ib). The percentage of
dead cells decreased to approximately 87% when IL-4 dosage was at
5
x105 jig/ml.
a)
so
45
4°
cs--- IL.4
35
c
20
1.
[IL-4] in LgI1il
b)
100
I
+
95
90
85
I
80
iE-0e iZ-0? 10-06 10-050.0001 0.001 001
DEI
[IL_4] in i.g/ni1
Figure 2.1 IL-4 rescues resting T cells from death by neglect and de
induced death in a dose-dependent manner. T cells from a B10.A mouse were
cultured in the presence of IL-4 at the indicated concentrations for 21 hours (a). T
cells from a DO 11.10 mouse were cultured in the presence of 1 x 10-6 M
dexamethasone and IL-4 at the indicated concentrations of IL-4 for 16 hours (b).
The T cells were stained with propidium iodide and the percent apoptosis was
assessed by flow cytometry (a). The numbers of viable T cells was plotted based
on Trypan Blue exclusion counts (open squares) and the percentage of non-viable T
cells was plotted based on Annexin V staining (closed squares) (b). Shown are the
means ± SEMs of the triplicate cultures. These data are from one experiment of
two similar experiments.
Doses of IL-4 at 5 x 10 pg/ml and 0.005 g/ml reduced apoptosis to
approximately 82%.
These finding show that IL-4 rescues T cells from death by neglect and
dexamethasone-induced death in a dose-dependent manner. These experiments
also provide an effective working concentration of IL-4 for further experiments.
59
IL-4
treatment leads to increased tyrosine phosphorylation of
IL-4Ra
Many studies have shown that IL-4 initiates a signaling cascade via the IL-
4R, triggering the tyrosine phosphorylation of the IL-4Ra itself, receptor associated
tyrosine kinases (JAKs), as well as a number of downstream molecules, thus
activating certain pathways (6). To determine if IL-4 treatment in our system leads
to IL-4Ra phosphorylation, indicating the initiation of a signaling cascade, we
examined tyrosine phosphorylation of the IL-4Ra. Activated T cells were isolated
from DO11.1O mice and cultured without or with IL-4 for the indicated times. Cell
lysates were immunoprecipitated with an anti-phosphotyrosine monoclonal
antibody and then separated by SDS-PAGE and blotted with an anti-IL-4Ra
antibody. The blot revealed that the 140 kD a chain becomes tyrosine
phosphorylated upon IL-4 treatment (Fig. 2.2). Phosphorylation was detected as
early as 30 minutes of IL-4 treatment with a densitometry measurement of 1354.66
arbitrary units (Fig. 2.2. lane 3). At 1 hour of IL-4 treatment, phosphorylation
appears to decrease slightly, giving a densitometry reading of 1235.39 units (Fig.
2.2, lane 5). The 140 kD band intensity peaked at approximately 2 hours of
treatment with a densitometry measurement of 3988.26 units (Fig. 2.2, lane 7).
Tyrosine phosphorylation was still detected at 6 hours of IL-4 treatment, resulting
Hours:
0.5
0
2
1
6
WCL
+
-
+
10
11
F.
11-4 Treatment:
-
-
Lane:
1
2
+ -
+ -
+ -
3
5
7
4
6
8
9
Figure 2.2 IL-4 treatment leads to increased tyrosine phosphorylation of the IL4Ra chain on activated T cells. Forty-eight hour SEB-activated I cells from
DO 11.10 mice were cultured in the absence or presence of IL-4 for 0, 0.5, 1, 2, or 6
hours. Cell lysates were immunoprecipitated (or not immunoprecipitated as a
control; designated whole cell lysate [WCL]) with an anti-phosphotyrosine
antibody and separated by SDS-PAGE as described in the Materials and Methods.
The blot was probed with a primary anti-IL-4Ra antibody and secondary goat antirabbit antibody conjugated to HRP. Bands were detected by chemiluminescence.
Densitometry measurements were taken for the 140 kD bands. These data are from
one experiment of at least three similar experiments.
in a densitometry measurement of 1008.89 (Fig. 2.2, lane 9). These data show that
activated T cells treated with IL-4 initiate a signaling cascade as demonstrated by
the tyrosine phosphorylation of the IL-4R a chain.
61
IL-4 inhibits the degradation
of poly
(ADP-ribose) polymerase
In an attempt to uncover the mechanism(s) of IL-4-mediated rescue of T
cells from apoptosis, we next examined caspase activity in cells cultured with or
without IL-4 by assessing the degradation of poly (ADP-ribose) polymerase
(PARP). PARP is a 116 kD protein associated with DNA repair that can be
cleaved into 85 kD and 24 kD fragments during the execution of apoptosis by
caspases-3, -7, and -9(7). As controls, thymocytes from a DO11.1O mouse were
cultured for 6 hours with 1 x 106 M dexamethasone to induce apoptosis in the
absence or presence of a pan-caspase inhibitor. Cell lysates were separated by
SDS-PAGE and then immunoblotted with an anti-PARP antibody that recognizes
the intact 116 kD band as well as the 85 kD degradation product. The control lanes
revealed that with dexamethasone treatment, the 116 kD PARP was degraded, as
indicated by the presence of the 85 kD degradation product (Fig. 2.3a, lane 10, 16).
However, caspase inhibition using a pan caspase inhibitor prevented the
degradation of PARP, as seen by the absence of the 85 kD degradation product.
Densitometric analysis of the PARP standards in the top panel of Figure 2.3a
revealed a 1.83 fold increase in the 116 kD band in lysate from cells treated with
both dexamethasone and a pan caspase inhibitor (Fig. 2.3a, lane 10) compared to
lysate from cells treated with dexamethasone only (Fig. 2.3 a, lane 11). Analysis of
the PARP standard in the bottom panel of Figure 2.3a revealed a 1.70 fold increase
63
Resting T cells were isolated from Balb/c mice and cultured under the
indicated treatments for various times. Quantitation of the intensity of the 116 kD
band was determined by densitometry. The 116 kD PARP protein was detected in
all 1 hour treated samples. Lane 1 (no treatment), lane 4 (IL-4 treatment), and lane
5 (IL-4 and dexamethasone treatment) showed a 1.11 fold increase, a 0.9 fold
reduction, and a 0.71 fold reduction, respectively, in the 116 kD band compared to
lane 3 (dexamethasone treatment). At 6 hours, IL-4 treatment (Fig. 2.3a, lane 8)
resulted a preservation of the 116 kD band with a 3.24 fold increase of the 116 kD
band compared to dexamethasone treatment (Fig. 2.3a, lane 7). Treatment with
both IL-4 and dexamethasone resulted in a 4.70 fold increase of the 116 kD band
(Fig. 2.3a, lane 9) compared to dexamethasone treatment (Fig. 2.3a, lane 7). At 12
hours, dexamethasone treatment resulted in the full degradation of PARP, as seen
by a complete absence of the 116 kD band as well as the detection of a small
amount of the 85 kD degradation product (Fig. 2.3a, lane 13). However, samples
treated for 12 hours with IL-4 showed a preservation of the 116 kD PARP band
with a densitometry reading of 2780.08 arbitrary units (Fig. 2.3a, lane 14).
Samples treated for 12 hours with both IL-4 and dexamethasone also showed a
preservation of the 116 kD band with a densitometry reading of 2956.05 arbitrary
units (Fig. 2.3a, lane 15). At 24 hours, IL-4 treatment continued to maintain the
116 kD band, giving a densitometry reading of 2241.27 (Fig. 2.3a, lane 20), while
treatment with both dexamethasone and IL-4 also resulted in the preservation of the
116 kD band, giving a densitometry reading of 940.42 (Fig. 2.3a, lane 21). Similar
to 12 hours, no 116 kD band was detected in 24 hour dexamethasone-treated cells
(Fig. 2.3a, lane 19).
Thus far, these data show that upon treatment with IL-4 the degradation of
PARP was inhibited, indicating that IL-4-initiated signaling resulted in an
obstruction of the pathway leading to PARP degradation.
,,
CP
t = 48hrs
t = 24hrs
I
IL-4 treatment:
Lane:
-
1
2
+
-
+
3
4
5
Figure 2.3b IL-4 inhibits the degradation of PARP in activated T cells. Forty-eight
hour SEB-activated T cells from DO 11.10 mice were cultured in the absence or
presence of IL-4 for 48 hours. Cell lysates were separated by SDS-PAGE and
blotted, as described in the Materials and Methods, and probed with a primary
polyclonal rabbit anti-PARP antibody and a secondary goat anti-rabbit antibody
conjugated to HRP. Bands were detected by chemiluminescence. Densitometry
measurements were taken for the 116 kD band. These data are from one
experiment of three similar experiments.
65
To observe PARP degradation in T cells undergoing AICD, 48 hour in vivo
SEB-activated T cells were isolated from DO 11.10 mice and cultured without or
with IL-4 for the indicated times. Upon IL-4 treatment for 24 and 48 hours, the
116 kD band was maintained (Fig. 2.3b, lane 3, 5). Treatment with IL-4 for 24
hours resulted in a 2.95 fold increase in the 116 kD band compared to no treatment,
as determined by densitometry. At 48 hours, untreated cells underwent full
degradation of the 116 kD band (Fig. 2.3b lane 4) while IL-4 treatment resulted in a
densitometry reading of 989.44arbitrary units (Fig. 2.3b, lane 5). These data show
that in activated T cells, IL-4 treatment inhibits PARP degradation, indicating that
IL-4-initiated signaling resulted in an obstruction of the pathway leading to PARP
degradation. Collectively, we have shown this to be the case in three apoptotic
scenarios: death by neglect (Fig. 2.3a), steroid-induced death (Fig. 2.3a), and AICD
(Fig. 2.3b).
Caspase-3, -4, and -6 are involved in the apoptosis of resting and activated T cells
PARP degradation occurred in T cells undergoing various forms of
apoptosis, showing that caspase activity is present in T cells in this model. In order
to identify specifically which caspases may be involved in T cell death, caspase
inhibitors with specificity for caspase-1, -3, -4, or -6 were added to cultures of
activated or resting T cells. Forty-eight hour SEB-activated T cells were isolated
from DO 11.10 mice and cultured without or with each inhibitor for 0, 24, or 48
hours. DNA content was assessed by propidium iodide staining and percent
apoptosis was determined. Figure 2.4 shows that inhibition of caspase-6
substantially decreased apoptosis, while inhibition of caspase-4 decreased the
levels of apoptosis to a lesser extent. Inhibition of caspase- 1 had a minimal effect
on apoptosis, while the inhibition of caspase-3 revealed virtually no effect on levels
of apoptosis. The control samples exhibited up to approximately 90% apoptosis by
48 hours, while the addition of caspase-4 or -6 inhibitor reduced apoptosis to
approximately 65% and 45%, respectively, after 48 hours of treatment.
100
-
C,,
25
0
24
48
Hours in culture
Figure 2.4 Caspase-4 and -6 are involved in AICD and death by neglect of T cells.
Forty-eight hour SEB-activated T cells from DO11.10 mice were cultured in the
presence of DMSO (vehicle control; open squares), caspase- 1 inhibitor (AcYVAD-CHO; 50.2 tM, open diamonds), caspase-3 inhibitor (Ac-DEVD-CHO;
181.4 jtM, closed squares), caspase-4 inhibitor (Ac-LEVD-CHO; 199 tM, open
triangles), or caspase-6 inhibitor (Z-VEID-FMK; 153 tM, open circles). The
percent of apoptosis was determined by propidium iodide staining. Shown are the
means ± SEMs of the triplicate cultures. Results are representative of three
independent experiments.
These results suggest that caspase-6, and to a lesser extent caspase-4, are
involved in AICD of T cells. It was unexpected to find virtually no inhibitory
effect of caspase-3 inhibition on levels of apoptosis since caspase-3 is heavily
involved in PARP degradation (7).
Our results using a caspase-3 inhibitor suggested that caspase-3 may not be
involved in the death pathway of T cells in this model. Because of this surprising
result, we tested the effects of two other caspase-3 inhibitors, one especially
constructed to be cell permeable, and another with an attached hydrophobic
fluoromethylketone (FMK) moiety. Resting T cells were isolated from DO11.1O
mice and cultured without or with the indicated caspase-3 inhibitors for 24 hours.
Figure 2.5a shows that caspase-3 inhibition by the FMK-bound caspase-3 inhibitor
II drastically reduced the levels of apoptosis after 24 hours, from 75% (with vehicle
treatment) to approximately 4%. Consistent with previous observations, caspase-3
inhibitors I and III tested did not appear to reduce the percentage of T cells
undergoing apoptosis compared to vehicle treatment, resulting in values similar to
vehicle treatment at approximately 75% apoptosis. These results emphasize the
need for caution when using reagents identified as performing specific functions
(variations exist that may not be obvious from the title given to the reagent).
In evaluating activated T cells, 48 hour SEB-activated T cells were isolated
from DO11.1O mice and cultured without or with the indicated caspase-3 inhibitors
for 24 hours. Figure 2.5b shows results similar to Figure 2.5a, where vehicle
treatment resulted in approximately 50% apoptosis while the FMK-bound
a)
100
75
50
25
vehicle
b)
C-3 inh.I
C-3
inh.II
C-3 inh.III
100
75
50
0
vehicle
C-3 inh.I
C-3 inh. II C-3 inh.III
Figure 2.5 Caspase-3 is involved in AICD and death by neglect of T cells. T cells
from a B10.A mouse (a) or 48 hour SEB-activated T cells from a DO 11.10 mouse
(b) were cultured in the presence of DMSO (vehicle control), or caspase-3 inhibitor
I (Ac-DEVD-CHO; 99.9 mM), caspase-3 inhibitor II (Ac-DEVD-FMK; 298.4
mM), or caspase-3 inhibitor III (Ac-DEVD-CMK; 362 mM) for 24 hours. The
percent of apoptosis was determined by propidium iodide staining. Shown are the
means ± SEMs of the triplicate cultures. Results from (a) and (b) are from one and
two independent experiments, respectively.
caspase-3 inhibitor II reduced apoptosis to approximately 5% (Fig. 2.5b). Again,
caspase3 inhibitors I and III had failed to reduce apoptosis. T cells treated with
caspase-3 inhibitor I showed higher levels of apoptosis (approximately 75%),
compared to the 50% value observed with vehicle treatment, while fates of I cells
treated with caspase-3 inhibitor III were almost identical to those treated with
vehicle, (50% apoptosis). The hydrophobic FMK moiety appeared to be key in
permitting a caspase-3 inhibitor to enter mouse T cells while the COH or
chioromethyketone (CMK) moieties appeared to interfere with entrance into the T
cells.
These results demonstrate that caspase-3, -6, and to a lesser extent, -4, play
a role in AICD, and caspase-3 also plays a role in death by neglect.
IL-4 blocks the activation of caspase-3
To determine whether or not IL-4 has the ability to modulate caspase-3
activity, we examined the effect IL-4 exerts on caspase-3 activation in T cells
undergoing death by neglect or dexamethasone-induced death. Cell lysates were
separated by SDS-PAGE and then immunoblotted with an anti-caspase-3 antibody
specific for the 17 kD and 11 kD active subunits of the caspase-3 enzyme. The
control lane containing 0.1 tg of recombinant active caspase-3 subunit (Fig. 2.6a,
lane 5) reveals a prominent 17 kD band.
Resting T cells were isolated from B 10.A mice and cultured with the
treatments indicated for 4 hours. The blot shows that dexamethasone treatment
results in the activation and detection of active caspase-3 (Fig. 2.6a, lane 2).
However, IL-4 treatment in addition to dexamethasone decreases the amount of
71
substrate molecule contains two rhodamine molecules that theoretically are
quenched and do not fluoresce (although the precleaved substrate is not completely
quenched). When the substrate is cleaved, the two rhodamine molecules separate
from each other and the substrate is excited at 505 nm and emits at 530 nm.
Resting T cells were isolated from B10.A mice and cultured with the
treatments indicated for 26 hours. Caspase-3 activity was assessed by measuring
the amount of processed substrate by flow cytometry. Figure 2.6b reveals that after
26 hours of dexamethasone treatment, approximately 99% of the cells contained
active caspase-3 and approximately 3% of these cells were viable as determined by
forward scatter (larger cells representing healthy, viable cells, and smaller cells
representing dying, apoptotic cells). However, the presence of active caspase-3
within cells treated with IL-4 in addition to dexamethasone was substantially less,
decreasing from 99% with dexamethasone only to approximately 67%.
Furthermore, IL-4 treatment in addition to dexamethasone treatment increased the
percentage of viable cells to approximately 29%. Approximately 14% of IL-4treated cells contained active caspase-3 compared to approximately 44% of
untreated cells. Nearly 79% of IL-4-treated cells appeared viable while only
approximately 51% of untreated cells were determined viable as measured by
forward scatter.
Q
JIII2U.O
Control
(- substrate)
11
,.,. I
i
hi1
hi1
hi1
1T
a4
thSflt
Wias
j
FiQun'
I
No treatment
"l
I4.r;a
4rwdS.Sw
O.41t121h?fl
Dexamethasone
(1 x 10.6 M)
,
i
D4WJ
p
0111211w004
.,IuIa,q
I
A
Ar',..T
a Ca P
Ill 1O
,i.v,.opasnt.
'1'
(0.5 tg/ml)
11
I
J
F..,wd aal'
auIu.aFr.,
IL-4ldex
lkWdpaflfl.
I
I
hi
Treatment
Control (- substrate)
No treatment
Dexamethasone
(lx 106M)
IL-4 (0.5 p.g/ml)
IL-4/dex
b
obi I
Percent of cells with
cleaved substrate
Percent of viable cells
0.03
43.74
99.15
50.99
53.08
2.09
13.74
66.82
79.33
29.28
Figure 2.6b IL-4 blocks caspase-3 activation during death by neglect or
dexamethasone-induced death, as detected in intact T cells. Resting T cells from
B 10.A mice were cultured in the absence or presence of 0.05 tg/m1 IL-4, 1 x 10.6
M dexamethasone, or both IL-4 and dexamethasone for 26 hours. The cells were
stained with the fluorogenic caspase-3 substrate as described in the Materials and
Methods. The amount of cleaved caspase-3 substrate was analyzed by flow
cytometry. Data are from one experiment of two similar experiments.
These findings demonstrate that IL-4 blocks the activation of caspase-3 in T
cells undergoing both death by neglect and dexamethasone-induced death.
73
IL-4 maintains the mitochondrial membrane potential ( W,,,) in resting T cells
undergoing death by neglect or dexamethasone-induced death.
Once we demonstrated that IL-4 blocks caspase-3 activation in T cells
undergoing various forms of cell death, we examined the mechanism(s) by which
this phenomenon occurs. During apoptosis, mitochondrial integrity is often
disrupted prior to caspase activation (7). Furthermore, dissipation of /.Wm results in
the release of pro-apoptotic factors from the mitochondria, including cytochrome c.
When complexed with Apaf-1, dATP, and procaspase-9, cytochrome c forms the
apoptosome, which, in turn, activates caspase-3, initiating apoptosis. To determine
whether IL-4 prevents caspase-3 activation by maintaining A'{'m, two sensors of
mitochondrial membrane potential, JC- 1 and DiOC6, were used to assess
mitochondrial integrity in response to various treatments. When the membrane
potential is high, or maintained, iC-i forms aggregates that emit at approximately
590 nm. conversely, when the membrane potential is low, or disrupted, JC- 1
returns to its monomeric form, resulting in a shift in emission down to 527 nm. On
the other hand, DiOC6 is able to accumulate within the matrix of mitochondria
when membrane potential is intact. However, as the membrane potential
dissipates, DiOC6 levels within the mitochondria decrease.
Resting T cells were isolated from B i0.A mice and cultured with the
treatments indicated for 0, 4, 8, and 24 hours. Maintenance of LWm was assessed
by analyzing the presence of aggregates and monomers within T cells stained with
iC-i. Figure 2.7a reveals that after 4 hours of treatment, virtually no change in
1'm could be measured. Results were similar at 8 hours. However, after 24 hours,
74
approximately 75% of T cells treated with dexamethasone contained monomers.
IL-4 treatment in addition to dexamethasone reduced monomer formation, from
approximately 75% down to approximately 25%. Moreover, approximately 25%
of untreated T cells cultured for 24 hours contained monomers, and IL-4 treatment
reduced this to approximately 10%.
In analyzing aggregate formation, approximately 25% of dexamethasonetreated T cells contained aggregates, while approximately 15% of untreated T cells
or T cells treated with both IL-4 and dexamethasone contained aggregates; 10% of
IL-4 treated T cells contained aggregates.
The accumulation of T cells is shown in Figure 2.7b. Over 75% of T cells
stained at the beginning of the time course exhibited high accumulation of DiOC6
(data not shown). At 4 hours, virtually no change in
AWm
could be detected as seen
by DiOC6 accumulation. However, at 8 hours dexamethasone reduced the
accumuulation of DiOC6 from approximately 75% to approximately 50%, and at
24, virtually no DiOC6 accumulation could be detected. When IL-4 was combined
with dexamethasone, approximately 10% of cells still exhibited DiOC6
accumulation at 24 hours. Also, at 24 hours there was almost no difference in
DiOC6 accumulation in untreated cells or cells treated with IL-4 alone.
Collectively, these findings suggest that IL-4 is able to prevent
dexamethasone-induced dissipation of
as determined by monomer formation
75
100
100
75
75
50
50
_25
25
cr
-
oj IL.1
I__:_r____.l ...i.
Enothing dex IL-41L-4/dex
1I.....j
II......
I
nothing dex
IL-41L-4/dex
ç 100
aggregates
1
D
50
monomers
25
o
C.:.:.]
I
I:::J
I
:::
I
i:::
I
nothing dex IL-41L-4/dex
Figure 2.7a IL-4 delays the dissipation of mitochondrial membrane potential in
resting T cells. T cell from a Bl0.A mouse were cultured in the presence or
absence of 0.05 pg/m1 of IL-4, 1 x 106 M dexamethasone, or both IL-4 and
dexamethasone. The A'Fm was assessed at 4, 8, and 24 hours by JC-1 staining.
The percentage of cells containing either JC- 1 monomers or aggregates was
determined by flow cytometry. Shown are the means ± SEMs of the triplicate
cultures. These data are from one experiment of four similar experiments.
with JC-1 staining and by DiOC6 accumulation. IL-4 is also able to prevent the
dissipation
ofzW1,,
in a small percentage of T cells undergoing death by neglect as
demonstrated by JC-1 staining.
76
L)
C
100
00
75
75
50
50
25
0
no treatment
x
LL-4
IL-4/dex
0
no treatment
x
IL-4
IL-4/dex
100
=
r'1
1
50
25
0
no treatment
dcx
IL-4
IL-4/dex
Figure 2.7b IL-4 maintains mitochondrial membrane potential in resting T cells.
T cells from a B l0.A mouse were cultured in the presence or absence of 0.05 tg/ml
IL-4, 1 x 106 M dexamethasone, or both IL-4 and dexamethasone. The AWm was
assessed at 4, 8, and 24 hours by DiOC6 staining. The percent of cells containing
DiOC6 was determined by flow cytometry. Shown are the means ± SEMs of the
triplicate cultures. These data are from one experiment of two similar experiments.
DISCUSSION
IL-4 rescues cells from apoptosis in a number of different cellular systems
(1, 8, 9). This chapter represents our attempt to uncover the mechanism(s) of IL-4mediated survival of T cells undergoing death by neglect, dexamethasone-induced
77
death, or AICD. Specifically, we examined the effects IL-4 might exert on events
occurring within several apoptotic pathways. Our data reveal that IL-4 indeed
rescues T cells undergoing spontaneous or dexamethasone-induced death in a dose-
dependent manner. Initiation of the IL-4-mediated survival signal involves tyrosine
phosphorylation of the IL-4Ra chain in activated T cells. We also found that IL-4
administered to resting and activated T cells blocks the degradation of the 116 kD
PARP protein. This demonstrated an interruption of caspase activity in response to
a death signal during AICD and spontaneous death of T cells because PARP
degradation is a reflection of caspase activity. Moreover, we showed, using
Western blot and flow cytometric analysis, that IL-4 treatment blocks caspase-3
activation in T cells undergoing spontaneous or dexamethasone-induced death.
In keeping with previous studies outlining a subsequent phosphorylation of
IL-4Ra upon ligation of IL-4 or IL-13 (10), our system demonstrates an IL-4-
dependent phosphorylation of IL-4Ra. Signal transduction involves the
recruitment and binding of downstream signaling molecules to a membrane-
proximal receptor. Often, conserved regions, located on both the recruited
molecule and the membrane-bound receptor, facilitate the binding of signaling
molecules. Depending on the system and signal, these regions may include Srchomology 2 (SH2) domains, pleckstrin homology (PH) domains, and
phosphotyrosine binding (PTB) domains, among others. In this case, the IL-4Rcz
possesses a number of tyrosine residues that, when phosphorylated, recruit
molecules with PTB domains able to bind phosphopeptides on the receptor. Thus,
IL-4-mediated IL-4Ra chain phosphorylation initiates and permits a signaling
cascade necessary to carry out IL-4-mediated cell survival. Although the events
downstream of IL-4Ra phosphorylation leading to cell survival have yet to be
identified in our system, our findings demonstrate that a classic signaling
mechanism may be employed to connect extracellular signals, in this case IL-4, to
intracellular molecules involved in a signaling cascade.
The data in Figures 2.4, 2.5, and 2.6 collectively demonstrate that caspases
are critical for AICD, spontaneous death, and dexamethasone-induced death. in
particular, Figure 2.4 and 2.5 show that caspase-3, -6, and to a lesser extent,
caspase-4, play major roles in apoptosis of T cells. As mentioned previously,
PARP is a major substrate of caspase-3. However, PARP can also be degraded by
caspase-6, -7 and -9 (7). Thus the PARP degradation experiment demonstrates that
IL-4 can block the activation of PARP-degrading caspases in T cells undergoing
spontaneous or dexamethasone-induced death. These data, along with data
obtained in Figure 2.5 and 2.6, collectively show that IL-4 blocks caspase-3
activation. Due to the fact that caspase-3 has other apoptotic functions, IL-4mediated blockage of caspase-3 activation has more implications than the blockage
of PARP degradation.
Once caspase-3 is active, it can, in addition to degrading substrates such as
the PARP protein, activate other caspases, including caspase-6 and -2 (11, 12).
Activated caspase-6 can, in addition to degrading substrates such as nuclear
proteins, cytoplasmic proteins, and focal adhesion kinase (7), can activate caspase-
79
8 and -10 (13). The processing of upstream caspases by a downstream effector
caspase is an indication of an amplification loop, driving the processing of more
caspases as apoptosis progresses. Thus, in addition to our demonstration that IL-4
prevents the degradation of PARP (presumably through blocking the activation of
PARP-degrading caspases like caspase-3), IL-4 may also be hindering the caspase-
3-mediated activation of caspase-6. The importance of this is seen in Figure 2.4,
since the inhibition of caspase-6 results in a drastic reduction in AICD. As stated
above, caspase-6 can drive an amplification of caspase activation by processing the
initiator caspases-8 and -10. In our system, then, this amplification may be central
in the breakdown of T cells undergoing AICD.
The data in Figure 2.4 also show that caspase-4 is involved in AICD of T
cells in our model. Though little is known about caspase-4, it has been reported to
activate caspase-1 (14). Both caspase-1 and -4 belong to the ICE subfamily of
caspases and are included in Group I with other caspases that are involved in
inflammation and cytokine maturation (15). Our data demonstrate that caspase-4
may be an activator of caspase- 1 in T cells undergoing AICD. Inhibition of
caspase- 1 can block AICD to some degree, yet inhibition of caspase-4 decreases
apoptosis to a further degree, indicating that caspase-4 may lie upstream of
caspase-1 in this apoptotic pathway. However, additional studies would be needed
to elucidate the role of caspase-1 and -4 in AICD.
The data in Figure 2.6 support the hypothesis that caspase-3 activation is
modulated by IL-4. Resting T cells undergoing spontaneous or dexamethasone-
induced death activate caspase-3 during the apoptotic process, and this is shown to
be blocked by IL-4. These data help to solidify our hypothesis that IL-4 modulates
the activation of caspases, specifically the activation of the executioner caspase-3.
However, it remains unclear how IL-4 mediates this process. Therefore, the events
upstream of caspase activation must be examined. Two major ways in which
caspases are activated include cell surface death receptor activation and activation
in response to mitochondrial alterations. The former involves the initiator caspase8 and direct or indirect subsequent activation of caspase-3. The latter, a
mitochondrial-mediated caspase activation pathway, involves the association of the
initiator caspase-9 with Apaf- 1 and cytochrome c, forming the apoptosome, which
drives caspase-9 activation. Activated caspase-9 in turn activates caspase-3. We
focused on mitochondrial integrity and hypothesized that IL-4 may maintain the
mitochondria, thus preventing the release of cytochrome c and subsequent initiation
of a caspase activation cascade.
The data in Figure 2.7 demonstrate that IL-4 treatment, in addition to
dexamethasone treatment, blocks the dissipation of the L'Pm of the mitochondria as
indicated by the probes JC-1 and DiOC6. Only at later time points tested (i.e., after
8 hours) does AWm dissipation become evident. However, we have shown in our
system that PARP is degraded in T cells as early as 6 hours after dexamethasone
treatment, indicating the activation of caspases: yet no Wm dissipation was
detected until 8 hours. Two interpretations are possible. Activation of other
EII
proteases that cleave PARP may occur in the early stages of apoptosis, or initiation
of a caspase activation cascade may occur prior to Z'I'm dissipation.
Indeed, some studies have shown that cytochrome c release and caspase
activation can occur before disruption of the LWm, implying that the permeability
transition pore complex (PTPC) may open after apoptosome-mediated caspase
activation (16, 17). In our system, caspase activation may occur before
dissipation since it is uncertain exactly when zWm began to dissipate.
A'Pm
IL-4
blocked
dexamethasone-induced PARP degradation as early as 6 hours indicating caspase
activity, while LPm dissipation was detected after 4 hours but before or at 8 hours,
making the sequence of events difficult to discern since we did not assess 1m at 6
hours. Nonetheless, it is possible that IL-4 may be preventing the activation of
caspases in a manner independent of LWm maintenance, since we detected caspase
dissipation. However, once caspases become activated and
activation before
amplification is initiated, a survival effect IL-4 may exert includes delaying tWm,
thus delaying a further amplification of caspase activation by other apoptotic
proteins released by
disrupted mitochondria. Our data suggest another IL-4-
mediated survival mechanism independent of the mitochondria, and centered on
blockage of caspase activation.
The data presented illustrate events involved in various forms of T cell
apoptosis. Caspase-3,
-4,
and -6 are involved in AICD, spontaneous death, and
dexamethasone-induced death of T cells.
IL-4
initiates a signaling cascade via the
IL-4Ra that leads to the blockage of caspase activation as shown by the prevention
of both PARP degradation and caspase-3 activation. Furthermore, IL-4 delays the
dissipation associated with apoptosis, although this effect is not drastic,
suggesting that mitochondrial maintenance is neither the primary mechanism nor
the only mechanism in IL-4-mediated survival. Therefore, IL-4 may block various
forms of T cell apoptosis through the maintenance of mitochondrial integrity and
through a yet to be detailed mechanism which blocks caspase activation.
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Nicholson, D. W., A. Au, N. A. Thornberry, J. P. Vaillancourt, C.
K. Ding, M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, Y. A. Lazebnik, and et
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Telford, W. G., L. E. King, and P. J. Fraker. 1992. Comparative
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Gessner, A., and M. Rollinghoff. 2000. Biologic functions and
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Mammalian caspases: structure, activation, substrates, and functions during
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Ueno, H., K. Sasaki, H. Honda, T. Nakamoto, T. Yamagata, K.
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Smerz-Bertling, C., and A. Duschl. 1995. Both interleukin 4 and
interleukin 13 induce tyrosine phosphorylation of the 140-kDa subunit of the
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Van de Craen, M., W. Declercq, I. Van den brande, W. Fiers, and P.
Vandenabeele. 1999. The proteolytic procaspase activation network: an in vitro
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Bratton, S. B., M. MacFarlane, K. Cain, and G. M. Cohen. 2000.
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Slee, E. A., M. T. Haste, R. M. Kiuck, B. B. Wolf, C. A. Casiano, D.
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D. Newmeyer, H. G. Wang, J. C. Reed, D. W. Nicholson, E. S. Alnemri, D. R.
Green, and S. J. Martin. 1999. Ordering the cytochrome c-initiated caspase cascade:
hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9dependent manner. J Cell Biol 144:28].
Faucheu, C., A. Diu, A. W. Chan, A. M. Blanchet, C. Miossec, F.
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Herve, V. Collard-Dutilleul, Y. Gu, R. A. Aldape, J. A. Lippke, and et al. 1995. A
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Nicholson, D. W. 1999. Caspase structure, proteolytic substrates,
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Bossy-Wetzel, E., D. D. Newmeyer, and D. R. Green. 1998.
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17.
EXPLORING IL-4-MEDIATED MECHANISMS IN MODULATING CASPASE
ACTIVITY
Cathleen M. Moscibrocki, Chen-Yao Chen, and Anthony T. Vella
Department of Microbiology
Oregon State University, Corvallis, OR 9733 1-3804 U.S.A.
INTRODUCTION
This chapter represents a compilation of attempts at uncovering the
mechanism involved in IL-4-mediated T cell survival. In review, apoptosisinduced mitochondrial changes trigger caspase activation, namely caspase-9,
leading to caspase-3 activation. We have previously shown that IL-4 blocks the
activation of caspase-3 in T cells undergoing AICD, death by neglect, and
dexamethasone-induced death. However, the mechanism by which
IL-4 prevents caspase-3 activation and mitochondrial deterioration has yet to be
understood.
We explored events within the apoptotic signaling pathway and the IL-4initiated signaling pathway in an attempt to pinpoint the mechanism(s) of IL-4mediated survival. We focused on the phosphatidylinositol-3 kinase (P1-3 K)
pathway, which has been implicated in survival in many systems (1). Finally, we
hypothesized that dexamethasone-induced death involved the proapoptotic Bcl-2
family protein Bax, and that IL-4 may exert its survival effects by regulating Bax
activity.
A previous study demonstrated that IL-4 prevents the downmodulation of a
Bax-associated protein (2). The downmodulation of the novel protein P16 resulted
in the release of a functional Bax molecule that could locate to the mitochondna
and disrupt the mitochondrial membrane potential. Thus, IL-4 may partly protect
dexamethasone-treated T cells by blocking the B ax-induced fall in mitochondrial
membrane potential, which, as mentioned previously, would block subsequent
caspase activation.
To determine whether P1-3 K was involved in IL-4-mediated survival, we
treated resting T cells with two different P1-3 K inhibitors, wortmannin or
LY294002 (Lily compound) and revealed that IL-4 mediated survival may not be
dependent on P1-3 K activity. In addition, we performed experiments using Bax -Imice to determine if Bax was indeed involved in dexamethasone-induced T cell
death, and if IL-4-mediated survival involved the regulation of Bax. These
experiments revealed that T cells still underwent dexamethasone induced cell death
and IL-4 still rescued T cells from death by neglect and dexamethasone-induced
cell death, regardless of the presence of Bax. We also performed cellular
fractionation to analyze the movement of cytochrome c. This involved separating
the mitochondria from the cytosol of T cells to detect changes in cytochrome c
levels within each fraction in response to IL-4 treatment.
Lastly, we developed a fluorometric assay to eventually measure and
compare caspase activity in IL-4-treated T cells and untreated T cells in our model.
We initially established parameters for the fluorometric assay by conducting a
series of control experiments using recombinant active caspase-3 and a caspase-3-
specific substrate. We then detected caspase-3 activity in resting T cells
undergoing dexamethasone-induced death.
These P1-3 K and Bax studies provide evidence for the idea that IL-4 does
not exert its survival effects through P1-3 K activity or through the modulation of
Bax activity.
MATERIALS AND METHODS
Mice
B 1O.A mice were purchased from the National Cancer Institute (Frederick,
MD) and Balb/c, C57BL/6, and C57BL/6Baxtm
(Bax-deficient) mice were
purchased from the Jackson Laboratory (Bar Harbor, ME). The mice were
maintained in our animal facility under specific pathogen-free conditions. In all
experiments, mice between the ages of 6 and 12 weeks were used.
Preparation and Culture of T cells
The axillary, bronchial, mesenteric, aortic, and popliteal lymph nodes and
spleen of the mouse were teased through a cell screen. T cells were isolated by
nylon wool purification as previously described (3) and resuspended in MEM
(Gibco-BRL, Grand Island, NY) supplemented with 10% FCS (Gibco-BRL),
essential and nonessential amino acids (Gibco-BRL), 5 x iO M 2mercaptoethanol, sodium pyruvate at 1.16 mg/mi, 2 x 1 o M glutamine, and
antibiotics (Gentamycin: 47.7 mg/i; Penicillin G: 56.7 mg/I; Streptomycin Sulfate:
99 mg/i). For preparation of cell lysates, cultures were set up in 24-well plates with
1 x 106 cells/well in a 1 ml volume. For flow cytometric analysis of caspase-3
activity, P1 staining, or staining with JC- 1 or DiOC6 of T cells were set up in 96-
well plates with 1 x
i05
cells/well in a 200 tl volume. For P1-3 K inhibition
experiments, cells were set up in 96-well plates with 2 x
i05
cells/well in a 200 j.tl
volume. For all T cell cultures, 0.05 pjg/mI IL-4 (Intergen, Purchase, NY), andlor
106 M dexamethasone were added to the appropriate wells at the beginning of the
culture period.
Analysis of Cell Death
For P1-3 K inhibition and Bax-/- experiments, T cells were isolated and
cultured as described above. Samples of the cells were stained to measure DNA
content as previously described (4). Briefly, the plate was spun and the supernatant
was removed from the wells. A DNA stain, comprised of 5 ig/m1 propidium
iodide (P1), 0.3% saponin, 5 mM EDTA, and 50 j.tg/ml RNAase, was added to each
well. The plates were incubated at room temperature for 30 minutes in the dark and
then stored on ice. The intensity
of P1 staining was assayed using a FACSCalibur
flow cytometer (Becton-Dickinson, San Jose, CA)
Fluorometric Analysis of Caspase Activity
One hundred units of recombinant active caspase-3 (Calbiochem, La Jolla,
Calit') was added to appropriate wells with buffer (100 mM HEPES, 10% sucrose,
10 mM DTI', 0.5 mM EDTA, pH 7.5) in the absence or presence of 2 nM of
91
caspase-3 inhibitor III (Ac-DEVD-CMK), caspase-4 inhibitor I (Ac-LEVD-CHO),
caspase-6 inhibitor II (Ac-VEID-CHO), or caspase-8 inhibitor I (cell permeable;
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-AIa-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-
Glu-Thr-Asp-CHO) (Calbiochem). The plates were incubated at room temperature
for 2 hours and then 40 tM (or concentrations indicated in the substrate titration
experiments) of a caspase-3 substrate V (fluorogenic; MCA-VDQMDGWK[DNP] -NH2; MCA-Val-Asp-Gln-Met-Asp-Gly-Trp-Lys-(DNP)-NH2) (Calbiochem)
was added. The plates were incubated at room temperature for an additional 90
minutes in the dark and then analyzed using a fluorometer (Perkin-Elmer, Norwalk,
CT) at 325nm1392nm. For lysate titration experiments, various volumes of T cell
lysates were added to appropriate wells with buffer. For caspase inhibitor titration
experiments, various concentrations of caspase-3, -6, -8, and -9 inhibitors were also
added. All plates were incubated at room temperature for 2 hours (if inhibitors
were added) and then 40 jiM of a caspase-3-specific substrate was added. The
plates were incubated at room temperature for various times in the dark and then
analyzed using a fluorometer (Perkin-Elmer, Norwalk, CT). For experiments
utilizing lysates as well as recombinant active caspase-3, 10 t1 of lysate containing
an equivalent of 1 x 106 cells was added to appropriate wells in addition to 100
units of recombinant active caspase-3. The lysate-enzyme mixture was incubated
at room temperature for 20 minutes and then a caspase-3-specific substrate was
added. The plate was incubated for 2 hours at room temperature in the dark and
then analyzed using a fluorometer.
92
Analysis of Caspase-3 activity via flow cytometry
T cells were isolated and cultured as described above. At the appropriate
times, the cells were spun down for 3 minutes and the supernatant was removed
from the wells. Then 50
tl
of caspase-3 substrate (PhiPhiLux, Oncolmmunin,
Gaithersburg, MD) was added to the 1 x iO cells in culture, followed by 5
tl
of
FCS (Gibco-BRL). The samples were incubated at 37°C with 5% CO2 for 60
minutes, uncovered. The intensity of the caspase-3 substrate staining was assayed
using a FACSCalibur flow cytometer (Becton-Dickinson).
Analysis of mitochondrial membrane integrity
T cells were isolated and cultured as described above. At appropriate times,
the cells were spun down for 3 minutes and the supernatant was removed from the
wells. Then the cells were stained with either JC-1 (Molecular Probes, Eugene,
OR; 1 pM in DMSO) or DiOC6 (Molecular Probes; 40 nM in DMSO). Cells
cultured at 1 x
i05
cells! well in a 200 jfl volume were stained with JC-1 or DiOC6
by incubation at 37°C with 5% CO2 for 15 minutes. The cells were then washed 3
times with ice cold PBS, brought up to a 400 pJ final volume with PBS, and
analyzed using a FACSCalibur flow cytometer (Becton-Dickenson).
P1-3 Kinase inhibition
T cells were isolated and cultured as described above. Indicated doses of
the Lily compound (BioMol, Plymouth, PA) or wortmannin (Sigma, St. Louis,
93
MO) were added at the beginning of the culture period, and fresh wortmannin was
added every 3 hours throughout the 21 hour culture period.
Cellular fractionation
Cellular fractionation was conducted as previously described (5). At
designated times, the cells were lysed on ice for 30 minutes in buffer A (20 mM
Hepes, pH 8.0, 10 mM KC1, 1.5 mM MgC1,, 1 mM EDTA, 1 mM EGTA, 250 mM
sucrose) containing freshly added protease inhibitors. The cells were then spun
through multiple centrifugation steps and the resulting supernant was designated
the cytosolic fraction. The pellet was resuspended in buffer A and further
centrifuged for 20 minutes at 13,000rpm (15,493g) at 4°C. The pellet was
resuspended in buffer B (10 mM Tris- Cl, pH 7.4, 150 mM NaC1, 5 mM EDTA)
with freshly added protease inhibitors and placed on ice for 30 minutes. The lysate
was spun for 30 minutes at 13,000 rpm (15,493g) at 4°C. The resulting supernatant
was designated the mitochondrial protein extract.
SDS-PAGE and Western Blot
Mitochondrial and cytosolic fractions of T cells were isolated as described
above. Fifty jig of protein from each fraction was separated by SDS-PAGE using a
15% mini-gel and blotted onto an Immobilon PVDF membrane (Millipore,
Bedford, MA) using a Trans-Blot apparatus (BioRad, Hercules, CA). Blots were
blocked overnight in a 4°C room with 1X TBS/5% non-fat dried milk. The
membrane was blotted with an anti-cytochrome c antibody (Santa Cruz
Biotechnology; 1 tg/ml in 1X TBS/5% non-fat dried milk) or an anti-cytochrome c
oxidase antibody (Molecular Probes; 3 tg/mI in 1X TBS/5% non-fat dried milk) as
a negative control for 2 hours at room temperature. The membrane was then
washed 3 times with 1X TBS/0.l% Tween-20 for 10 minutes and 2 times with 1X
TBS for 10 minutes. The secondary antibody, goat anti-rabbit Ig conjugated to
horseradish peroxidase (BioRad; 1:3000 dilution in 1X TBS/5% non-fat dried milk)
was added to the blot and incubated for 1.5 hours at room temperature. Washes
were performed as described above and the protein bands were detected by
chemiluminescence using Supersignal kit (Pierce, Rockford, IL).
RESULTS
Recombinant active caspase-3 cleaves afluorometric caspase-3 substrate
In an attempt to solidify our findings that caspases are involved in the
apoptotic signaling pathways of T cells, we explored another method of detecting
by fluorometry caspase activity in T cells undergoing various forms of apoptosis.
We aimed to establish controls by utilizing a recombinant active caspase-3 enzyme
to illustrate that caspase-3 indeed cleaves its cognate substrate, and to demonstrate
the effectiveness and specificity of caspase inhibitors used in these experiments.
125
100
75
.
E
©
;©
50
0
60
120
180
240
Time (in minutes)
Figure 3.1 Recombinant active caspase-3 cleaves a caspase-3-specific substrate.
An inhibitor to caspase-3 (open circles), caspase-4 (open diamonds), caspase-6
(open triangles), or caspase-8 (open squares with crosshairs) was added at a
concentration of 2 nM to wells containing 100 units of recombinant caspase-3 and
incubated up to 4 hours. At 2 hours, 40 .tM of a caspase-3-specific substrate was
added and cleaved substrate was assessed by a fluorometer at the timepoints
indicated. Shown are the means ± SEMs of the triplicate cultures. These data are
from one experiment of five similar experiments.
A caspase-3 specific substrate was added to wells containing recombinant active
caspase-3 enzyme with or without various caspase inhibitors (Fig. 3.1). The
amount of cleaved caspase-3 substrate was determined by fluorometry. The
addition of caspase-3 inhibitor blocked recombinant caspase-3 activity as seen by a
marked reduction in fluorometric signal at 120, 180, and 240 minutes (Fig. 3.1).
Furthermore, other caspase inhibitors, namely caspase-4, -6, and -8 inhibitors, did
not inhibit recombinant active caspase-3 (Fig. 3.1), demonstrating the specificity of
the inhibitors and the effectiveness of this assay.
These findings demonstrate that recombinant active caspase-3 cleaves the
caspase-3 substrate specifically, and the inhibitors function to inhibit caspases
specifically, as well.
Cells treated with IL-4 do not contain endogenous inhibitors of
recombinant active caspase-3
In order to determine if T cells treated with IL-4 promoted the expression or
activation of endogenous agents that may inhibit caspase activation or activity, we
treated T cell lysates from cells cultured in the presence or absence of IL-4 with or
without 100 units of recombinant active caspase-3 and analyzed caspase-3 activity
via fluorometric assay. Lysates from T cells cultured in the absence or presence of
IL-4 exhibited low levels of cleaved caspase-3-specific substrate and there was no
detectable difference between IL-4-treated and untreated cells (Fig. 3.2). However,
when active caspase-3 was added to lysates from IL-4-treated or untreated cells, the
amount of cleaved caspase-3-specific substrate increased substantially, from less
than 100 units detected in samples without the recombinant enzyme to over 700
units detected in samples treated with the recombinant enzyme (Fig. 3.2).
Conversely, no difference in units could be detected between IL-4-treated or
untreated cells with added recombinant enzyme. Lastly, the recombinant active
caspase-3 enzyme alone reveals approximately 400 units detected.
97
lysis buffer only
comb.C-3
omb.C-3
lysate from IL-4-treated cells
lysate from untreated cells
0
200
400
600
800
Fluorometric Units
Figure 3.2 The activity of recombinant caspase-3 is not blocked by T cell lysate.
Resting T cells from B 10.A mice were cultured in the presence of 0.005 tg/mI of
IL-4 for 4 hours and cell lysate was generated as described in the Materials and
Methods. One hundred units of recombinant active caspase-3 was added to the cell
lysate and incubated at room temperature for 20 minutes. A caspase-3-specific
substrate at 40 .tM was added and caspase-3 activity was assessed 120 minutes
later by fluorometry. These data are from one experiment and treatments were
performed singly.
Results shown in Figures 3.1 and 3.2 demonstrate that recombinant enzyme
is able to cleave the caspase-3 substrate. However, when the recombinant enzyme
is added to T cell lysates from IL-4-treated or untreated cells, the activity increased
substantially, indicating a potential synergistic effect between the already active
enzyme and components within the lysates. However, the substantial increase of
activity was the same in both IL-4 treated and untreated cells. These results also
suggest that the lysates do not contain any endogenous inhibitors of recombinant
caspase-3. Moreover, these findings show there is no detectable difference in
caspase-3 activity in cells treated with or without IL-4, suggesting that the
threshold of detection of this assay when applied to T cells in our model is too low.
This experiment shows that IL-4 treatment does not promote the expression or
activation of endogenous inhibitors of recombinant active caspase-3.
Optimization
of conditions
for analyzing T cell lysates
To determine the optimum volume of cell lysate and the optimum caspase-3
substrate concentration, we conducted a series of titration experiments. Resting T
cells were cultured for 4 hours with a physiological dose of dexamethasone to
induce apoptosis, and lysates were generated as described in the Materials and
Methods. Various volumes of lysates were tested using a fixed concentration of a
caspase-3-specific substrate.
The data from Figure 3.3a reveal 20 jil of lysate gave optimal readings.
However, lysate volumes as low as 5 tl provided sufficient readings. A 40 tM
concentration of a caspase-3-specific substrate gave optimal readings when used
with 5 tl of lysate (or an equivalence of 5 x iO cells) (Fig. 3.3b). Thus, 5 jtl or
more and a substrate concentration of 40 jiM provide sufficient detection of
fluorescence that can be applied to further experiments using T cell lysates.
Various caspase inhibitors fail to inhibit caspases in T cell lysates
We found it necessary to use caspase inhibitors as controls for the
fluorometric assay, effectively establishing which caspases cleave the fluorogenic
caspase-3 substrate in our model. Although the substrate is termed a caspase-3
substrate, sensitivity to other proteases was a possibility. Thus, inhibiting various
caspases would aid in identifying which were responsible for processing the
caspase-3 substrate. We hypothesized that inhibiting caspases-6-, -8, and -9 would
not affect substrate processing, while inhibiting caspase-3 would affect the amount
of processed substrate, thus defining caspase-3 as the key caspase in substrate
cleavage.
To optimize caspase inhibitor concentrations for use in the fluorometric
assay, resting T cells were cultured for 4 hours with a physiological dose of
dexamethasone and lysates were generated as described previously. When 10 tl of
lysate and 40 p.M of caspase-3 substrate were used, all concentrations of caspase-3,
-6, -8, and -9 inhibitors were ineffective in inhibiting casapse-3 activity (Fig. 3.4).
A repeat analysis involving the concurrent titration of caspase-1, -3, -4, -6, -8, and
-9 inhibitors revealed similar results (data not shown). These findings demonstrate
that all caspase inhibitors tested at all examined concentrations were generally
ineffective. Notably, we were unable to effectively inhibit caspase-3 at the time
points tested.
100
a)
300250200150100-
50-
60
b)
E
I
120
180
240
120
180
240
125-
.E ioo
7550
250
60
Time in minutes
Figure 3.3 Titration of cell lysate and caspase-3 substrate. T cells from B10.A
mice were cultured in the presence of 1 x 106 M dexamethasone for 4 hours and
cell lysate was generated as described in the Materials and Methods. a) Caspase-3
substrate at a concentration of 40 pM was added to a 96-well plate in combination
with either 20 tl (2 x 106 cells equivalent; open squares), 10 jil (1 x 106 cells
equivalent; open diamonds), 5 tl (5 x i05 cells equivalent; open circles), or 2.5 t1
(2.5 x i05 cells equivalent; open triangles), 1.25 t1 (1.25 x i05 cells equivalent;
open squares with crosshairs), 0.625 tl (6.25 x iO cells equivalent; open diamonds
with crosshairs), or 0 jfl (open circles with crosshairs) of lysate. b) Five 1.11 of
lysate (as determinted by results in (a)) was added to a 96-well plate in combination
with 40 pM (open squares), 20 pM (open diamonds), 10 j.tM (open circles), 5 p.M
(open triangles), 1 p.M (open squares with crosshairs), or 0.1 p.M (open diamonds
with crosshairs) of caspase-3 substrate. Caspase-3 activity was assessed at 90, 180,
and 240 minutes by fluorometry. Each treatment was performed in duplicate.
These data are from one experiment of four similar experiments.
101
41--- tOn.n
300
250
200
150
100
50
300
250
200
150
100
50
0
0
IE-05 0.0001 0.001
0.01
0.1
IE-05 0.0001 0.001
10
[C-6
[C-3 inh] in pmol
E
300
250
200
300
50
00
150
100
50
150
0.01
0.1
10
1
inhj in pmol
100
50
0
0
IE-05 0.0001 0.001
[C-8
0.01
0.1
inh] in pmol
10
IE-05 0.0001 0.001
0.01
[C-9 inh]
0.1
1
10
in pmol
Figure 3.4 Titration of caspase-3, -6, -8, and -9 inhibitors. Resting T cells from
B 10.A mice were isolated and cultured with 1 x 106 M dexamethasone for 4 hours.
Cell lysate was generated as described in the Materials and Methods. Ten p.! of
lysate was added to a 96-well plate in combination with various concentrations of
caspase inhibitors and incubated for 2 hours at room temperature. Caspase-3
substrate at a concentration of 40 p.M was added and caspase-3 activity was
measured at 90 and 120 minutes by fluorometry. These data are from one
experiment of at least five similar experiments. Each treatment was performed
singly.
Inhibition of P1-3 K has no effect on IL-4-mediated survival of T cells
Many studies have shown that the phospholipid products of P1-3 K promote
survival downstream of extracellular stimuli (6). IL-4 stimulation results in the
association of P1-3 K with the IL-4Ra chain (7). A key point illustrated in studies
using the 32D cell line showed wortmannin or Lily compound to partially inhibit
102
IL-4-mediated survival (8). Thus, P1-3 K may play a crucial role in the survival
signaling pathway initiated by IL-4. To test this hypothesis, T cells were isolated
from B10.A mice and cultured under indicated treatments for 21 hours in the
presence or absence of various concentrations of wortmannin or Lily compound.
Fresh wortmannin was administered 3 hours post-culture to eliminate the
possibility of skewed results due to breakdown of the labile inhibitor. DNA
content and percent apoptosis was measured by propidium iodide staining.
Regardless of the presence of P1-3 K inhibitors, no significant difference in
apoptosis could be observed between treatment groups (Fig. 3.5). The experiment
was repeated by isolating T cells from B10.A mice and culturing the cells for 21
hours with or without IL-4 and 100 nM of wortmannin. Fresh wortmannin was
administered every 3 hours. These results show that IL-4-mediated reduction in
apoptosis was unchanged upon wortmannin treatment (Fig. 3.6). Nevertheless, in
the presence of wortmannin the percent of apoptotic cells increased, supporting the
efficaciousness of the dose used. Also, the notion exists that survival of some cells
is dependent on P1-3 K activity (8). In conclusion, these findings indicate that
resting T cells undergoing spontaneous death were still rescued by IL-4, and active
P1-3 K was not required for this event.
103
100
100
75
75
50
50
25
25
0
dex
nothing
i
IL-4 IL-4/dex
0
nothing
100
1 uM
dex
IL-4 IL-4/dex
100 nM Wortmannin
75
50
50
25
25
0
nothing
100
dex
IL-4 IL-4/dex
5 uM Lily Compound
0
nothing
100
75
75
50
50
25
25
0
nothing
dex
IL-4 IL-4/dex
dex
IL-4 IL-4/dex
500 nM Lily Compound
0
nothing
dex
IL-4 IL-4/dex
Figure 3.5 IL-4 rescues T cells from death by neglect and dexamethasone-induced
apoptosis when P1-3 K is inhibited. T cells from B 10.A mice were isolated and
cultured for 21 hours with or without 0.05 tg/m1 of IL-4, 1 x 106 M
dexamethasone, or both IL-4 and dexamethasone. Lily compound at either 5 pM or
500 nM was added at the beginning of the culture period. Wortmannin, at either 1
or 100 nM, was added at the beginning of the culture period and at t=3 hours
(due to short half-life of Wortmannin). The T cells were stained with propidium
iodide and the percent apoptosis was assessed by flow cytometry. Each treatment
was performed in duplicate. These data are from one experiment of two similar
experiments.
104
100
nt
ly (DMSO)
Tortmannin
50
25
0
-IL-4
+IL-4
Figure 3.6 IL-4 rescues T cells from death by neglect in the absence of active
P1-3 K. T cells from B 10.A mice were isolated and cultured without or with 0.05
ig/ml of IL-4. At the beginning of the culture period and for every 3 hours up to
21 hours, fresh Wortmannin at 100 nM was added to the culture. The I cells were
stained with propidium iodide and the percent apoptosis was assessed by flow
cytometry. Each treatment was performed in duplicate. These data are from one
experiment of two similar experiments.
IL-4 rescues Bax deficient T cells from apoptosis
A number of BcI-2 family member proteins possess apoptotic functions,
including the ability to disrupt mitochondrial integrity. The activation of the proapoptotic protein Bax involves subcellular translocation and dimerization (9).
After a death stimulus Bax translocates to the mitochondria to become a
105
homodimeric integral membrane protein (9). Conversely, anti-apoptotic proteins
such as Bcl-2 or Bcl-XL prevent the activation of Bax following a death signal (9).
One study has revealed that IL-4 can modulate Bax activity by blocking the down-
modulation of a novel Bax-associated protein, P16 (2). Upon dissociation from
P16, Bax becomes an active protein possessing pro-apoptotic functions (2). IL-4
treatment may, however, prevent this dissociation from occurring. Thus, we aimed
to determine if the dexamethasone-induced signaling pathway involved the Bax
protein in our model and if so, to determine if IL-4 mediates a survival signal by
modulating Bax activity.
Resting T cells from wildtype C57B116 or Bax deficient mice were isolated
and cultured as described in the Materials and Methods. Approximately 5% of
cells from both C57B16 and Bax deficient mice were apoptotic at time 0 as
determined by propidium iodide staining (Fig. 3.7). Eighteen hours later, both
wildtype and Bax -I- cells exhibited similar percentages of apoptosis in response to
no treatment, dexamethasone, or dexamethasone and IL-4 (Fig. 3.7). However,
dissimilar results were between wildtype and Bax -I- cells treated with IL-4 for 18
hours. Approximately 85% of wildtype and B ax-deficient cells underwent
apoptosis in response to dexamethasone treatment, while IL-4 treatment in addition
to dexamethasone reduced apoptosis to approximately 60% for wildtype and 45%
for Bax deficient cells.
106
100
75
50
25
100
C57B16
Bax-/-
0
75
C57B16
50
25
0
no treatment dex
IL-4
IL-4&dex
Figure 3.7 IL-4 rescues T cells from death by neglect and dexamethasone-induced
death in the presence or absence of Bax. T cells from C57B16 or Bax-deficient
mice were isolated and cultured without or with 0.005 p.g/ml of IL-4, 1 x 106 M
dexamethasone, or both IL-4 and dexamethasone. At time 0 and t=18 hours T cells
were stained with propidium iodide and the percent apoptosis was assessed by flow
cytometry. Shown are the means ± SEMs of the triplicate cultures. These data are
from three separate experiments combined.
As for untreated cells, approximately 40% of untreated wildtype cells were
apoptotic compared to 30% of untreated Bax-deficient cells. Lastly, approximately
45% of IL-4 treated wildtype cells were apoptotic, significantly higher than IL-4
107
treated Bax-deficient cells, of which only 10% were apoptotic. Collectively, these
results suggest that regardless of the presence or absence of the proapoptotic Bax
protein, T cells were able to undergo dexamethasone induced apoptosis, and IL-4
treatment was able to rescue T cells from both dexamethasone induced death and
death by neglect. However, IL-4-mediated rescue was significantly higher in Baxdeficient cells from dying by neglect.
As a control for mitochondrial staining, sample wells were treated with or
without menadione, an agent that disrupts the
"m The membrane potential was
determined by JC-1 staining. Samples were also analyzed for apoptosis via
propidium iodide staining and for caspase-3 activity via flow cytometric analysis.
JC-1 staining of wildtype or Bax deficient cells treated with or without menadione
at the beginning of the culture period reveals cells from both wildtype and Bax -Icontaining a high percentage of monomers upon menadione treatment, indicating
an equivalent dissipation of the
LPm
(Fig. 3.8a). Conversely, a high percentage of
untreated wildtype or Bax deficient cells contained aggregates at the beginning of
the culture period, indicating a maintained
(Fig. 3.8a). At 18 hours, over 75%
of both wildtype and Bax deficient T cells contained monomers in response to
dexamethasone treatment while IL-4 treatment in addition to dexamethasone
treatment reduced the percentage of cells containing monomers to approximately
50% in wildtype cells and 40% in Bax deficient cells (Fig. 3.8b). Furthermore,
approximately 25% of both wildtype and Bax deficient cells undergoing death by
a)
ioo.
-
o......
75- :::::::
..
100
Menadione
50
75
50
25
25
C57B16
Bax-/-
C57B16
Bax-/aggregates
b)ioo
El
100
C57B16
50
0II...1
Ir.-.
no treatment
dex
E1
IL-4
monomers
75
T T
°
::ili: ±
25
:::j
IL-4&dex
no treatment
dex
IL-4
IL-4&dex
Figure 3.8 IL-4 prevents mitochondrial membrane dissipation in the presence or
absence of Bax. T cells from wildtype or Bax-deficient mice were isolated and
cultured with or without 0.005 tg/ml of IL-4, 1 x 106 M dexamethasone, or both
IL-4 and dexamethasone for 18 hours (b). At time 0, cells were left untreated on
ice or treated with menadione for 0.5 hours (a). At both time points, T cells were
stained with JC- 1 and the percent of cells containing either the monomeric or
aggregate form of JC-1 was assessed by flow cytometry. Shown are the means ±
SEMs of the triplicate cultures. These data are from three separate experiments
combined.
i[,i,J
neglect contained monomers, significantly fewer compared to dexamethasone
treatment at approximately 85%. IL-4 treatment reduced the percentage of
monomers in spontaneously dying cells to approximately 15% in wildtype cells and
10% in Bax-deficient cells. These findings reveal that IL-4 is able to prevent the
dissipation of the
lWm
in some wildtype and Bax-deficient cells undergoing death
by neglect or dexamethasone-induced death. Furthermore, the IL-4-mediated
prevention of
LWm
dissipation in cells undergoing both forms of apoptosis seemed
to be more effective in Bax-deficient T cells.
In analyzing aggregate formation, approximately 75% of untreated T cells
or IL-4 treated T cells from both wildtype and Bax-deficient animals appeared to
contain aggregates, while approximately 10% of dexamethasone-treated T cells
from both wildtype and Bax-deficient animals contained aggregates; approximately
50% of T cells from both animals treated with dexamethasone and IL-4 contained
aggregates. JC- 1 analysis revealed maintenance of mitochondrial membrane
potential in response to IL-4 treatment, regardless of the presence of the Bax
protein.
In analyzing caspase-3 activity, fluorometric analysis of caspase-3 activity
in both wildtype and B ax-deficient cells at time 0 was approximately 5%, which
are background levels (Fig. 3.9). Both wildtype and Bax-deficient cells revealed
similar levels of caspase-3 activity after 18 hours in culture. Approximately 95%
of dexamethasone treated cells contained active caspase-3, while IL-4 treatment
110
100
75
50
c-)
.- C,,
0
=
C57B16
Bax-/-
Cl,
100
L)
D
50
C57B16
25
0
no treatment
dex
IL-4
IL-4&dex
Figure 3.9 IL-4 blocks caspase-3 activity is the presence or absence of Bax. T
cells from C57B116 or Bax-deficient mice were isolated and cultured without or
with 0.005 tg/m1 of IL-4, 1 x 106 M dexamethasone, or both IL-4 and
dexamethasone. T cells were stained at time 0 or t= 18 hours with 50 l of a cell
permeable fluorometric caspase-3 substrate at a stock concentration of 10 iM and
caspase-3 activity was assessed by flow cytometry. Shown are the means ± SEMs
of the triplicate cultures. These data are from three separate experiments combined.
111
in addition to dexamethasone treatment reduced caspase activity to approximately
75% in wildtype cells and 70% in Bax -I- cells. Approximately 45% of untreated
cells from both groups exhibited caspase-3 activity compared to IL-4 treatment that
resulted in a reduction to approximately 35% in wildtype and no reduction in Bax-
deficient cells. These results show that IL-4 is able to block caspase-3 activity in a
comparable fashion in cells that do or do not express the pro-apoptotic Bax protein.
However, our previous results show that IL-4 treatment appeared to rescue a
slightly higher percentage of Bax-deficient cells from death by neglect compared to
wildtype cells, yet these results indicate that IL-4 appeared to have little effect in
blocking the caspase-3 activity in Bax-deficient cells undergoing death by neglect.
Considering the entire set of experiments, it appears IL-4 is able to rescue
Bax-deficient T cells from death by neglect and dexamethasone-induced death.
Interestlingly, more Bax-deficient cells were rescued from apoptosis by IL-4
compared to wildtype cells, and IL-4 treatment in Bax-deficient cells resulted in a
decrease in caspase-3 activity in cells undergoing steroid-induced death.
Nonetheless, IL-4 generally reduced caspase activity and delayed the mitochondrial
changes associated with cell death.
Analysis of cytochrome c levels in mitochondrial and cytosolic fractions
of T
cells
Cytochrome c exists as a mitochondrial inner membrane protein that is
important in oxidative phosphorylation and electron transport. Upon receiving a
112
death trigger, mitochondria may release cytochrome c into the cytosol, where it
can, in combination with Apaf- 1 and dATP or ATP, activate the initiator caspase-9.
To determine whether IL-4 prevents cytochrome c relocation, resulting in a
caspase activation cascade in T cells undergoing spontaneous death, mitochondrial
and cytosolic fractions from cells cultured in the presence or absence of IL-4 were
analyzed for cytochrome c content via Western blot.
O.1ig
cytochiome c
IL-4 Treatment:
-
+
I
I
unto
fiBctlon
I
+
I
cytosolic
fraction
Figure 3.10 Tracking the translocation of cytochrome c in T cells treated with or
without IL-4. Resting T cells were cultured in the absence or presence of IL-4 for
24 hours. Cells were lysed and fractionated as described in the Materials and
Methods. Resulting lysates were separated by SDS-PAGE and blotted, as
described in the Materials and Methods, with a primary anti-cytochrome c antibody
and a secondary goat anti-rabbit antibody conjugated to HRP. Bands were detected
by chemiluminescence.
113
T cells were isolated from Balb/c mice and cultured with or without
IL-4
for 24
hours. Mitochondrial and cytosolic fractions were prepared as described in the
Materials and Methods. Equivalent fractions were separated by SDS-PAGE and
then blotted with a polyclonal anti-cytochrome c antibody or an anti-cytochrome c
oxidase antibody as a negative control.
As seen in Figure 3.10, no cytochrome c was observed in either the
mitochondrial or cytosolic fractions of T cells treated with or without
IL-4,
although control cytochrome c appeared on the Western blot. Thus, we could not
define the effect IL-4 may have exerted on the movement of cytochrome c in cells
undergoing death by neglect through this experiment.
DISCUSSION
This chapter reports a closer analysis of the events that occur upstream and
downstream of mitochondrial alterations in response to apoptosis. In addition to
attempting to design another assay that could aid in the detection of caspase activity
via fluorometry, we also tried to pinpoint a "location" along the apoptotic pathway
where
IL-4
may exert its survival effects, thus revealing the mechanism of IL-4-
mediated T cell survival.
In further demonstrating the caspase-3 activity in T cells undergoing
dexamethasone-induced apoptosis, we utilized a fluorometric assay. The control
experiments using recombinant active caspase-3 instead of cell lysate, with or
114
without various caspase inhibitors, defined the processing of the fluorometric
casapse-3 substrate as a specific event. It solidified the expectation that indeed the
active caspase-3 enzyme cleaves its cognate substrate, and the use of a caspase-3
inhibitor prevents this process. This could ultimately be applied to our model when
utilizing T cell lysates; we aimed at analyzing the endogenous active caspase-3 in
resting T cells treated with dexamethasone, to eventually compare the effects of a
caspase-3 inhibitor against IL-4. However, the introduction of lysates into the
assay in combination with various caspase inhibitors, including the caspase-3
inhibitor, resulted in a failure of all tested inhibitors to prevent caspase-3 substrate
processing, although we did observe a slight inhibition with caspase-3 inhibitor, as
expected. These results demonstrate that, for reasons we have not yet elucidated,
the inhibitors, most importantly the caspase-3 inhibitor, were not totally ineffective
in preventing the processing of the caspase-3 specific substrate, despite extensive
titrations of the inhibitors. In our earlier studies, lysates were analyzed for PARP
degradation from cells cultured with a caspase-6 inhibitor or a caspase-3 inhibitor
identical to the caspase-3 inhibitor we used in the fluorometric assay (data not
shown). These results showed a decrease in PARP degradation, indicating the
inhibition of caspase activity. However, the addition of these inhibitors to cell
lysates, rather than intact cells in culture, appears to exert an effect that renders the
inhibitors ineffective, as demonstrated by this fluorometric assay.
Our findings also suggest the T cells in our model do not contain
endogenous inhibitors of caspase-3 when treated in the presence or absence of IL-4
115
for 4 hours in culture. In Figure 3.2, recombinant active caspase-3 added to cell
lysates remained active and able to process a fluorometric caspase-3-specific
substrate. The amount of cleaved substrate was substantially more in cell lysates
with added recombinant caspase-3, demonstrating a possible amplification between
the recombinant active caspase-3 and endogenous procaspase-3 possibly activated
by the recombinant enzyme in trans. Moreover, in the absence or presence of IL-4
the cultured cells showed no difference in measured caspase-3 activity, suggesting
that this method of analysis was below the threshold of endogenous active caspase-
3 detection. It is possible that endogenous inhibitors of caspases may indeed be
present, but an overwhelming amount of recombinant caspase-3 masked any
possible effect exerted by these endogenous inhibitors. Therefore, it is difficult to
determine whether untreated or IL-4-treated cells express inhibitors of caspases,
specifically inhibitors of apoptosis (TAPs), which, when upregulated, can temper
the caspase cascade.
An area of interest in relation to IL-4-mediated survival includes the P1-3 K
pathway. Many studies show P1-3 K as part of a survival pathway initiated by
various stimuli, including IL-4 (7). Although our data demonstrate that IL-4 is able
to rescue cells from death by neglect and dexamethasone-induced death when P1-3
K is inhibited, the data leave room for discrepancy. While performing the
experiments, we did not include controls to show that the inhibitors were fully
functional in our hands. Thus it is impossible to determine whether our results
demonstrated that IL-4 can rescue T cells from apoptosis in a manner independent
116
of active P1-3 K, or that P1-3 K was simply not inhibited due to ineffective
wormannin or Lily compound. Nevertheless, the dose used was effective in other
published reports (8).
Keeping this in mind, we feel confident that IL-4 mediates survival via a P13 K-independent mechanism, or P1-3 K is not required for IL-4-mediated survival.
Alongside our interest in P1-3 K was the downstream effector Protein Kinase B
(PKB) or Akt. Akt is activated shortly after P1-3 K activation, and has been
implicated in the survival of many cell systems (10). Despite our finding that IL-4mediated survival does not appear to require P1-3 K activation, the possibility that
Akt may play a role in IL-4-mediated survival still exists. Akt can be activated by
a P1-3 K-independent mechanism, involving a calmodulin-dependent enzyme CaM-
KK (11). Further studies need to be conducted to reveal whether Akt is in fact
involved in IL-4-mediated survival.
We aimed to explore events upstream of death-associated mitochondrial
alterations, including the involvement of the pro-apoptotic Bcl-2 family protein
Bax. Our idea that dexamethasone-induced death may exist as either a Baxdependent or B ax-independent event promted us to explore IL-4 and its possible
role in rescuing T cells from dexamethasone-induced death by modulating Bax
activity. He et al. (1998) showed that IL-4 treatment of T lymphocytes resulted in
blocking the down-modulation of a novel Bax-associated protein, namely P16,
which, when down-modulated, would liberate Bax and allow it to exert its pro-
apoptotic effects. We asked whether this mechanism could be applied to our
117
model, specifically in response to dexamethasone treatment. Studies using Bax
deficient mice would definitively answer the question whether dexamethasone-
induced death is Bax-dependent or Bax-independent. Therefore, the question is
whether IL-4 blocks dexamethsone-induced death by altering death-induced Bax
activity. Our findings show that Bax-deficient T cells still undergo
dexamethasone-induced death, demonstrating that this apoptotic pathway does not
require Bax, or, that in the absence of Bax, compensatory pathways are activated.
IL-4 rescued B ax-deficient cells from dexamethasone-induced death and death by
neglect to a better extent than wildtype cells, as measured by P1 staining,
demonstrating that IL-4-mediated rescue of dexamethasone-treated cells is exerted
in a Bax-independent fashion. These results also suggest that if indeed alternate
dexamethasone-induced death pathways were activated in the absence of Bax, IL-4
could accommodate alternate death pathways by exerting its survival effects.
Interestingly, IL-4 treatment reduces apoptosis of resting T cells to a further
degree in Bax deficient cells compared to wildtype cells, suggesting that Bax
deficient cells are more receptive to IL-4-mediated signaling. Further complicating
the issue, however, is the fact that IL-4 did not block caspase-3 activation in Baxdeficient cells undergoing death by neglect after 18 hours of culture, as shown in
Figure 3.9. These particular findings indicate an IL-4-mediated survival effect
downstream of caspase-3 activation in Bax-deficient cells. It may be possible that
at later time points lAPs are upregulated in response to IL-4 treatment, thus
118
inhibiting apoptosis by quelching the caspase cascade through distinct lAPmediated mechanisms.
Furthermore, Figure 3.9 shows a virtual absence of IL-4-mediated rescue in
wildtype cells undergoing death by neglect. The results are part of at least three
independent experiments and a substantial error margin exists which may mask the
actual decrease in spontaneous death in response to IL-4. Although it is not as
apparent in this set of experiments, we have repeatedly shown IL-4 to rescue
resting cells from death by neglect.
As mentioned previously, Bax and other pro-apooptotic proteins can insert
themselves into the mitochondria where they become integral membrane proteins,
possibly forming selective channels that facilitate the exit of cytochrome c (12). In
our analysis of the change in
in wildtype or Bax-deficient cells treated with
IL-4 we found that IL-4 was not able to significantly block the dissipation of the
in T cells undergoing death by neglect. This indicates that death by neglect
may not involve mitochondrial disruption. Significant mitochondrial disruption
still occurred in wildtype or Bax-deficient cells, the latter possibly through the
action of other pro-apoptotic proteins such as Bak or Bid, and IL-4 tempers this
event. Studies involving cell-free systems or recombinant Bak or Bid have
demonstrated that these proteins can insert themselves into lipid membranes (12).
Furthermore, the Bcl-2 proteins have considerable homology to membrane-
insertable bacterial toxins, such as the diptheria toxin (13). Thus, as seen by our
119
results, IL-4 can substantially delay mitochondrial alterations triggered by
dexamethasone and instigated by pro-apoptotic protein(s) other than Bax.
Considered together, the data presented thus far illustrate IL-4-mediated
survival as a part of a Bax-independent pathway that involves the blockage of
dexamethasone- and death by neglect-triggered mitochondrial membrane
disruption, apoptosis, and (not including Bax-deficient cells undergoing
spontaneous death) caspase-3 activation. Furthermore, IL-4 may exert its effects in
a P1-3 K-independent manner, suggesting that signaling is taking place along
alternate survival pathways that may or may not involve downstream second
messengers of P1-3 K, namely Akt. The mechanism by which IL-4 maintains the
remains unclear, and it appears that in our system caspases are activated in a
fashion independent of, or more likely, upstream of mitochondrial disruption, since
we observed PARP degradation hours before we detected the dissipation of
A'Pm.
Nonetheless, IL-4 preserves mitochondrial integrity, indicating the complexity of
apoptotic processes and the possibility that IL-4-mediated survival is exerted
through a number of mechanisms that ultimately lead to the blockage of caspase-3
activation and/or caspase-3 activity. Further examination is necessary to provide
insight into the exact mechanism(s) of IL-4-mediated survival.
120
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122
CLOSING THOUGHTS AND CONCLUSIONS
IL-4 has been identified as a multi-functional cytokine that plays a central
role in influencing the immune response by controlling the differentiation of naïve
CD4 T cells into Th2 helper cells (1). IL-4 mediates a number of events; Ig class
switching to IgE, the induction of CD23 expression, enhancement of MHC II
expression, the activation of IL-4-responsive genes, as well as the proliferation of B
and T cells (1). There is relatively little information concerning IL-4 as a survival
factor. So far, IL-4 has been shown to protect 32D myeloid cells against apoptotis
through two different pathways, one of which involves the IL-4Ra-associated
signaling molecule insulin receptor substrate-i (IRS-i) (2). IL-4 was shown to
rescue human peripheral T cells from apoptosis and induce Bcl-2 and
Bcl-xL
activation (3). Moreover, in resting and activated mouse T cells, IL-4 decreases
death by neglect and AICD in addition to maintaining the levels of Bcl-2 and Bcl-
x (4), (5). The latter data served as a foundation for our attempt to uncover the
signaling mechanisms involved in IL-4-mediated survival.
Nakayama et al. (1995) demonstrated that IL-4 still rescued lymphocytes
derived from Bcl-2 deficient mice, indicating that Bcl-2 is not required for IL-4-
mediated survival. Taking this into consideration, we did not conduct any studies
using Bcl-2 -I- animals. Nonetheless, keeping in mind the preliminary data
showing IL-4 to maintain the levels of anti-apoptotic proteins Bcl-2 and Bcl-XL, we
focused our attention on the mitochondria since these proteins may modulate
survival through mitochondrial interaction. After establishing the fact that IL-4
123
modulates caspase-3 activation, we hypothesized that, by maintaining levels of
anti-apoptotic proteins, IL-4 blocked mitochondrial membrane disruption.
Despite the data that we have gathered thus far, a precise and detailed
mechanism has yet to be revealed. We have demonstrated clearly that IL-4 blocks
caspase-3 activation in peripheral mouse T cells undergoing spontaneous,
dexamethasone-induced or activation-induced cell death. This fact confirms an IL-
4-mediated uncoupling of the apoptotic signaling pathways. However, our
difficulty has been to decipher the IL-4-mediated events that prevent caspase-3
activation and subsequent death. The IL-4-mediated events that do exist serve as
potential sources of information toward an understanding of the fundamental
requirements of T cell survival, as well as how T cells that are required to undergo
AICD instead avoid this process and survive, thus posing obvious detrimental
effects to the host. Also, uncovering the mechanism(s) by which IL-4-treated T
cells avoid dexamethasone-induced death would allow for an improvement of
widely used glucocorticoid-based therapies.
Our findings that illustrate IL-4 as a survival factor for T cells may lead to
crucial insights toward treating illnesses, such as asthma, that affect a significant
proportion of the population. Despite an increase in the understanding of the
pathogenesis of asthma, the prevalence and mortality rate due to this disease is still
rising (6). It appears that Th2 helper cells are predominantly involved in asthma
and the roles of Th2-type cytokines, such as IL-4 and IL-5, have been given more
attention in terms of their respective signaling pathways (6). Furthermore, IgE-
124
mediated mechanisms and the protein tyrosine kinase signaling cascade are key in
mediating the release of pro-inflammatory agents from inflammatory cells (6).
However, host mechanisms exist to temper the inflammation that occurs, which in
turn has encouraged researchers an outlet to explore therapies. More specifically,
therapeutic agents that boost host-mediated anti-inflammatory mechanisms, or
attenuate the pro-inflammatory processes in asthma, have recently been developed,
including inhibitors of IL-4 and IL-5 (6).
IL-4 commits naïve T cells to the Th2 phenotype (7) and promotes isotype
switching towards IgE (8). Interestingly, IL-4 represents an important candidate
asthma gene not only because of its functions but because the IL-4 gene is located
in the 5q31-5q33 cluster (9), where several other candidate asthma genes exist.
These genes have been linked to total serum IgE levels as well as to bronchial
hyperresponsiveness (10, 11, 12). Promoter polymorphisms within the IL-4 gene
have also been associated with enhanced IL-4 activity in asthma (9). Also, IL-4 is
important in regulating the recruitment of pro-inflammatory cells such as
eosinophils (eosinophilia), airway hyperresponsiveness and mucus production, yet
it is not mandatory for these events (13). To date, two different types of IL-4R
complexes have been described, one that utilizes the 140 kD IL-4Ra chain and the
yc (Type I complex) or one that utilizes the IL-4Rct chain and the IL-l3Ral chain
(Type II complex) (14). It remains unclear whether IL- 13 acts alone (as a factor
required for airway hyperresponsiveness and mucus production (15), and the
125
regulation of lung eosinophilia (16)), or in combination with IL-4 in the
pathophysiological effects described above (17).
Since IL-4 has been recognized as one of the important factors involved in
asthma, inhibitors have been created as therapeutic agents for treating asthma.
Inhibitors include soluble receptors and antibodies to IL-4 and the IL-4R, as well as
an IL-4 mutant protein (6). Since it has been recognized that small molecular
inhibitors may not completely block IL-4-mediated signaling due to the multichain
binding domains of the IL-4 receptor, a mutant variant form of this cytokine has
been developed (6). The mutant protein, termed IL-4.Y124D, binds the IL-4R with
high affinity and has been shown to inhibit IL-4-mediated T cell proliferation (18)
and IL-4/IL-13-mediated IgE synthesis (19). Furthermore, a genetically engineered
soluble IL-4R (sIL-4R) has been developed and acts as an effective anti-
inflammatory agent (20). Lastly, antibodies against IL-4 have been developed in
animal models that effectively blocked IgE production, airway hyperreactivity, and
attenuated eosinophilia (21). However, administration of murine-derived
antibodies in humans unavoidably elicits an immune response, forcing the
development of a humanized, non-immunogenic antibody. Indeed such an
antibody, termed SCH 55700, has been developed against IL-5, and has significant
effects against the pathogenesis of asthma in animal studies (22). However, an
equivalent anti-IL-4 has yet to be created.
Collectively, these therapies represent logical and effective ways to combat
asthma. Related to our interest, it may be beneficial to consider the survival effects
126
of IL-4 in light of such diseases as asthma. Considering the responses and events
that occur during asthma as a whole, IL-4 may be important in T cell-mediated
pathogenesis primarily by skewing the T cells involved to survive rather than die,
thus perpetuating a harmful cell population within the host. As the signaling
pathway of IL-4-mediated survival becomes increasingly clear, therapies can be
designed to purposely block events that occur along the survival pathway and arrest
IL-4-driven maintenance of undesirable T cells. This approach is complicated in
that the elimination of specific, damaging T cells alongside a simultaneous
preservation of beneficial T cells is daunting in the least. Nonetheless, by studying
primary T cells to uncover IL-4-mediated survival, this pathway may eventually be
defined, allowing us to better understand the basic survival requirements for T cells
as well as how cancerous cells, autoreactive T cells, or pathogenic T cells avoid
death in vivo.
127
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