Download The role of apoptosis in systemic lupus erythematosus

Rheumatology 1999;38:1177–1183
Systemic Lupus Erythematosus/Series Editors: D. Isenberg and C. Gordon
The role of apoptosis in systemic lupus
erythematosus
M. Salmon and C. Gordon
Division of Immunity and Infection, The University of Birmingham, Birmingham
B15 2TT, UK
When it was described by Kerr et al. [1], the phenomenon of apoptosis, or programmed cell death, was
greeted with a resounding lack of interest. The importance of this phenomenon is such that it is now very
difficult to pick up any journal in a biomedical field
without finding at least one paper on the subject. It has
become apparent that the process is ubiquitous, representing a vital part of growth and differentiation in all
tissues. The fundamental mechanisms of apoptosis and
the genes that control it are highly conserved between
species as diverse as worms and humans [2, 3].
Immunological research in apoptosis has focused on
three areas in particular: (1) the selection of new
lymphocytes, both B and T cells, before antigen challenge [4–6 ]; (2) the regulation of clonal expansion and
resolution, including the elimination of apoptotic cells
[7–10]; (3) the selection of memory cells [7–9, 11–14].
About 10 million new lymphocytes are produced in the
bone marrow each day [11] and during a viral infection
the total number of lymphocytes may easily double,
mostly with a vast number of activated cytotoxic T cells
[11]. These cells must be eliminated at the end of the
response, to avoid an exponential increase in the size
of the immune system [7]. However, this homeostatic
balance must be achieved within a framework that
accommodates the generation and persistence of
memory cells, to permit a more potent response on
subsequent re-challenge. Rescue from apoptosis is the
crucial selection event in immunological memory for
both B and T lymphocytes.
Apoptosis
Apoptosis is an active process that leads to the ordered
destruction of cells, avoiding the release of intracellular
contents into the extracellular microenvironment, where
they have a powerful inflammatory effect. Apoptotic
cells undergo a series of distinct physical changes, including alteration of the surface lipid membrane, cytoskeletal
disruption, cell shrinkage and a characteristic pattern of
DNA fragmentation.
Submitted 1 June 1999; revised version accepted 17 June 1999.
Correspondence to: M. Salmon, Division of Immunity and
Infection, Rheumatology Research Group, The Medical School, The
University of Birmingham, Birmingham B15 2TT, UK.
Apoptosis can be induced actively, through ligation
of specific receptors such as Fas or TNFR, or passively,
through lack of essential survival signals [8]. All cells in
the body require continuous positive signals to stay alive
[15]. In the case of T lymphocytes, for example, these
signals include interleukin (IL)-2 and interferon beta
(IFN-b), which act by upregulating expression of antiapoptotic molecules such as Bcl-2 and Bcl-x [16, 17].
L
These molecules, in turn, suppress the release of
cytochrome c from mitochondria [18, 19]. In the absence
of a positive signal for survival, cytochrome c forms a
complex with APAF-1 and caspase 9, to form a caspase 3
activating unit [20]. Caspase 3 is the fulcrum between
regulation of apoptosis and execution of the cell. When
the activation of caspase 3 reaches a threshold level, its
autocatalytic activity sets off a catastrophic chain reaction that leads to the activation of all the caspase 3 in
the cell [21]. This is the point of irrevocable commitment
to apoptosis. The active caspase 3 subsequently activates
a range of downstream mediators that co-ordinate the
ordered destruction of the cell [22]. Active induction of
apoptosis may either facilitate the release of cytochrome
c, or directly activate caspase 3 by a different route. In
each case, caspase 3 activation defines the point of
commitment and the convergence point for all apoptosis
induction pathways.
In vitro, the terminal phase of apoptosis is ‘blebbing’.
Minute balloon-like membrane extrusions bud off from
the surface of the cell, incorporating nuclear and cytoplasmic material. In vivo, this rarely happens, because
apoptotic cells are very effectively removed by phagocytes, which use a range of recognition systems to
identify them [10, 23].
A question of tolerance
Immunological tolerance is a compromise that works
for most people most of the time. Tolerance to self
operates through a number of mechanisms, but is principally the domain of T lymphocytes. T cells are selected
in the thymus from a population of immature thymocytes expressing randomly generated antigen receptors.
Unlike B cells, which use surface-expressed antibody
as a receptor and consequently can recognize native
antigens, T cells only recognize short peptide antigens
presented in the groove of class I (CD8 cells) or class II
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© 1999 British Society for Rheumatology
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M. Salmon and C. Gordon
(CD4 cells) MHC molecules. T cells are selected in the
thymus so that they will bind to self-MHC with selfpeptides in the groove (positive selection), but not
sufficiently strongly to activate the cell (negative selection). The cells which fail these tests are, predictably,
deleted by apoptosis [24]. Cells which fail positive
selection are useless, those which fail negative selection
are autoimmune and consequently dangerous. The
importance of negative selection is ably illustrated by
graft rejection reactions.
Positive selection is a highly effective process, but
negative selection is limited by the impracticality of
presenting all possible peptides from every protein
expressed in the body, in all available MHC molecules
(anywhere from six to about 14) to every thymocyte
produced throughout life. The reality seems to be that
many antigens are not used for selection in the thymus,
so central (thymic) tolerance is only partially effective.
In the periphery, if antigens are presented by nonprofessional antigen-presenting cells (APC ) which lack
appropriate co-stimulatory molecules such as CD80/86
or CD40, then T cells are switched off (anergy) or
deleted by apoptosis.
Systemic lupus erythematosus as a disease of
defective apoptosis
B-cell differentiation and antibody production are highly
dependent on T-cell help, as we shall discuss later, so
the crucial role of apoptosis in immunological tolerance
(somewhat overestimated until quite recently) led to the
first model to link apoptosis and systemic lupus erythematosus (SLE ). For many years, SLE was considered
to be a disease of B-cell hyper-reactivity [25]. Autoantibodies are produced in patients with this disease to
a bewildering variety of cellular components, at least 40
and probably more like 2000 target antigens in the
nucleus, cytoplasm and membranes [26 ]. The MRL lpr
mouse, which likewise produces autoantibodies to such
antigens, was considered by many to be an excellent
model for the disease [27, 28]. The discovery that the
lpr gene is a defective form of Fas (CD95), which is a
surface receptor on the surface of cells (particularly
lymphocytes) that transduces an active signal for
apoptosis [29], led to much speculation that SLE would
similarly have defective Fas-mediated apoptosis at the
core of the failure of self-tolerance [30]. Support for
this hypothesis came when the gld abnormality (another
mouse gene that produces a similar phenotype to lpr)
was identified as a non-functional Fas ligand gene [31].
Unfortunately, there were severe problems with this
model: firstly, the lpr and gld mice suffer from a severe
lymphoproliferative condition. When these mice die, at
a few months of age, up to half their body weight
consists of abnormal lymphocytes [27, 28]. This is not
a characteristic of patients with SLE, who are often
profoundly lymphopenic. Secondly, both the expression
and function of Fas and its ligand are normal in SLE
[32–34]. Indeed, some reports suggest that Fas expression is a little higher than normal [32]. Actually, Fas-
mediated apoptosis does not play a significant role in
thymic selection of T cells anyway [35]. So, when a
small number of individuals were found who had an
lpr-type genetic abnormality and they had a lymphoproliferative disease, much like the MRL mouse, but
not much like SLE, no one was too surprised [36–38].
Too much apoptosis?
In fact, far from being a disease of too little apoptosis,
the evidence has been available for a long time that SLE
is quite the opposite. Under normal physiological circumstances, apoptotic cells are cleared very effectively
by phagocytes [10, 23]. If this were not the case, we
would all be in quite serious trouble. Up to 1011 neutrophils are produced in the bone marrow and subsequently
die each day. These cells have a half-life in the blood
of about 8 h, yet they contain toxic granules that produce
significant inflammation if they are released into the
extracellular microenvironment. Patients with SLE often
have high levels of circulating DNA. This is usually
present as short (162 base pair) stretches, wound around
a histone core, i.e. as nucleosomes [39] which are the
classical cleavage product of apoptosis. Furthermore,
haematoxylin bodies, which are often found in the
glomeruli of patients with lupus nephritis, are actually
‘blebs’ from fragmented apoptotic cells [46 ]. The LE
cell, which has only recently been removed from the
ACR criteria for SLE (because no one tests for it
anymore, not because it was not specific), is itself a
neutrophil that has phagocytosed apoptotic material of
this sort. Apoptotic blebs can be processed by professional APC as foreign antigen and directed to class II
MHC molecules. In addition to nucleosomes, they contain proteins such as Ro and La, which are clearly
recognized autoantigens in SLE [40, 41].
Unfortunately, the major compromise operating in
the generation of immunological tolerance means that
we are not really functionally tolerant to the inside of
our cells, just ignorant. Obligate intracellular proteins
do not encounter the immune system very often in
significant quantities, because phagocytes are so good
at removing them intact. However in SLE, where this
process appears to be mildly deficient, the peripheral
immune system is challenged by these proteins. The Tcell dependence of autoantibodies in SLE is suggested
very strongly by their high-affinity, isotype-switched
nature, as we shall discuss in the next section. However, the evidence at this point suggests a new model
for antigen-driven autoantibody production in SLE.
Deficient phagocyte-mediated clearance of intact apoptotic cells leads to their fragmentation and the release
of intracellular antigens (to which the immune system
is not tolerant) in a form that can trigger an immune
response. Before we can consider the implications of
this further, we need to discuss recent developments
in our understanding of T-cell-dependent antibody
production.
Role of apoptosis in SLE
Somatic hypermutation and B-cell memory
Primary antibody responses are exclusively IgM and
predominantly low affinity. Athymic individuals are very
good at producing such responses, but repeated challenge does not yield the characteristic features of a
secondary antibody response, which are high-affinity
IgG antibodies. These features are T-cell dependent. As
the antibodies characteristic of SLE are mostly highaffinity IgG antibodies, they are very likely to result
from T-cell-dependent responses.
High-affinity memory B cells are produced in germinal
centres by somatic hypermutation [9] ( Fig. 1). A germinal centre requires B cells and T cells activated in a
primary response in the paracortex of secondary
lymphoid tissue, and follicular dendritic cells that have
the relevant antigen bound to their surface. The B cells
(centroblasts) start to divide rapidly and spontaneously
mutate the variable region of their antibody genes. As
long as they keep dividing, they are safe from apoptosis,
but as soon as they leave the dark zone and stop
dividing, they have ~8 h to live. This is rather important, because random mutations to the immunoglobulin
variable genes are as likely to produce an improvement
in affinity as random tinkering with a spanner is to
improve the performance of a Formula 1 racing engine.
In both cases, blind chance will occasionally do the
trick. However, in both cases, most such changes will
be either ineffective or dangerous. Spontaneous mutation may well turn a useful anti-pathogen antibody into
an autoantibody, but it is much more likely to produce
a useless immunoglobulin. The germinal centre functions
to select the fortuitous beneficial mutation from the
garbage. To do this, the somatically hypermutated B cell,
now termed a centrocyte, must pass a series of tests
(Fig. 2). Firstly, the centrocytes must remove surface-
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bound antigen from the follicular dendritic cells in the
light zone, then they must pinocytose the antigen, process it and incorporate it into class II MHC molecules
on their surface. As they try to escape the germinal
centre, the centrocytes encounter T cells which recognize
the same antigen. If a T cell recognizes the antigen
presented by a centrocyte, it releases onto its surface a
preformed CD40 ligand, which binds to CD40 on the
surface of the centrocyte. This cognate interaction overrides the apoptosis programme in the centrocyte, which
then becomes a stable mature memory B cell or, with
appropriate signals, it can differentiate into a plasma
cell and produce large quantities of antibody. Clearly,
only a centrocyte that has produced a high-affinity
mutation will be able to retrieve antigen from the
follicular dendritic cells and thereby survive. This process defines the central role of T cells in self-tolerance.
A conundrum
Improved understanding of the mechanisms leading to
the selection of memory B cells in germinal centres poses
an obvious problem. Somatically hypermutated, classswitched B cells can only survive if they can present a
T-cell epitope from their pinocytosed antigen to a T cell.
The conundrum is this: T cells can only recognize
peptides, but the most characteristic antibodies in SLE
are to DNA and phospholipids.
This is easily solved in the context of the model we
describe. Nucleosomes, as described above, consist of
short stretches of DNA wound around a histone core,
rather like a cotton reel. In an SLE patient, in whom
high levels of circulating nucleosomes are found, a
germinal centre can form in which the B cells are specific
for double-stranded DNA, but effectively mutated centrocytes will use their anti-DNA antibody to remove
F. 1. Somatic hypermutation and selection of B-cell antigen receptors (antibody) in a germinal centre. CB, centroblast; CC,
centrocyte; mB, memory B cell; PC, plasma cell; T, T cell; FDC, follicular dendritic cell.
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M. Salmon and C. Gordon
F. 2. The process of selecting memory B cells in a germinal centre. This interaction, where mutated antigen receptors are used
by centrocytes (B cells) to retrieve antigen from follicular dendritic cells, which they process and present to T cells, allows the
selection of high-affinity clones for the initiating antigen. It also defines the fundamental interaction responsible for tolerance in
T-cell-dependent antibody responses.
whole nucleosomes from the follicular dendritic cells.
When this is processed, the protein core will be incorporated into the grooves of class II MHC molecules and
presented to histone-specific T cells ( Fig. 3). Experimental evidence for this model has now been reported
[42]. Similarly, membrane phospholipids such as phosphatidyl-serine are linked to lipid-binding proteins which
can act as carriers. This provides a simple hapten-carrier
model similar to that used in the production of conjugate
vaccines.
Why is the clearance of apoptotic cells
deficient in patients with SLE?
A complete failure of the clearance mechanisms that
remove apoptotic cells would be fatal in days, so any
such deficiency in patients with SLE could only be
partial, reducing the threshold for overload of the
system. Patients with homozygous deficiencies in the
early complement proteins (C2, C4 and especially C1q)
develop a severe lupus-like disease early in life [43].
Strangely, deficiencies of C3 produce a much less severe
phenotype than deficiencies in C1q [43]. Yet C3 is the
first point of convergence for all three complementactivating pathways (rather like caspase 3 in apoptosis)
and consequently will have the most marked effect on
complement activation. Deficiencies in components of
the membrane attack complex are relatively minor.
Recent data have shown that C1q receptors on the
surface of phagocytes are an extremely important mechanism in clearing apoptotic cells [44]. Patients, or mice,
with homozygous C1q deficiency develop autoantibodies
and a lupus-like syndrome. This is apparently because
they cannot clear apoptotic cells effectively and con-
sequently present antigens derived from them to the
immune system [45–47]. However, the levels of antinuclear and anti-dsDNA antibodies are not particularly
high, and the clinical manifestations tend to be restricted
to the skin and kidneys.
Quite why patients with SLE develop defective clearance of apoptotic cells is less clear. Antibodies to C1q
are present in a large proportion of patients, particularly
those with renal disease [48–50]. This probably results
in a functional deficiency of the protein. It is unlikely
that C1q antibodies represent the primary abnormality
in most patients with SLE, but it would certainly provide
a mechanism for persistent disease and flaring. Exposure
to UV irradiation and infection are the most common
triggers of flares in lupus patients. Both will produce a
sharp increase in the number of apoptotic cells in the
skin [39, 40, 51] and in the blood [8, 11], respectively.
If this overloads the inefficient clearance mechanisms of
the reticuloendothelial system, large amounts of antigen
will be released. This will have two effects: firstly, to
drive the immune response to produce more antibodies
and, secondly, to bind existing antibodies in the form
of immune complexes. This will produce inflammatory
reactions in highly vascularized tissues, such as the skin,
kidneys and lungs. The immune complexes will also
cause massive consumption of classical pathway complement, including C1q, thus exacerbating the initial problem. The relationship between complement deficiency
and SLE will be developed further in a future article in
this series.
A vicious circle of C1q
In summary, the most plausible current model for the
perpetuation of SLE involves deficient clearance of
Role of apoptosis in SLE
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F. 3. The hapten-carrier mechanism responsible for selecting high-affinity IgG antibodies to non-protein autoantigens in SLE.
Nucleosomes derived from apoptotic cells are pinocytosed by centrocytes, which bind to the DNA wound around the outside.
The protein core is processed and presented to the T cell, which delivers a rescue signal through CD40.
apoptotic cells under conditions of stress, at least partly
due to a functional deficiency of C1q. This produces
large amounts of antigen to which the immune system
is not tolerant. Periodic episodes of infection or UV
exposure produce high levels of apoptotic cells which
will fragment and drive the disease process by the
formation of immune complexes, thus causing localized
microvascular inflammation and consumption of C1q,
rendering the protein even more deficient and less able
to clear apoptotic cells. In individuals with homozygous
C1q deficiency, provision of recombinant C1q may have
therapeutic benefit, but in SLE patients with antibodymediated functional deficit, provision of C1q in this way
may well exacerbate the disease by providing antigen to
drive the specific immune response.
The model we describe here is useful for explaining
the persistence of SLE and also the strong association
between sunlight/infection and disease flares, but it does
not really address the causes of the disease. Functional
C1q deficiency is unlikely to be a primary factor in
causing SLE. It is far more likely to be a secondary
development of the disease process that leads to persistence, analogous to stromal proliferation in rheumatoid
synovitis [52]. Intriguingly, in severe infections such as
infectious mononucleosis, where the reticuloendothelial
system is overloaded by a vast excess of apoptotic
immune cells [11], autoantibodies are very commonly
found, but they resolve when the infection is eliminated.
In contrast, an autoimmune disease process such as SLE
that actively inhibits the clearance of apoptotic cells is
likely to be very difficult to resolve.
On a cautionary note, perhaps we should end with a
quotation from Oscar Wilde that history has shown to
be highly applicable to theories of autoimmunity and to
those associated with SLE in particular: ‘Fashion is a
form of ugliness so intolerable that we have to alter it
every six months’.
Acknowledgements
This work was supported by the Arthritis Research
Campaign (SO190, SO624) and by Lupus UK. We
would like to thank Darrell Pilling, Chris Buckley and
Nick Henriquez for invaluable discussion.
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