Download Apoptosis and cell penetration by autoantibody may represent

Clinical and Experimental Rheumatology 1999; 17: 643-647.
Apoptosis and cell
penetration by
autoantibody may
represent linked
processes
R.C. Williams Jr., E. Peen
Division of Rheumatology and Clinical
Immunology, Department of Medicine,
University of Florida Health Sciences
Center, Gainesville, Florida, USA.
Supported in part by a grant from the
Florida Chapter of the Arthritis Foundation, and in part by the Marcia Whitney
Schott Endowment for Research in
Rheumatic Diseases to the University of
Florida. Dr. Elisabeth Peen was supported
by a grant from the Norwegian Council of
Research Project No. 111159/320.
Please address correspondence and reprint
requests to: Ralph C. Williams Jr., MD,
Division of Rheumatology, University
of New Mexico Health Sciences Center,
Department of Medicine, 5 ACC Building,
2211 Lomas Boulevard NE, Albuquerque,
New Mexico 87131-5271, USA.
Received on August 16, 1999; accepted on
August 30, 1999.
© Copyright CLINICAL AND
EXPERIMENTAL RHEUMATOLOGY 1999.
Key words: Antibody cell penetration,
apoptosis.
ABSTRACT
An hypothesis is presented in which
the natural physiologic process of
apoptosis may sometimes be linked
to cell penetration by autoantibody.
Apoptotic blebs as residuals of the
prograramed cell death process have
recently been demonstrated to contain
clusters of disparate autoantigens
which have previously been implicated
in the generation of autoimmune
reactions. In parallel, there now exists
a mounting body of evidence that
certain autoantibody molecules are
capable of living cell penetration and
even nuclear membrane ingress. We
suggest that these two processes,
apoptosis and cell penetration by
autoantibody, may be associated with
one another in certain circumstances.
Hypothesis
Apoptosis and cell penetration by autoantibody may represent naturally linked
events in the same cycle. A series of recent observations by many different
workers have suggested that the cellular
debris which are presented on the residual outpouchings of the apoptotic cell
could account for the process of diverse
intracellular antigens being presented to
the immune system and subsequently
stimulating the production of autoimmunity. In parallel it has also been suggested that certain autoantibodies are
somehow capable of penetrating living
cells or even subcellular barriers such as
nuclear membranes and, in some cases,
of initiating cell death and apoptosis. One
of the problems in attempting to analyze
whether indeed such processes of apoptosis and cell penetration by antibody are
actually different stages of the same linked process is that currently our insight
into which comes first - antibody cell
penetration or apoptosis - is not yet clear.
This review will attempt to provide information currently available and then
let the reader decide for himself.
Specific mechanisms actually involved
in the generation of what is looked on as
an autoimmune response have been examined by a number of workers and are
the subject of several major hypotheses
concerning how autoantibodies are produced and how such antibodies escape
the usual presumed scrutiny and disci643
HYPOTHESIS
pline of tolerance mechanisms (1-4).
Recent reports by Casciola-Rosen,
Anhalt and Rosen (5) have presented an
interesting hypothesis which perhaps can
explain how a number of diverse molecules such as DNA, histones, phospholipids and various cytoplasmic antigens cluster together extended on the cell
surface during the terminal phases of the
complex process of apoptosis (6-9).
The basic hypothesis, simply stated, is
that seemingly diverse and apparently
unrelated structures such as DNA and
phospholipid moieties suddenly find
themselves as closely packed bedfellows
in the cell surface during the final phase
of apoptotic regurgitations outward to the
cell margins before complete dissolution
of the dying cell. Such an hypothesis may
provide a convenient and rational explanation for the simultaneous generation
of an extremely diverse immune response and ultimately autoantibodies to
antigens on DNA, phospholipid moieties, histones, components of nucleosomes and other former intracellular
structures during the course of active
systemic lupus erythematosus (SLE).
One of the major theoretical problems
involved in accepting the “apoptotic
belch” of cell contents as a universal
truth to help explain immune system
antigenic presentation of such diverse
intracellular autologous structures as
DNA, phospholipids and histones is that
it may be difficult to document similar
antigenic presentation of all antigens
which react with lupus or other diseaserelated autoantibodies - including various conformational determinants on IgG
(in the case of rheumatoid factors), thyroglobulin, thyroid gland cell peroxidase,
erythrocyte membrane antigens (including the I, i system) and autoantibodies
reacting with platelet components. There
are a number of autoantigens which thus
far are unaccounted for, or at least have
not yet been definitely identified within
the apoptotic ladder of DNA fragments
or associated with the complex process
during which various apoptosis-related
cleaving enzymes called caspases appear
to be very active in breaking down cell
constituents. Even through apoptosis
provides a unifying concept for how diverse structures within the cell may
present themselves to the immune sys-
HYPOTHESIS
tem, it still does not adequately explain
how, in parallel, such a process can account for loss of tolerance to these many
self components.
Cell penetration by antibody - the
other process which could initiate
apoptosis
An inverse, contrasting process to
apoptosis may also eventually be linked
to the intracellular events leading up to
apoptosis or modifying its cycle in some
as yet undetected way. The actual spectrum of signals, which normally initiates
apoptosis, is still being defined. One conceivably could relate to cell penetration
by whole antibody molecules. The first
report that whole antibody molecules
were actually capable of directly entering living cells was published more than
20 years ago by Alarcón-Segovia et al.
in 1978 (10). Subsequently this group
along with a number of other workers
made similar confirmatory observations,
which have accelerated and become
more abundant over the subsequent
twenty years (11-29). Our own laboratory with the work of Okudaira (18)
showed that monoclonal anti-DNA antibody could enter living cells and in
concurrent studies that polyclonal antibodies from SLE patients could enter
lymphocytes. Recently this subject was
the focus of a special “conference within
a conference” at the Fifth International
Conference on Systemic Lupus Erythematosus held at Cancún on April 20-24,
1998 (30).
The phenomenon of whole antibody
molecules actually penetrating not only
the outer membranes of cells but their
cytoplasmic interstices and eventually
their nuclear membranes is now coming
into its own - having been confirmed and
extended by a number of other groups
(19-29). However, apparently there are
still a substantial number of workers who
regard this subject with disdain or consider it fringe science - somewhat akin
to acupuncture, melatonin, or the megavitamin hysteria.
A number of additional new bits of information have recently been added to
the antibody cell penetration story. Initially, Alarcón-Segovia and coworkers
felt that the cell entry must be accomplished by engagement of antibody IgG
Apoptosis and cell penetration by antibody / R.C. Williams Jr. & E. Peen
molecules by cell surface Fc-receptors
(10). However, Yanase, Madaio and coworkers heve recently defined a distinct
cell surface myosin-1 molecule as being
directly involved with cell membrane
penetration and ultimate delivery into the
living cell (28). Moreover, the same phenomenon of cell penetration has now
also been described and studied by
Reichlin and coworkers (25, 29), particularly as associated with anti-DNA antibody with anti-Sm(D) cross reactivity
(31). Initial observations of intracellular
events by this latter group appear to indicate that intracytoplasmic residence of
the internalized IgG antibody coincides
with the rather abrupt cessation of protein synthesis and yet the cells do not
perish. Some 24 to 48 hours later, whole
antibody molecules inside the cell have
disappeared or are undetectable and protein synthesis recommences. One could
wonder at this point whether cell penetration by antibody could initiate a sort
of general cellular dyspepsia, culminating in a final apoptotic event ? As a matter of record, it is fascinating to note that
more than two decades ago AlarcónSegovia and coworkers reported that
anti-ribonucleoprotein IgG was capable
of penetrating Tγ lymphocytes and thereby abrogating their presumed suppressor function (12).
Several other examples of cell penetration by antibodies of other unique specificities have also now been described.
One of the most interesting are the reports by Adamus and coworkers of retinal rod cells being entered and ultimately
dying after penetration by IgG antibody
to recoverin, the retinal protein implicated in cancer-associated retinopathy
(CAR) (34, 35). In this example, antibody with anti-recoverin specificity from
a human patient with small cell lung carcinoma and CAR was instilled into the
ocular posterior chamber of an experimental animal. Serial studies of the
events thereafter indicated that the IgG
anti-recoverin antibody entered the retinal rod cells and subsequently was associated with the apoptotic death of these
same cells with permanent blindness of
the eye thereafter (36). Clear evidence
for apoptosis of rod cell nuclear DNA
was evident in this experimental model.
This finding represents the first demon-
644
stration that specific antibody cell penetration can induce apoptosis of the target cells directly. A similar disease mechanism is presumed to be operative in
naturally occurring CAR in the case of
some patients with small cell carcinoma.
Thus, in this particular dramatic example, the autoantibody with anti-recoverin
specificity was directly responsible for
apoptosis and target cell death.
There are also several other fascinating
new observations related to antibody cell
penetration. The first is that there appear
to be unique or distinetive V-region antibody primary amino acid sequence
motifs, which may actually regulate or
be important for the ability of the antibody to cross the outer cell membrane.
Madaio and coworkers studied the primary amino acid sequences of the V-region complementarity-determining regions (CDRs) in a panel of monoclonal
anti-DNA antibodies demonstrated to be
capable of cell penetration and found that
a basic sequence motif within the heavy
chain complementarity region (CDR3)
of VSRKRPPRPA was somehow linked
to this biological activity (37, 38) This
observation was of great interest since it
indicated that portions of the anti-DNA
antibody intimately involved in making
up the antibody combining site, namely
the CDR3 of the heavy chain, were directly involved in intracellular localization. Along the same lines Buttin and
coworkers from the Pasteur Institute reported that small peptides synthesized
from the third complementary-determining region of the V-region heavy chain
sequence of cell penetrating monoclonal
anti-DNA antibodies were themselves
capable of cell and eventual nuclear
membrane penetration (39). However,
contrary to the anti-recoverin story, cell
penetration by anti-DNA antibodies or
peptides from the latter was not uniformly followed by cell death or apoptosis.
This entire concept has now also been
ingeniously adapted by Weisbart and
coworkers (40) as a possible method to
transport molecules of interest inside the
cell and, of course, eventually to the nuclear chromatin. These workers demonstrated that a very large molecule such
as alkaline phosphatase (245 Kd) linked
to the recombinant Fv portion of 3D10,
Apoptosis and cell penetration by antibody / R.C. Williams Jr. & E. Peen
a mouse monoclonal anti-DNA known
to be capable of cell penetration (27),
was transported piggy-back with the
monoclonal IgG antibody across the nuclear membrane and still stayed attached
to the antibody to which it had been
linked. Since the antibody or even the
recombinant Fv portion of the antibody
can deliver molecules directly to the
chromatin structures, this mechanism
also conceivably might provide an alternative pathway for either gene therapy
or even chemotherapeutic molecules directed at tumors.
The investigative work in the general
area of cell penetration by various autoantibodies is now only in its infancy. It
seems possible that other connective tissue disorders associated with autoantibody production such as C-ANCA in
Wegener’s granulomatosis may also
somehow be involved in similar phenomena. In Wegener’s granulomatosis,
Csernok and coworkers demonstrated an
overexpression of the autoantigen proteinase-3 (PR-3) on the cell surface of
neutrophils (PMNL) when PMNL from
Wegener’s patients were compared to
healthy controls (41). In this study, intracytoplasmic IgG antibodies were observed in PMNL from Wegener’s patients. Along a similar line, Gilligan and
coworkers (42) showed that apoptosis of
unprimed PMNL may also be associated
with the translocation of cytoplasmic
granules to the cell surface and aligament
just beneath the intact cell membrane.
By FACS analysis, they demonstrated
reactivity of anti-PR3 positive sera with
apoptotic but not with viable PMNL.
Although these observations do not directly support cell penetration by antiPR3 antibody, they do emphasize that
apoptotic cells may be involved in the
antigen presentation of neutrophil granule antigenic constituents to the immune
system.
One of the most critical aspects of apoptosis and autoantigen presentation to the
immune system certainly relates to how
self antigens exposed within cell apoptotic blebs can escape built-in indigenous
tolerance. One interesting mechanism
recently suggested by Utz and Anderson
(43) is that a host of self components may
be cleaved by caspases or cysteine proteases with aspartic acid substrate speci-
ficity and that novel epitopes uncovered
by such proteolytic cleavage might thereby be exposed. In such cases, no tolerance to these previously buried determinants would be present and an immune
response would follow. In addition, the
very process of apoptosis may be associated with the phosphorylation of various proteins such as serine arginine-rich
splicing factors known to be contained
in certain autoantigens such as the U1
snRNP autoantigen complex. Moreover,
phosphorylation has also been shown to
affect the immunogenicity of some proteins and these alterations as a product
of apoptosis could produce new antigens
to which tolerance has never been established.
Thus various post-translational protein
modifications engendered by the apoptotic process could bypass tolerance and
initiate an autoimmune response. Additional evidence for post-translational
modifications of various autologous proteins has also recently been presented by
Schellekens and coworkers (44), who
demonstrated that autoantibodies in the
sera of patients with rheumatoid arthritis (RA) reacted with synthetic peptides
containing the unusual amino acid, citrulline, with a post-translationally modified arginine residue. Moreover, affinity
purified antibodies were positive in the
immunofluorescence-based anti-perinuclear factor assay.
The presence of autoantibodies in RA
sera which react with citrulline-containing epitopes might suggest that deimination of arginine residues to produce
citrulline could represent a post-translational alteration inducing a neoantigenic
determinant capable of stimulating an
immune response. The enzyme which
modified arginine to citrulline is peptidylarginine, which is normally expressed
in a wide range of tissues and cell types
(45, 46). However, activation of these
enzymes capable of the deimination of
arginines to citralline has not been specifically demonstrated to be linked to
apoptosis and more work on this subject
needs to be completed.
The apoptotic process itself has been
demonstrated to be dysregulated or abnormal primarily in SLE, as evidenced
by elevated levels of soluble Fas in some
SLE patients’ sera as well as increased
645
HYPOTHESIS
expression of bcl -2 (47). Abnormal
apoptosis in SLE could contribute to tolerance of T cells to self antigens since it
could allow the persistence of autoreactive T cells (48). It is not yet clear
whether cell penetration by autoantibodies such as anti-DNA could also
somehow distort the normal apoptotic
process.
Thus one may be able to begin to look at
the process of immune processing and
generation of autoantigens by cells as a
product of apoptosis and possibly also
linked to cell penetration by autoantibodies. Which process comes first and
which initiates which is still not clear
because cell penetration by antibody is
not always followed by cell death. Clearly one could envisage a scenario in which
the presence in plasma of autoantibodies of various specificities and bearing
various key sequence motifs essential to
cell penetration in their VH CDRs capable of cell membrane and subsequent nuclear membrane penetration, could enter the cell and shortly thereafter initiate
various degrees of cellular indigestion or
what a rheumatologist might term “intracellular fibromyalgia”. In some susceptible cell populations this could then
lead to the induction of apoptosis through
the activation of caspases and other enzymes, leading eventually to apoptotic
extrusion of a number of diverse autoantigens and presentation of the latter to
antigen presenting cells of the immune
system. The various steps involved in
apoptosis are illustrated in Figure 1.
How much of the basic engine driving
autoimmune reactivity could be relegated to such an apoptosis-antibody cell
penetration concept now remains to be
determined. Moreover, it raises another
interesting question as to whether cell
penetration by autoantibody could actually represent a natural function driving
the apoptotic process. In this respect it
has long been apparent that there exist
in normal mice and in many normal humans a number of so-called natural autoantibodies (50, 51). These have been
extensively documented and studied in
detail by Avreamas, Coutinho and coworkers (51, 52). Could it be that this
general pool or spectrum of natural autoantibodies might in some circumstances
also be capable of initiating cell death
HYPOTHESIS
Apoptosis and cell penetration by antibody / R.C. Williams Jr. & E. Peen
Fig. 1. Schema proposed by Korsmeyer (52) to indicate various checkpoints, key elements and phases, which are involved in apoptosis. The pro-apoptotic
BAX pro-death family member is known to have the capability of forming ion conductive pores. In situations where cell penetration by antibody somehow
might activate BAX, the inevitable progression to cell death follows. Exactly how cell penetration by antibody could initiate apoptosis is not known but
intranuclear penetration by antibody could possibly activate the endonuclease implicated in cell death (point of no return), or interfere with DNA repair
pathways and initiate the death cascade. Activation of the caspase enzymatic systems is shown to the lower right. An alternative pathway to the point of no
return is shown at the lower left.
as a natural phenomenon ? Presently, of
course, there is no direct experimental
data to support such a notion. Much remains to be done and re-examined in this
general area and we all look forward to
a final balanced synthetic assessment.
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