By
From the * Department of Immunology, The Scripps Research Institute, La Jolla, California 92037;
and Advanced Research Technologies, Inc., San Diego, California 92121
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Abstract |
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Large numbers of neuritic plaques (NP), largely composed of a fibrillar insoluble form of the
-amyloid peptide (A
), are found in the hippocampus and neocortex of Alzheimer's disease (AD) patients in association with damaged neuronal processes, increased numbers of activated
astrocytes and microglia, and several proteins including the components of the proinflammatory complement system. These studies address the hypothesis that the activated complement
system mediates the cellular changes that surround fibrillar A
deposits in NP. We report that
A
peptides directly and independently activate the alternative complement pathway as well as
the classical complement pathway; trigger the formation of covalent, ester-linked complexes of
A
with activation products of the third complement component (C3); generate the cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory
C5b-9 membrane attack complex, in functionally active form able to insert into and permeabilize the membrane of neuronal precursor cells. These findings provide inflammation-based mechanisms to account for the presence of complement components in NP in association with
damaged neurons and increased numbers of activated glial cells, and they have potential implications for the therapy of AD.
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Introduction |
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We and others (1) have noted that the pathological changes which characterize Alzheimer's disease (AD)1 could all result from complement activation in neuritic plaques (NP), since this effector system has the ability to activate various cell types with release of cytokines and secondary mediators; to induce directed migration of these cells toward the complement activator; to alter cellular functions; and to damage cells (4, 5). Potential complement involvement in the brain is not dependent on disruption of the blood-brain barrier, since neurons, astrocytes, microglia, and oligodendrocytes synthesize most, and likely all, of the proteins of the complement system (6).
Since activation is a prerequisite for manifestation of all
of the biological activities of the complement system, the
-amyloid peptide (A
) or another component of NP
must possess the ability to activate complement in order for
complement to be involved in mediating the pathologic
cellular characteristics of AD. In this regard, we and others
previously showed that fibrillar forms of A
bound the first
reacting factor of the classical complement pathway (CCP),
C1q (3, 7), and depleted the activity of the fourth complement component (C4) as well as whole complement activity (CH50), when incubated with human serum as a complement source in vitro (3, 7). Residues 14-26 of the
collagen-like portion of the A polypeptide chain of the
C1q molecule were implicated in binding fibrillar A
(7).
Recent studies have confirmed this suggestive evidence of
CCP activation by aggregated A
(8), and have also
emphasized the critical role of the
-pleated structure of
A
in mediating these effects (9, 11). Inhibition studies have implicated A
residues 1-11 in C1q binding (11, 12). The complement depletion, inhibition, and cleavage assays
used in these various studies have provided suggestive evidence for CCP activation by fibrillar A
; however, as indirect assays, they are subject to other interpretations. In this
context, we recently presented preliminary evidence suggesting that A
forms complexes with C3 after incubation
of fibrillar A
with a complement source (9).
In the course of these studies, we found that the addition
of fibrillar A to a complement source led to the generation of covalent ester-linked complexes of A
with C3 activation fragments, providing unequivocal evidence for
complement activation by A
, since covalent attachment
of C3 activation fragments to complement activators represents a fundamental tenet of complement action. We also
found that fibrillar A
possesses the ability to activate the
alternative complement pathway (ACP) in serum, as well as in mixtures of the six purified proteins of the alternative
pathway in physiologic concentrations, providing the first
indication that A
independently activates both complement pathways. Additionally, we observed that such activation is highly specific for A
and completely independent
of oxidative processes. These studies are described here. Finally, we also report for the first time that A
-mediated
complement activation is biologically significant, as it leads
to generation of the cytokine-like C5a complement-activation fragment, and mediates formation of the proinflammatory C5b-9 membrane attack complex (MAC), in functionally active form able to insert into and permeabilize the
membranes of neuronal precursor cells.
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Materials and Methods |
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A-Mediated Complement Activation and Complement Activation
ELISA Assays.
Studies with Hydroxylamine.
After capture of complexes onto 10D5-coated wells, replicate wells were treated with 0.1 M Tris, pH 9.5, or 1 M hydroxylamine in 0.1 M Tris, pH 9.5, for 2 h at 37°C. After washing, remaining bound C3 was detected as described above. Residual AMass Spectroscopy.
After solubilization in 70% formic acid, samples were analyzed by MALDI spectroscopy (Perseptive Voyager ELITE; Perseptive Biosystems, Inc., Framingham, MA).C5a.
C5a (and C5a des-Arg) was detected in diluted samples with the Biotrak radioimmunoassay kit (Amersham Corp., Arlington Heights, IL); the samples were subjected to acid precipitation before analysis (16). Values were back-calculated to the concentrations in undiluted NHS.C5b-9 Membrane Insertion.
Ntera2/D1 (NT2) cells (Stratagene Inc., La Jolla, CA) were grown to subconfluence, released with nonenzymatic cell dissociation solution (GIBCO BRL, Gaithersburg, MD), washed, and resuspended (2 × 107 cells/ml). NT2 cells (100 µl) were incubated with 50 µl NHS in the presence or absence of EDTA and 50 µl preaggregated A ![]() |
Results and Discussion |
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Sandwich-type ELISAs showed that complexes containing A and C3b/iC3b were generated in NHS, as a complement source, after incubation with aggregated A
1-42.
Complexes were demonstrable after capture with mAbs to
activation-dependent neoantigens in the first (C3b) or second (iC3b) C3 cleavage products and detection with rabbit
Ab to A
(Fig. 1, a and b), as well as after capture with mAb
to A
and detection with rabbit Ab to C3 (Fig. 1 c) or C3d
(not shown). EDTA, which blocks complement activation
by chelating calcium and magnesium, prevented complex
formation (Fig. 1 c). ELISAs in which complexes were captured with mAb to A
and detected with Ab to C3 were used
for most of the studies, since such ELISAs permitted quantitation by reference to included standard curves generated with
purified C3 captured on wells coated with mAb to C3 and detected with rabbit Ab to C3 (Fig. 1 d). Complement activation was detectable to ~1 µM A
1-42 (Fig. 1 d). 10 different
preaggregated A
1-40 and 20 different preaggregated A
1-42 preparations from 5 manufacturers generated such
complexes. A
1-42 was generally 5-10-fold more active
than A
1-40 in this regard. The cation-dependent formation of A
-C3b/iC3b complexes after incubation of aggregated A
with NHS provides unequivocal evidence for
complement activation by aggregated A
.
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A-C3b/iC3b complex formation was evident after incubation of aggregated A
1-42 with NHS lacking factor
B, an essential component of the ACP (Fig. 1 e); such sera
contain an intact CCP, but do not permit ACP activation.
A significant reduction in complex formation was also evident in C1q-depleted serum compared with NHS (Fig. 1 e).
These data show that the CCP mediates complex formation by aggregated A
, findings that were anticipated from the results of the complement depletion assays described
earlier. Unexpectedly, however, the ACP also mediated
the formation of complexes, since they were also generated
after the addition of aggregated A
1-42 to NHS lacking
factor C1q, and complex formation was reduced in factor
B-depleted serum compared with NHS (Fig. 1 e), a result
replicated in four additional experiments with different A
1-42 preparations. The ability of A
1-42 to activate the
ACP was confirmed in three studies in which preaggregated A
1-42 was incubated with a mixture of the six purified
proteins of the ACP (factors B, D, H, and I, properdin, and
C3) in physiological ratios (reference 13; Fig. 1 f ). These
data document the ability of aggregated A
not only to activate the CCP, but also to independently activate the ACP.
This is the first indication that aggregated A
activates the
ACP; it had been presumed that complement activation by
A
was exclusively via the CCP because of the absence of
ACP components (factor B and properdin) in NP (1, 17,
18). The failure to detect ACP components in NP may be
due to the extreme lability of the ACP C3 convertase.
Multiple different aggregated A 1-42 preparations activated complement, as determined by the classical CH50
complement consumption technique (Fig. 1 g). The aging
procedure used to aggregate A
generates
-pleated fibrils
(9). Nonfibrillar "amorphous" aggregates of A
are devoid
of complement-activating ability (9). In contrast to the aggregated preparations, A
used immediately after dissolution had limited ability to activate complement (Fig. 1 g).
These data document the important role of fibril formation
for complement activation by A
in vitro. Amylin, another
peptide which spontaneously forms
-pleated fibrils, was
also tested for complement-activating ability in these studies. The 37-residue amylin polypeptide represents the principal constituent of the amyloid deposits in type 2 diabetes. On SDS-PAGE gels, aged amylin migrated primarily as
large SDS-insoluble stained bands. However, this fibrillar
peptide did not significantly activate complement at a concentration of 100 µM (7% CH50 consumption).
The specificity of complement activation by A was also
evaluated by determining whether complement was activated by other small peptides (20-50 amino acids) containing multiple residues able to mediate covalent linkage to
the glutamate residue of the hydrolyzed thioester of C3
(serine, tyrosine, threonine, lysine) and expressing similar
overall charge to A
. All of the peptides were processed
and aged in the same manner as A
. None of the peptides,
including the insulin B chain (30 residues), neuropeptide Y-porcine (36 residues), urotensin I (41 residues), exendin
3 (39 residues), amyloid precursor peptide 657-676 (20 residues), and the adenovirus penton base fragment (50 residues) significantly activated complement at a concentration
of 20 µM, as assessed by the classical CH50 technique (Fig.
1 g). The peptides also showed little or no ability to activate
complement in other assays, including the ability to deplete
residual functional C3 and form the SC5b-9 complex (not
shown). To determine whether peptide aggregation would
increase complement-activating ability, urotensin I, neuropeptide Y-porcine, and exendin 3 were covalently cross-linked with the primary amine reactive reagent BS3 before
evaluating their ability to activate complement by the CH50 technique. Cross-linked urotensin I and neuropeptide Y-porcine gave a ladder of Coomassie-stained bands
on SDS-PAGE analyses, but exendin 3 gave no stained
bands, possibly due to the formation of very large aggregates.
These three cross-linked peptides did not significantly activate complement (<10% CH50 depletion) at a concentration of 20 µM. In another study, cross-linked urotensin I exhibited 7% CH50 consumption at a concentration of 100 µM, whereas aggregated A
1-42 showed 45% consumption. These data cumulatively demonstrate the marked specificity of complement activation by fibrillar A
.
C3 preferentially binds to activators via ester bonds, although amide linkage has been described (19, 20); such
bonds form between the reactive -carbonyl group of the
glutamate residue of the activation-cleaved internal thioester
bond in C3, and hydroxyl (ester) or amino (amide) groups
on the activator (19). To evaluate possible ester linkage,
complexes were captured with mAb to A
and incubated
with Tris buffer containing 1 M hydroxylamine at pH 9.5 for 2 h at 37°C, a treatment which disrupts ester but not amide bonds (19, 20). Approximately 50% of the bound
C3, but none of the A
, was removed from the captured
complexes by this treatment (Fig. 2, a and b), a result replicated in two additional studies with A
1-40 and 1-42.
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The formation of covalent complexes of A with C3 activation products was also independently demonstrated using a
Western blotting approach. In these studies, aggregated A
1-40 was incubated with serum, and the complexes of insoluble fibrillar A
with C3 activation fragments were then
sedimented, washed, and incubated with either 1 M hydroxylamine at pH 9.5 or control buffers. After washing, a
prominent band with a molecular mass of ~180 kD, the molecular mass of C3, as well as several higher molecular
mass bands, were detected with Ab to C3 (Fig. 2 c). After
stripping, the same bands were also found to react with Ab
to A
, although the gels were darker due to the presence of
large amounts of aggregated A
(Fig. 2 c). Bands of the same
molecular masses reactive with Abs to both A
and C3
were also observed when the blotting studies were performed in the reverse direction, i.e., blotting first with either mAb or polyclonal Ab to A
followed, after stripping, by blotting with rabbit Ab or mAb to C3 (not shown). The
180-kD band and the larger bands, which contain both C3
and A
, undoubtedly represent complexes of A
monomers with C3b monomers and oligomers, since they were
not evident in the reactions carried out in the presence of
EDTA or in the absence of fibrillar A
(Fig. 2 c).
C3 was also detected in the large A aggregates on the
top of the gels (except for the EDTA lane) on longer exposure (not shown). The lesser reactivity of C3 in the larger
A
aggregates at the top of the gels, compared with the C3
monomers and oligomers within the gels, indicates that not
all A
monomers bear a molecule of C3b; this is not surprising, since A
is in large aggregates and, in addition, in
molar excess over C3. It may also be that A
molecules
bearing covalently bound C3b dissociate from the aggregates, in analogy to the dissociation of immune complexes by the covalent binding of C3b (21, 22).
Hydroxylamine treatment disrupted approximately half of
the complexes (Fig. 2 c). Quantitative scanning of the C3
Western blot showed that the treatment with 1 M hydroxylamine at pH 9.5 removed 42% of the bound C3 compared with the pH 9.5 control; the pH 9.5 buffer treatment
removed only trivial amounts (5.1%) of the bound C3 compared with the pH 7.4 treatment. The A Western blot
could not be satisfactorily scanned due to the large background, but visual inspection reveals the same pattern (Fig.
2 c). Identical results were obtained with A
1-42 (not
shown). These two independent assay systems both show
that ester bonds, in part, mediate covalent attachment of C3
activation fragments to A
. A
1-42 contains two serines,
at positions 8 and 26, and a tyrosine, at position 10, which
could mediate ester linkages with C3 activation fragments.
With regard to the bond(s) responsible for the nonester-linked A-C3b/iC3b complexes, A
alone has been reported to generate free radicals (23) upon incubation in
aqueous solution, and oxidative processes have been associated with A
denaturation, fragmentation, and oxidation
(24, 25). Because of the potential relevance of these processes to the formation of complexes of A
with C3 activation fragments, A
1-42 was assessed by MALDI mass
spectroscopy after aging from 0 to 10 d. Aggregates are not detected in these assays, since the samples are dissolved in
70% formic acid for mass spectroscopic analysis. The molecular mass of the major peak in the various samples
ranged from 4510 to 4514, and no other peaks were
present, ruling out significant oxidation, fragmentation, and
covalent cross-linking of A
. To determine whether oxidative processes mediated the formation of complexes of
A
with C3 activation fragments, complement activation
was carried out in the presence of deferoxamine, glutathione, dimethylthiourea, catalase, SOD, and catalase plus
SOD. Since none of these antioxidants or free radical scavengers inhibited the formation of or interfered with the detection of complexes (Fig. 2 d), it is unlikely that free radical-mediated or oxidative processes are involved in the formation of complexes of A
with C3 activation fragments. In all likelihood, amide bonds are responsible for
the remaining A
-C3b/iC3b complexes. A
1-42 contains two lysine residues, at positions 16 and 28, which
could mediate such linkages.
Additional studies showed that A triggered activation of
the terminal, proinflammatory portion of the complement-reaction sequence in NHS. C5a, a cytokine-like activation
cleavage product of C5 with numerous biological properties,
was efficiently generated by aggregated A
1-42 in NHS, as
determined by a specific radioimmunoassay which detects
C5a and C5a des-Arg (lacking the COOH-terminal arginine residue) (reference 26; Fig. 3 a). A sandwich ELISA in
which an mAb to a C5b-9 neoantigen located in poly C9
served as the capture Ab, and polyclonal Ab to C6 served
as the detection Ab, showed that A
-mediated complement activation led to formation of the C5b-9 complex
(Fig. 3 b). This ELISA detects C5b-9 as well as SC5b-9
complexes; the latter are formed in NHS in the absence of
cells, as a consequence of the binding of S protein, a complement control protein, to the complex. A
1-42 was
generally more efficient in generating C5b-9 than A
1-40
(Fig. 3 b). In contrast, another group recently reported that
A
-mediated complement activation does not lead to generation of the C5b-9 complex (10). The reason(s) for their
failure to demonstrate C5b-9 formation is not known. One
possibility is the well-known variability in the properties of
different A
preparations. In this regard, we have observed more variability in the ability of various A
1-40 and A
1-42 preparations to trigger C5b-9 complex formation,
than in their ability to generate A
-C3b and A
-iC3b
complexes. Other explanations could lie in slight differences in experimental conditions. For example, their C5b-9
formation experiments were carried out in NHS diluted
1:10 in phosphate-buffered NaCl; this combination provides
suboptimal concentrations of calcium and magnesium,
which are required for CCP and ACP activation. In this
regard, we obtained 10-fold higher levels of SC5b-9 formation than they obtained with 1 µM aggregated IgG in
their control studies (not shown).
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The C5b-9 complex generated by A 1-42-mediated
complement activation was able to insert into the membranes of NT2 cells, a committed neuronal precursor cell
line, when such cells were included in reaction mixtures
with aggregated A
and NHS (Fig. 4 a). Depicted are flow
cytometric analyses with rabbit Ab to activation-specific
neoantigens in the C5b-9 MAC. C5b-9 membrane insertion was likely proportional to the extent of complement activation, since it was dependent on the concentration of
A
1-42. mAb to C5b-9 neoantigens gave the same result
(not shown). Identical A
1-40 concentrations mediated
lower levels of C5b-9 membrane insertion (not shown),
probably because of the significantly lower levels of C5b-9
formation with A
1-40. NT2 cells and other neuronal
cell lines are resistant to complement-dependent cytolysis, likely because of the presence of CD59 (27), a complement
regulatory protein, a finding confirmed here. Nevertheless,
C5b-9 insertion into NT2 cell membranes mediated an increase in the permeability of the cells to propidium iodide
that was dependent on the concentration of A
1-42 (Fig.
4 b). These data indicate that C5b-9 generated by A
-mediated complement activation is functionally competent, since it inserts into the membranes of neuronal precursor
cells and renders them permeable to small molecules.
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Thus, A directly and independently activates the ACP
as well as the CCP, leading to the formation of covalent
A
-C3b and A
-iC3b complexes; generates C5a; and mediates assembly of functionally active C5b-9 complexes in
vitro. These findings have potential implications for understanding the mechanisms which lead to continuing neuronal damage and altered glial functions in the vicinity of NP, and thus to the progression of AD. First, they provide
an explanation for the association of bound C3 with A
in
NP (1), since covalently bound C3b molecules in NP
would remain bound and provide a nidus for chronic complement activation. Second, C5a generated by A
-mediated complement activation could be responsible for the
increased numbers of activated astrocytes and microglia around NP compared with diffuse A
plaques (28), since
these cells possess C5a receptors and are activated and migrate in response to C5a (6, 29). C5a could also trigger
the release of proinflammatory cytokines (IL-1, IL-6, IL-8,
and TNF-
) from glial cells, as it does from other cell types
(26, 32); proinflammatory cytokines are increased in the
AD brain (2, 28, 33). These cytokines could further activate glial cells and alter neuronal and glial functions (28,
32). Third, incoming activated glial cells could bind and
remain adherent, via their complement receptors, to C3
activation fragments attached to A
(6). Fourth, C5b-9 insertion into cell membranes provides an explanation for the association of this complex with dystrophic neurites in NP
(2, 3). Although not likely to be directly cytotoxic for neurons, since they bear CD59 (6, 34), C5b-9 as well as C5b-7
and C5b-8 complexes could alter neuronal functional
properties over time by chronic low-level triggering of various cellular signaling pathways (35). If this inflammation-based scenario is verified, complement inhibitors should be
evaluated for use in AD. Such inhibitors would need to
pass the blood-brain barrier, target both complement activation pathways, and prevent C5b-9 activation.
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Footnotes |
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Address correspondence to Neil R. Cooper, Department of Immunology, IMM-19, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: 619-784-8152; Fax: 619-784-8472; E-mail: nrcooper{at}scripps.edu
Received for publication 30 December 1997 and in revised form 27 May 1998.
We thank T. Hugli, D. Isenman, J. Rogers, and S. Webster for helpful discussions, G. Nemerow for PB50, and Athena Neurosciences, Inc. (South San Francisco, CA), for 10D5. We also thank Todd S. Bixby for his expert technical assistance.
This work was supported by National Institutes of Health grant NS-34682, and grant SFP-1141 from Novartis.
Abbreviations used in this paper
ACP, alternative complement pathway;
AD, Alzheimer's disease;
A,
-amyloid peptide;
CCP, classical complement pathway;
dd, double distilled;
MAC, membrane attack complex;
NP, neuritic plaques;
NHS, normal human serum;
SOD, superoxide dismutase.
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