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* Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110; and
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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Membrane cofactor protein (MCP)3 (CD46) is a widely expressed, intrinsically acting, 51–68-kDa regulator of the complement system (4, 5, 6). MCP and CR1 (CD35), as well as factor H (fH) and C4b-binding protein (C4BP) of plasma, serve as cofactors for the factor I-mediated cleavage of C4b and C3b. In contrast to MCP, CR1 has a limited tissue distribution, being expressed by peripheral blood cells and in tissues by B lymphocytes, follicular-dendritic cells, macrophages, and kidney epithelial cells (3, 7, 8, 9). MCP and CR1 are cofactor proteins for both C4b and C3b, while the plasma proteins fH and C4BP are specific for C3b and C4b, respectively. The resulting cleavage fragments are incapable of forming a convertase, thereby halting further complement activation. The proteolytic degradation of C4b leaves only the covalently attached C4d fragment on the target (a so-called immunologic scar as C4d possesses no known biologic activity). In contrast, the cleavage of C3b produces iC3b, which is a ligand for CR2 (CD21) (2, 10, 11), CR3 (CD11b/CD18) (12), and CR4 (CD11c/CD18) (13). These receptor-ligand interactions promote immune adherence, phagocytosis, Ag localization, and B cell signaling (12, 14).
Further dissection of these regulatory events may foster a better understanding of autoantibody-mediated tissue damage (15), ischemia-reperfusion injury (16), and apoptosis (17, 18). In these settings, complement activation occurs on host cells bearing regulatory proteins. Opsonization and clearance of immune-complexes and apoptotic cells by complement may be critical in preventing tissue deposition of proinflammatory complexes (15), and in maintaining tolerance to self-Ags (17, 18, 19). Thus, an enhanced understanding of these complement clearance mechanisms could lead to a means to reduce damage in immunopathologic reactions and to prevent loss of self-tolerance.
We previously developed a classical pathway (CP) activation model system in which C4 cleavage products were analyzed (20, 21). The goal of the present experiments was to investigate the role MCP plays vs that of the plasma regulatory proteins in modulating the deposition and degradation of C4b and C3b. To accomplish this, we activated complement on Chinese hamster ovary (CHO) cells transfected with human MCP. The results point out distinct and nonoverlapping roles for MCP vs that of the plasma regulators in the regulation of C4b and C3b deposited on host tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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CHO cells were stably transfected with the BC1 isoform of human MCP and cloned as previously reported (21). CHO cells, transfected with MCP cDNA in reverse orientation, served as a control (MCP-). Abs were from standard sources (noted for each Ab in the sections below) except for the mAb to fH. This mouse mAb (MH10) was generated using purified human fH as the Ag (J. Lambris, unpublished observation).
Quantitation of MCP expression
The quantity of MCP expressed per cell line was assessed by FACS (21). Briefly, cells were trypsinized, harvested, and washed in 1% FCS-PBS before transfer to 96-well microtiter plates (5 x 105 cells/well). TRA-2-10 (a murine mAb to MCP; Ref. 22) was added (10 µg/ml) and incubated with the cells for 30 min at 4°C. Following centrifugation and washing, FITC-goat anti-mouse IgG was added (Sigma-Aldrich, St. Louis, MO). After a 30-min incubation at 4°C, cells were resuspended in 1% FCS-PBS and analyzed by FACS. These results were compared with an ELISA (21). Clones and expression levels were designated as follows: M1 (clone 23.10; 90,000 MCP/cell), M2 (clone 23.9; 200,000 MCP/cell), and M3 (clone 23.1; 234,000 MCP/cell).
Initiation of complement activation
Standard procedure for initiation of the CP has been described (21). Briefly, CHO cells were grown to 70–80% confluency and collected by trypsinization into 1% FCS-PBS. Rabbit anti-hamster lymphocyte IgG (8.5 mg/ml; Sigma-Aldrich) was added to the cells and incubated for 30 min at 4°C. Following two washes with 1% FCS-PBS, 100 µl of C7-deficient (C7d) serum (donated by P. Densen, University of Iowa, Iowa City, Iowa) in gelatin veronal buffer was added. To block the CP, gelatin veronal buffer was used containing 10 mM EGTA and 7 mM magnesium chloride (Mg2+-EGTA). Cells were harvested at indicated time points and washed twice in 1% FCS-PBS before C4 and C3 fragment analyses. Antigenic levels of fH, C4BP, and factor I in C7d serum (measured at National Jewish Medical and Research Center, Denver, CO) were 117, 152, and 119% of normal values, respectively. C4BP functional activity in the C7d serum was comparable to that of normal human serum as assessed by monitoring cofactor activity for C4b. C6-depleted serum (Advanced Research Technologies, San Diego, CA) was also used.
FACS analysis of complement fragment deposition
Following complement deposition and washes, murine mAbs to the human complement component fragments C4c, C4d, C3c, C3d, or C5 (Quidel, San Diego, CA) were added (20 µg/ml). After a 30-min incubation at 4°C, FITC-conjugated goat anti-mouse IgG (preadsorbed with rabbit serum and rabbit-IgG agarose; both from Sigma-Aldrich) was added for 30 min at 4°C. Cells were then stained with propidium iodide and analyzed with a BD Biosciences FACSCalibur system, (BD Biosciences, Mountain View, CA). The anti-iC3b neoantigen mAb (Quidel) recognized both iC3b and C3b by FACS and Western blotting (data not shown); and thus, was not suitable for separating these fragments. C3 fragment deposition was also characterized by Western blotting.
Western blotting and fH blocking
For blocking studies, C7d serum was incubated at 4°C for 30 min with 1 mg/ml anti-fH mAb MH10 and this serum then incubated with sensitized CHO cells for 5 min. Cells were washed and then lysed with the nonionic detergent Nonidet P-40 (21) and the supernatants applied to gels containing 1 x 105 cell equivalents/lane. After SDS-PAGE, transfer to a nitrocellulose membrane, and blocking, a polyclonal goat anti-C3 Ab (Advanced Research Technologies) was added at a dilution of 1/7500 for 30 min at 37°C. This was followed by a 30-min 20°C incubation with rabbit anti-goat IgG-HRP at a dilution of 1/7500. Detection, using Super Signal West Pico Chemiluminescent substrate, was performed according to the manufacturer’s directions (Pierce, Rockford, IL).
Statistical analysis
The kinetics of cleavage by the three cell lines expressing MCP vs that of control CHO cells was compared by a hierarchical linear model analysis of variance using the statistical software Stata (Release 7) (StataCorp, College Station, TX).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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CP activation
For most experiments, CHO cells expressing human MCP were sensitized with 8.5 mg/ml of IgG and incubated in 10% human serum deficient in C7. The fluid phase complement regulators fH and C4BP were in the C7d serum.
MCP is the cofactor for the cleavage of deposited C4b.
The appearance and processing of C4b was determined by FACS using mAbs to
its C4c and C4d derivatives (Fig. 1
). No
C4b cleavage was detected on the MCP- cells as
deduced from the C4c:C4d ratio which was 
1.0. However, in the case
of cells bearing MCP, a majority of the C4b was cleaved to C4c and C4d
by 1 h. These findings indicate that MCP (and not C4BP) is
responsible for most of the C4b cofactor activity. Comparable results
were obtained with C6-depleted serum (data not shown) and with serum
concentrations from 20 to 0.05%).
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A). However,
subsequent C4b cleavage was slow, being approximately linear over a
30-min period (Fig. 2, A and B). After 45 min, no
further cleavage was observed, although 18–30% of the C4b remained
intact (Figs. 1
and 2B). Cells expressing 234,000 copies of
MCP/cell (cell line M3) produced more rapid C4b-cleavage compared with
cells expressing 90,000 copies of MCP/cell (cell line M1) (Fig. 2A), but this trend was not statistically significant
(p = 0.46).
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). C3b and C5b deposition were
similar in the MCP- and
MCP+ cell lines. Thus, the presence of MCP did
not influence the formation or stability of CP C3 or C5 convertases
because the inactivation of C4b by MCP occurs too slowly to impact the
activity of the CP C3 and C5 convertases.
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).
First, the cleavage of C3b was much more rapid than that of C4b; that
is, at 5 min the C3 
' fragment was barely detectable
(lanes 6 and 7). Second, as also shown in lane
6, the 1 and 2
fragments of iC3b were generated by the MCP-
cells. Therefore, the generation of iC3b was likely due to cofactor
activity of the serum protein fH. To test this, the effects of a mAb to
human fH were examined. As observed in Fig. 5, lanes 5 and 7,
the anti-fH mAb inhibits the appearance of iC3b fragments in the
MCP- and MCP+ cells,
indicating the prime importance of fH in this process. Interestingly,
under conditions in which fH is inhibited, some MCP-dependent
processing is observed (compare ' in lane 5 vs
7).
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1 fragment of
iC3b. As visualized in Figs. 4 and 5, a substantial quantity of
1 is present at its native m.w.,
indicating attachment to the cells either noncovalently or through
small molecules. However, scans of these Western blots demonstrated
4-fold less native-sized 1 than predicted from
the amount of 2, suggesting that a majority of
1 is attached to higher m.w. membrane
constituents. Lastly, the Western blot analysis indicates nearly
identical quantities of C3b deposited on MCP+ and
MCP- cells (compare the 
-,
1-, and 2-chains in lanes 4 and 6 of Fig. 5). This finding confirms
those made in the analyses by FACS.
MCP reduced C3b deposition at low serum concentrations.
As
the concentration of serum was reduced, C4b and C3b deposition also
decreased (Fig. 6). C4b deposition was
detectable at 
0.05% serum, while C3b deposition was detectable at
0.25% serum. At serum concentrations of 1 and 0.5% (where the AP
components are too dilute to be active), MCP+
cells did reduce C3b deposition. We interpret this to mean that in the
setting of limiting amounts of C4b deposition and C2 activation, the
activity of CP C3 convertase can be inhibited by MCP.
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MCP inhibits the AP C3 convertase. As the Ab used to sensitize the
cells was reduced (Fig. 7), CP
activation, as monitored by the quantity of C4d deposited,
progressively decreased. At lower Ab concentrations (1 mg/ml IgG), the
deposition of C3 fragments was inhibited >95% in the
MCP+ compared with the
MCP- cells. Here C3d was monitored because like
C4d, it reflects all of the parent C3b that had been deposited.
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, compare A and
C), C3b was deposited on the MCP-
cells to nearly the same extent as in the standard CP-driven activation
condition. In contrast to the CP (Fig. 8A), AP activation
was severely inhibited by MCP, as C3b deposition was 10-fold less on
the MCP+ cells (Fig. 8C). Thus, MCP is
a potent inhibitor of the AP C3 convertase. Furthermore, at a lower
level of sensitization (1 mg/ml), MCP inhibited C3b deposition by
>90% (Fig. 8B). In this case, the feedback loop is
responsible for most of the C3b deposition. If the CP is also blocked
(Fig. 8D), there is no C3b deposition on
MCP+ cells, indicating complete inhibition of the
AP by MCP at a lower level of cell sensitization.
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Regulation of the complement activation pathways is designed to allow unimpeded activation on an appropriate target, but to minimize attachment of C4b and C3b on self (1). Although the complement system’s main evolutionary goal is to optimize interactions with a microbial target, activation on self-tissue occurs at low levels continuously and, more substantially, in the setting of autoantibodies, ischemia-reperfusion injury (16), apoptosis (17, 18), and inflammation. In these situations, the host membrane inhibitors are present.
The goal of this study was to enhance our understanding of the regulation and inactivation of C4b and C3b deposited on self-tissue. Therefore, following Ab-mediated activation of complement, the quantity and nature of complement fragments on cloned cell lines transfected with human MCP were analyzed. Cleavage of C4b by factor I releases C4c, while C4d remains surface-bound. This reaction is promoted by a cofactor, either MCP expressed on the same cell or C4BP in the serum or a combination thereof. Cleavage of cell-bound C3b by factor I requires MCP, fH, or both, and produces iC3b, which remains target bound. Because CHO cells display no intrinsic regulatory activity on their cell surface for human C4b or C3b, we could address which cofactor assists factor I in cleaving C4b and C3b.
C4b fragment deposition and degradation
First, there was no difference in the quantity of C4b deposited
between the MCP+ and MCP-
cells. This result was expected because MCP cleaves already deposited
C4b (6). Second, on MCP+ cells, C4b
cleavage continued in a linear fashion for 30–45 min. The rate of C4b
cleavage was relatively slow, especially compared with C3 and C5
activation by their CP convertases. Except in dilute serum, MCP did not
influence the quantity of C3b and C5b deposited. Third, there was no
detectable cleavage of C4b by the MCP- cells.
This result indicates that C4BP does not serve as a cofactor for
cleavage of C4b in this system. Fourth, C4b cleavage was incomplete,
even by the high MCP-expressing lines, as 18–30% remained after
2 h. How this deposited C4b escapes inactivation is under
investigation. One possibility, as has been demonstrated for C3b and
fH, is that such C4b may be attached to the sensitizing IgG, and
thereby be less accessible to a cofactor protein (24).
Despite the presence of C4BP in the serum, as noted above, there was no cleavage of C4b on the surface of MCP- cells. This was surprising because C4BP accelerates the decay of the CP C3 and C5 convertases and is a cofactor for the factor I-mediated cleavage of C4b (25, 26, 27, 28, 29, 30, 31).
Four possible explanations were considered and ruled out to account for this lack of an effect of C4BP on cell-bound C4b. First, in the serum source used, C4BP was present in the expected quantities and was appropriately active against fluid phase C4b. Thus, the protein was antigenically and functionally intact. In addition, experiments using a second (C6-depleted) serum source produced the same result. Second, factor I was present in the expected antigenic amounts and was functionally active, as indicated by its cleavage of C4b and C3b with other cofactors. Third, the quantity of C4b deposited as documented by FACS was of such a magnitude that C4BP binding should have occurred. Fourth, competitive inhibition by C2 (30, 31) was inconsequential, as there is 15–25% more C4BP than C2 in human serum and, in the presence of factor I, C4BP is not consumed in its reaction with C4b. Consequently, MCP, and not C4BP, was responsible for cleavage of C4b on CHO cells. We suggest that this is the case with most host cells.
C3b deposition and degradation by CP activation
Except for very dilute serum where a modest effect was observed,
deposition of C3b was unaffected by MCP expression in the setting of CP
activation. The reason for this is that the rate of C4b cleavage by MCP
was too slow to alter the activity of the C3 convertases. This result
is also in concert with those of Devaux et al. (32), who
found no effect of MCP (5000 copies/cell) expression on CP-mediated
lysis. In our experimental system, we used MCP cell lines expressing
90,000, 200,000, and 234,000 copies/cell, and also saw no effect.
However, these results appear to be in conflict to earlier experiments
in which an inverse relationship between MCP expression and C3b
deposition in mouse fibroblast cells exposed to human serum
(6) and the inhibition of CP-mediated lysis by MCP in
several other experimental systems (6, 32). In one of
these (6), cell lines were used with much more MCP (1
x 106 copies/cell), and lysis was monitored, but
not C4b and C3b deposition. More importantly, normal human serum was
used as a source of Ab compared with the high titer anti-CHO Ab
used in this study (32). Using human serum in this manner,
the feedback loop of the AP contributes to C3b deposition (see next
section) and MCP has been shown by several groups to be
efficacious in inhibiting the AP C3 and C5 convertases
(32, 33, 34, 35).
Western blots indicated that C3 fragments on the surface of
MCP- and MCP+ cells
consisted predominantly of iC3b. Therefore, fH and not MCP was the
responsible cofactor. The experiments using a function-blocking mAb to
fH further established this conclusion. These results parallel
observations using a human melanoma cell line expressing moderate
amounts of decay-accelerating factor and low amounts of MCP
(5 x 103 copies/cell) in which the CP was
activated by IgG and purified components served as the complement
source (36, 37). In these studies, fH was responsible for
iC3b generation. These data are also consistent with several reports
that, once cells are heavily coated with C3b, fH binds efficiently
(37, 38). We conclude that following deposition of
relatively large amounts of C3b, fH is primarily responsible for the
cleavage of C3b to iC3b on host tissue. Of note, this reaction occurs
more rapidly than C4b cleavage by MCP. Our studies suggest that some of
the higher m.w. fragments represent C3b -chain fragments (' or
1) bound to the sensitizing IgG or other
membrane constituents (39, 40, 41). Interestingly, a
surprisingly large fraction of ' or 1 were
either not covalently attached or bound to small molecules.
Role of MCP in complement regulation: surveillance and inhibition
of the AP and the feedback loop
In prior reports, MCP was shown to protect only the cell on which
it is bound, so-called intrinsic regulatory activity (6, 35). MCP has a preferential ability to inactivate C3b bound to
large protein complexes, including the AP C5 convertase (34, 35, 42). In studies reported herein, MCP was shown to be a potent
inhibitor of AP activation and of the feedback amplification loop.
Taken together, these data suggest a surveillance role for MCP on host
cells. For example, we envision that MCP patrols the surface of host
cells where it seeks out C4b and C3b deposited during an
inflammatory/immune reaction or from the spontaneous turnover of C4 and
C3 (1, 6). In this regard, MCP continuously monitors the
cell surface to prevent the AP and its feedback loop from being
engaged. MCP’s role in the regulation of the AP has been repeatedly
suggested (32, 33, 34), especially its ability to inhibit the
feedback loop (32). A role for CP-mediated cytoprotection
or inhibition of lysis by MCP also has been shown (6, 21, 34, 43, 44). However, our results indicate that this effect on the CP is
likely to only be operative if the AP feedback loop is engaged or
perhaps if very large quantities of MCP are expressed. In the present
studies, we directly show that if the AP or feedback loop is required
for C3b deposition, MCP is a potent inhibitor of this reaction.
A new role for MCP is suggested by these investigations; namely, that MCP is responsible for inactivating most of the C4b bound to self-tissue. In contrast, the plasma protein fH is the responsible cofactor if large quantities of C3b are deposited. However, as noted, our data also strongly support an important role for MCP in the inactivation of low-level C3b deposition to prevent AP engagement (32, 33, 34, 35).
In summary, the striking frequency of autoimmunity, especially systemic lupus erythematosus, in early complement component deficiency states implies a critical role for these proteins in maintaining homeostasis in the immune system. The deposition and degradation of C4b and C3b may not only be pivotal in triggering the innate immune response and in instructing adaptive immunity, but also in the negative selection of self-reactive B cells (45). Consequently, further analysis will focus on experimental systems using both decay-accelerating factor and MCP with human autoantibodies and autoantigens. These types of studies should shed light on the role played by C4b and C3b and their limited degradation products in normal and aberrant immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to John P. Atkinson, Division of Rheumatology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8045, St. Louis, MO 63110-1093. E-mail address: jatkinso{at}im.wustl.edu
3 Abbreviations used in this paper: MCP, membrane cofactor protein; C4BP, C4b-binding protein; fH, factor H; CHO, Chinese hamster ovary; CP, classical pathway; AP, alternative pathway; C7d, C7 deficient.
Received for publication June 20, 2001. Accepted for publication April 17, 2002.
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References |
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1H in proteolysis of C4b and C3b. J. Exp. Med. 150:267.This article has been cited by other articles:
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, C4c; 
, C4d. M1, M2, and M3 represent CHO
cell lines expressing 90,000, 200,000, and 234,000 copies of MCP,
respectively. Data represent mean ± 1 SE for five
experiments.
