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The Journal of Immunology, 2007, 179, 4101 -4109
Copyright © 2007 by The American Association of Immunologists, Inc.
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Pathogenic Complement Activation in Collagen Antibody- Induced Arthritis in Mice Requires Amplification by the Alternative Pathway1

Nirmal K. Banda*, Kazue Takahashi{dagger}, Allyson K. Wood*, V. Michael Holers* and William P. Arend2,*

* Division of Rheumatology B115, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045; and {dagger} Developmental Immunology, Pediatric Services, Massachusetts General Hospital, Boston, MA 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune complex-induced inflammation can be mediated by the classical pathway of complement. However, using mice genetically deficient in factor B or C4, we have shown that the collagen Ab-induced model of arthritis requires the alternative pathway of complement and is not dependent on the classical pathway. We now demonstrate that collagen Ab-induced arthritis is not altered in mice genetically deficient in either C1q or mannose-binding lectins A and C, or in both C1q and mannose-binding lectins. These in vivo results prove the ability of the alternative pathway to carry out pathologic complement activation in the combined absence of intact classical and lectin pathways. C3 activation was also examined in vitro by adherent collagen-anti-collagen immune complexes using sera from normal or complement-deficient mice. These results confirm the ability of the alternative pathway to mediate immune complex-induced C3 activation when C4 or C1q, or both C1q and mannose-binding lectins, are absent. However, when all three activation pathways of complement are intact, initiation by immune complexes occurs primarily by the classical pathway. These results indicate that the alternative pathway amplification loop, with its ability to greatly enhance C3 activation, is necessary to mediate inflammatory arthritis induced by adherent immune complexes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Renewed interest in the role of B cells in the pathogenesis of rheumatoid arthritis (RA)3 has been stimulated by the recent observation that depletion of CD20-bearing B cells with a specific mAb led to substantial clinical improvement and occasional remissions (1). The importance of autoantibodies in RA has been further established by recent studies describing the presence of Ab to citrullinated peptides (anti-cyclic citrullinated peptide (CCP)) in the sera of RA patients for many years before the onset of clinical disease. The role of Ab to citrullinated epitopes in induction of synovitis in RA has not yet been clearly established, but these Ab can amplify injury in the setting of mild synovitis in collagen-induced arthritis (CIA) and in collagen Ab-induced arthritis (CAIA) in mice (2).

Abs to type II collagen (CII) are found in RA and may form immune complexes (IC) in the joint that are involved in local initiation of inflammation early in the disease process in a subset of patients (3). Epitopes of CII are present on the cartilage and synovium of RA patients and Ab to CII are expressed by synovial tissue and synovial fluid cells in RA, indicating local production (4, 5, 6). Extracts from rheumatoid articular cartilage demonstrate primarily rheumatoid factor activity and Ab to native and denatured CII (7). Autoantibodies to native CII are also present in RA sera but IgG binding to denatured CII is probably due to IgG-fibronectin complexes (8). Adherent immune complexes (adIC) formed from anti-CII Ab from early RA patients are biologically active as they bind to Fc{gamma}RIIa on monocytes in vitro and induce the production of TNF-{alpha}, IL-1beta, and IL-8 (9).

Complement activation is thought to play an important proinflammatory role in the joints of patients with RA (10). Immunoglobulins and complement components are adherent to articular cartilage, hyaline cartilage, and periarticular collagenous tissues (7, 11, 12). Activation fragments of both the classical pathway (CP) of complement as well as the alternative pathway (AP) are found in rheumatoid synovial fluids (10, 13). Local production of C3, factor B, C3aR, and C5aR can be demonstrated in rheumatoid synovial tissues, with mRNA for these components and receptors found in all parts of the tissue (14). Thus, it is likely that complement components and receptors are synthesized locally in the rheumatoid joint with adIC at least partially responsible for local complement activation.

Although the CP is assumed to play a major role in RA, involvement of the AP and the lectin pathway (LP) have also been suggested (15). All three pathways of complement activation result in the cleavage of C3 into C3a and C3b (16). The CP of complement activation is initiated by binding of the C1 complex (containing C1q, two molecules of C1r, and two molecules of C1s) through C1q to an Ab bound to a cell surface Ag. The association between C1q, C1r, and C1s in the C1 complex requires Ca2+. The sequential proteolysis of C1r and C1s follows with activated C1s then cleaving C4 and C2. The active fragments of these components bind to sites on the target surface and generate a C4b2a enzyme complex, designated the CP C3 convertase, that cleaves C3. The AP exhibits spontaneous generation of C3b in vivo through low-grade hydrolysis in a process called the "tickover" mechanism. C3b generated in the fluid phase through the tickover mechanism can covalently bind to hydroxyl groups on cell surface carbohydrates or on IgG. Factor B subsequently binds this C3b to form a C3bB complex, and factor D then cleaves the bound factor B to form the AP C3 convertase C3bBb. The binding of factor B to C3b requires Mg2+. The effects of the AP are amplified greatly by binding of properdin to C3b, stabilizing the enzyme and enabling cleavage of many molecules of C3 to C3b that bind covalently to the target surface around the initial site of complement activation. In fact, the AP may function primarily as an amplification loop after generation of C3b by all three pathways. Some newly formed C3b then binds to C4b and C3b in the CP and AP C3 convertases, forming C5 convertase enzymes. CP and AP C5 convertases cleave C5 and generate the potent anaphylatoxin C5a as well as the target-bound C5b that initiates formation of the C5b-9 membrane-attack complex. The LP is initiated by carbohydrates containing mannan or N-acetylglucosamine expressed on microorganisms or on other targets including altered self in the context of apoptotic and necrotic cells. These targets are bound by a complex of mannose-binding lectin (MBL) and MBL-associated proteases (MASP 1, MASP 2, and sMAP). Upon target binding, the associated proteases cleave C2 and C4 to generate the CP C3 convertase C4b2a, in a similar fashion as with C1r and C1s.

The roles of Ab and IC in mouse models of RA have recently been reviewed (17, 18), as have the roles of complement activation and inhibition in these models (19). The role of IC in the induction of inflammatory arthritis has been characterized in two different experimental murine models of disease, the K/BxN (KRN TCR transgenic mice on the C57BL/6 x NOD background) model and CIA. Acute arthritis can be induced in naive mice by the passive transfer of sera from diseased mice containing IgG Ab to glucose-6-phosphate isomerase (GPI, K/BxN model), by sera containing IgG Ab to collagen (CAIA), or by a cocktail of mAb to collagen (CAIA) (20, 21). In CAIA, transferred Abs to CII bind to specific epitopes expressed on the superficial layer of articular cartilage to form adCII-IC. Arthritis in the K/BxN model is mediated primarily by soluble IC, as extracellular GPI is found in most body fluids, whereas CIA and CAIA are mediated solely by adCII-IC. Patients with RA demonstrate both soluble and adherent IC. However, these two types of IC may exhibit different requirements for induction of inflammation and tissue damage.

Using mice genetically deficient in factor B (Bf–/–), it has been demonstrated by two different laboratories, including our own, that the AP of complement is required for mediation of inflammation and tissue damage in CAIA (22, 23). The present experiments have further explored the role of the AP in CAIA using mice deficient in C3, C1q, MBL, or in both C1q and MBL. The results show the development of robust arthritis in the absence of C1q, MBL, or both C1q and MBL, indicating for the first time, to our knowledge, that the AP alone can carry out inflammation and tissue destruction in vivo in the absence of the CP or LP, or of both pathways. To further understand the mechanisms of this role of the AP, we developed an in vitro assay for C3 activation by adCII-IC containing bovine CII and mAb to CII. Results of the in vitro studies substantiate that the AP is capable of activating C3 in the absence of C1q or C4, or of both C1q and MBL. However, C3 activation in vitro does not require the AP as the CP is primarily responsible for initiation of C3 activation when all three pathways of complement are intact. The possible reasons for the observed differences in AP requirement for C3 activation induced by adCII-IC in vivo vs in vitro are discussed. These observations may clarify the mechanisms whereby adIC initiate inflammation and tissue damage in human diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and sera

Ten- to 12-week-old C57BL/6 male or female mice were used for this study. C57BL/6 mice homozygous for gene deletions of MBL-A and -C (MBL–/–), C1q (C1q–/–), and both C1q and MBL-A and -C (C1q–/–/MBL–/–) were bred by Dr. Kazue Takahashi at the Massachusetts General Hospital (Boston, MA). C57BL/6J mice homozygous for a gene deletion of C3 (C3–/–) were originally obtained from Dr. Michael Carroll (Department of Pediatrics, Harvard Medical School, Boston, MA). Age-matched wild-type (WT) C57BL/6 male or female mice were used as controls (Jackson Laboratories). Sera from gene deleted mice were obtained from the following sources: MBL–/– and C1q–/–/MBL–/– from Massachusetts General Hospital or from a colony at University of Colorado at Denver and Health Sciences Center (UCDHSC); C1q–/– from Dr. Marina Botto (Imperial College of Medicine, London, U.K.); and C3–/–, C4–/–, and Bf–/– from breeding colonies at UCDHSC. All animals were kept in a barrier animal facility at UCDHSC with a climate-controlled environment having 12-h light/dark cycles. Filter top cages were used with three mice in each cage. During the course of this study, all experimental mice were fed breeder’s chow provided by the Center for Laboratory Animal Care, UCDHSC.

Collagen Ab-induced arthritis

CAIA was induced by the i.p. injection of mAb to CII (Arthrogen-CIA), as recently described (23). Arthrogen-CIA (Chondrex) is an arthritis-inducing mixture of four mAb to anti-CII resuspended in sterile Dulbecco’s PBS. All four mAb in this mixture recognize the conserved epitopes (CB11) shared by various species of CII and cross-react with homologous and heterologous CII. Three of these four mAb (IgG2a: clones F10-21, A2-10, D8-6)) recognize antigenic epitopes clustered within an 84 aa residue fragment, LyC2, of CB11, and the fourth mAb (IgG2b: clone D1-2G) reacts with LyC1. Eight milligrams of Arthogen-CIA per mouse were injected i.p. in each study. Four separate experiments were performed with C3–/–, MBL–/–, C1q–/–, and C1q–/–/MBL–/– mice, each with age- and sex-matched control C57BL/6 mice. These experiments were performed as recently described, with an i.p injection of 50 µg of LPS (from Escherichia coli strain 011B4) at day 3 to synchronize the onset of arthritis (23). No toxicity of anti-CII Ab or LPS was observed during the course of this experiment. Female C57BL/6 mice developed disease equivalently to male mice with this procedure. All mice started to develop arthritis at day 4 and were sacrificed at day 10. This procedure has been approved by the institutional animal review committee at UCDHSC.

Assessment of clinical disease activity

The severity of clinical disease activity both in gene deleted and WT mice was determined every day by two trained laboratory personnel acting independently and blinded to the mouse type, as recently described (23). The clinical disease activity was scored on a 3-point scale per paw: 0 = normal joint; 1 = slight inflammation and redness; 2 = severe erythema and swelling affecting the entire paw with inhibition of use; and 3 = deformed paw or joint with ankylosis, joint rigidity, and loss of function. The total score for clinical disease activity was based on all 4 paws and was a maximum of 12 for each mouse.

Histopathology of knee joints

At day 10, both forepaws and the entire right hind limb, including the paw, ankle, and knee, were surgically removed from all mice and fixed immediately in 10% buffered formalin (Biochemical Sciences). The preparation of tissue samples and histological analysis were performed as previously described (23). The histological sections were read by a trained observer who was also blinded to the mouse types and to the clinical disease activity score of each mouse. The joint sections were scored for the changes in inflammation, pannus, cartilage damage, and bone damage, on a scale of 0–5. Each parameter was represented as the mean value for 5 joints per mouse, and the overall histological damage score was calculated as the total of the four individual parameters.

Immunohistochemistry for C3 deposition

At sacrifice on day 10 the left ankle and paw were excised and fixed in formalin. C3 deposition in the joints of gene deleted and WT mice joints was immunohistochemically localized using polyclonal goat anti-mouse C3 Ab (ICN Pharmaceuticals), as recently described (23). Scoring for C3 staining was performed on the synovium and surrounding tissues combined and separately on the cartilage. The synovium and surrounding tissue were scored using a 3-point scoring system in which 0 represented no staining and 1, 2, and 3 represented low, moderate, and high staining, respectively. The criteria for cartilage staining was as follows: 0, no staining present; 0.5, one area of minimal staining of chondrocytes in one joint; 1, one area of moderate staining of chondrocytes in one joint; 2, multiple areas of moderate staining of chondrocytes with multiple joints affected; and 3, multiple areas of intense staining of chondrocytes and/or diffuse multifocal staining of articular cartilage lesions.

Complement protein levels in mouse sera

For determination of C3 levels, 96-well Costar (Corning) plates were coated with 1.25 µg/ml a rat mAb to mouse C3 (Cell Sciences) in 0.1 M sodium carbonate buffer (pH 9.5) at 4°C overnight. The wells were then washed three times with 1x PBS containing 0.05% Tween 20 and incubated with 200 µl of 1% BSA in PBS for 1 h at room temperature. The wells were washed three times and 100 µl of serum samples diluted 1/2000 in sodium barbital buffer was added to each well and incubated at room temperature for 1 h. The wells were washed three times and 100 µl of a secondary Ab, HRP-conjugated goat anti-mouse C3 (Cappell), diluted 1/2000 was added to each well and incubated for 1 h at room temperature. The wells were then washed five times for 30 s each and 100 µl of a 1/1 solution of tetramethylbenzidine (TMB) with 1% H2O2 was added to each well. The reaction was stopped after 3–7 min with 50 µl of 2 N H2SO4. Plates were read at 450 nm subtracting the values at 550 nm.

C1q levels were measured using 96-well Costar plates coated with 10 µg/ml polyclonal sheep anti-human C1q Ab (The Binding Site) in 0.1 M sodium carbonate buffer at 4°C overnight. The procedure was conducted as described above for the C3 ELISA except the serum samples were diluted 1/100. The secondary Ab, a rat mAb to mouse C1q (Cell Sciences), was used at a dilution of 1/2000, followed by a tertiary Ab, HRP-conjugated goat-anti rat IgG (Cell Sciences), diluted 1/4000.

C4 levels were measured by coating 96-well Costar plates with 10 µg/ml polyclonal sheep anti-human C4 Ab (Abcam) in 0.1 M sodium carbonate buffer at 4°C overnight. The procedure was again conducted as described above for the C3 ELISA except the serum samples were diluted 1/45. The secondary Ab, a rat mAb to mouse C4 (Cell Sciences), was used at a dilution of 1/2000, followed by a tertiary Ab, HRP-conjugated goat-anti-rat IgG (Cell Sciences) diluted 1/4000.

MBL-A and -C levels in sera were measured using commercial ELISA kits containing standards with values expressed as micrograms per milliliter (Cell Sciences). Factor B levels were estimated by Western blot analysis using a mAb specific for mouse factor B, which neutralized AP activity in vitro and in vivo (24).

C3 deposition onto zymosan

AP-mediated C3 activation was determined by measuring the deposition of C3 onto zymosan-coated microtiter wells. 96-well Costar plates were coated with 100 µl of 2 x 107 particles/ml zymosan (Sigma-Aldrich) in 0.1 M sodium carbonate buffer and incubated at 4°C for 24 h. Plates were then washed four times with 1x PBS containing 0.5% Tween 20. Serum samples were diluted 1/100 in 1x PBS, 5 mM MgCl2, 10 mM EGTA, and 100 µl was added to each well and incubated for 45 min at 37°C. The wells were then washed as previously described. C3 deposition was detected by addition of 100 µl of a 1/2000 dilution of HRP-conjugated goat anti-mouse C3 Ab (Cappell) to each well and incubation at room temperature for 1 h. The wells were washed five times for 30 s each and 100 µl of a 1/1 solution of TMB with 1% H2O2 was added to each well. The reaction was stopped after 15–25 min with 50 µl of 2 N H2SO4. Plates were read at 450 nm subtracting the values at 550 nm. The specificity of the assay was determined by the addition of 0.4 µg of a monoclonal anti-mouse factor B Ab to the dilution buffer and incubation with the serum samples on ice for 10 min before addition to the wells.

C3 activation induced in vitro by adCII-IC

The levels of C3 activation induced in vitro by adCII-IC were measured by ELISA. 96-well ELISA plates (Nunc) were precoated for 24 h with ELISA grade bovine CII (25 ug/ml) in collagen dilution buffer (Chondrex). ELISA plates were washed six times with PBS/0.05% Tween 20, then blocked for another 24 h with 300 ul of 20% BSA (ELISA grade; Sigma-Aldrich) in PBS/0.05% Tween 20. After washing again for six times, plates were incubated for 1 h at room temperature with 25 ug/ml anti-CII Ab (Arthrogen-CIA) in 1% BSA/PBS/0.05% Tween 20. Again after washing six times, serum samples were added to the wells diluted in veronal saline buffer (VSB) plus 1 mM MgCl2 and 2 mM CaCl2 and incubated at 37°C for 1 h. Following washes in PBS/0.5% Tween 20, HRP-conjugated goat anti-mouse C3 Ab (MP Biomedicals) was added to the wells. The anti-C3 Ab was diluted (1/4000) in freshly prepared PBS/0.05% Tween 20 to minimize the background levels. Plates were incubated at room temperature for another 1 h. After six more washings, the color reaction was developed for 8 min by adding 100 ul/well TMB substrate reagent mix (1/1) (BD Pharmingen). The reaction was stopped by adding 50 ul/well 2 N H2SO4 solution. Absorbance was read at 450 nm subtracting the values at 550 nm. In all experiments, C3 activation was also measured under identical conditions by using adCII alone without anti-collagen Ab; these values varied between experiments but were usually ~10% of the total OD using the adCII-IC. In some experiments, the AP was blocked by incubating the serum samples on ice with 0.4 µg of a specific inhibitory mAb to murine factor B (1379) for 15 min before addition of samples to the ELISA plate. Other experiments were conducted in the absence of Ca2+, using PBS with 5 mM MgCl2 and 10 mM EGTA, and sera from WT or various gene-deficient mice. Data were expressed by using the following formula: mean OD of adCII-IC minus OD of adCII alone. Either adCII alone or adCII-IC using all buffers in the absence of sera exhibited minimal background under these ELISA conditions.

Statistics

Statistical analyses were conducted as previously described with Student’s t test used to determine levels of significance (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CAIA in WT, C3–/ –, C1q–/ –, MBL–/ –, and C1q–/–/MBL–/– mice

Complement factor B is required in CAIA as mice genetically deficient in factor B (Bf–/–) failed to develop more than minimal arthritis (22, 23). These results indicated that in the presence of intact CP and LP, the AP was essential for induction of clinical disease. All three pathways of complement activation require C3 for subsequent cleavage of C5, with the simultaneous generation of the inflammatory fragment C5a and stimulation of the C5b-9 membrane attack complex. To confirm a requirement for C3 in CAIA, a mixture of 4 mAb to CII were administered i.p. to normal C57BL/6 mice or to C3–/– mice. At day 3 all mice were injected i.p. with LPS to cycle the development of arthritis. The mice were examined daily for disease activity and sacrificed on day 10. All WT mice developed disease beginning on day 5, reaching a disease activity score of 11.8 ± 0.3 (mean ± SEM, n = 4) on day 10 (Fig. 1, A and B). The C3–/– mice exhibited a slight delay in onset, although the incidence was also 100% (Fig. 1A). However, the clinical disease activity score was significantly reduced in C3–/– mice at days 6 through 10, with a 70% decrease at day 10 in comparison to WT (Fig. 1B).


Figure 1
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  		FIGURE 1. CAIA in WT and complement-deficient mice. A mixture of four mAb to CII were injected i.p. on day 0 followed by an i.p injection of LPS on day 3 to cycle the development of arthritis. The data are expressed as days after Ab injection vs the incidence (%) of arthritis in the left panels and disease activity score in the right panels. A and B, WT and C3–/– mice; C and D, WT and C1q–/– mice; E and F, WT and MBL–/– mice; G and H, WT and C1q–/–/MBL–/– mice. The data represent the mean ± SEM based on: n = 4 for WT and C3–/–, WT and C1q–/–, and WT and C1q–/–/MBL–/– mice; and n = 7 for WT and MBL–/– mice. In B, C3–/– vs WT disease activity score, p < 0.02 at day 6, and p < 0.005 at days 7 through 10.

 
Our previous results showed no reduction in the incidence or activity of arthritis in CAIA in C4–/– mice (23). Because both the CP and LP require C4, this result suggested that neither pathway was required for disease. To further explore the role of the CP in this model, anti-CII Ab and LPS were similarly administered to C1q–/– mice. Similar to C4–/– mice, the C1q–/– mice demonstrated no decrease in the incidence of arthritis or in the disease activity score in comparison to WT mice (Fig. 1, C and D).

Next, we examined possible involvement of the LP as this pathway also generates the CP C3b convertase. To explore the role of the LP of complement activation in CAIA, anti-CII Ab were administered to mice lacking both MBL-A and -C (two forms of MBL are present in mice whereas humans possess only MBL-A (25). The MBL–/– mice demonstrated no change in the onset or incidence of inflammatory arthritis (Fig. 1E) or in the clinical disease activity score in comparison with WT mice (Fig. 1F). Last, to examine whether the AP alone was capable of mediating arthritis in this model, mice deficient in both the CP and LP (C1q–/–/MBL–/–) were studied. These results indicated no change in the incidence or clinical disease activity of arthritis in comparison to WT mice (Fig. 1, G and H).

Histological examination of the joints from the WT, C3–/–, C1q–/–, MBL–/–, and C1q–/–/MBL–/– mice used in these experiments showed changes that were consistent with the clinical disease activity scores (Table I). Significant decreases in the scores for inflammation, pannus, cartilage damage, and bone damage were observed in the C3–/– mice in comparison to WT. However, the joints from mice deficient in C1q or MBL-A/C, or in both C1q and MBL-A/C, exhibited no differences in pathological changes in comparison with WT. In addition, levels of C3 deposition in the joints paralleled the degrees of inflammation and tissue destruction. Mice lacking C3 showed no C3 deposition, whereas C1q–/–, MBL–/–, and C1q–/–/MBL–/– mice demonstrated no change in the levels of C3 deposition in comparison to WT (Table II).


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  		Table I. Histopathology scores in WT and complement-deficient micea

 

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  		Table II. C3 deposition in the joints of WT and complement-deficient micea

 
These results indicate that neither the CP nor the LP are essential for the development of arthritis in CAIA in the presence of an intact AP. In addition, the AP alone is capable of mediating arthritis in this IC model in the single or combined absence of the CP and LP.

Levels of complement components

As a foundation for these studies, and to investigate any unanticipated complement deficiency states following gene targeting, we determined the absolute levels of C3, C4, C1q, MBL-A, MBL-C, and factor B in WT sera and in sera from mice where the genes for specific complement components were deleted. All of the complement component values were expressed in OD units, because of a lack of recombinant proteins for use as standards, except for MBL-A and MBL-C that were expressed in micrograms per milliliter. The levels of C1q in the WT sera were heterogeneous, varying from 0.35 to 2.19 OD units (data not shown). The levels of C1q were absent in the sera from C1q–/– and C1q–/–/MBL–/– mice and 70% decreased in the C4–/– sera, all in comparison to WT (Table III). C3 was not detected in the C3–/– sera and the levels were slightly higher in each of the remaining sera from mice deficient in other complement components in comparison to WT. C4 was not detected in the C4–/– sera. The C4 levels were 20% decreased in the C3–/– sera, 80% decreased in the C1q–/– sera, and 90% decreased in the C1q–/–/MBL–/– sera, all in comparison to WT. The levels of C4 in the MBL–/– sera were quite heterogeneous, varying from 0.25 to 2.26 OD units (data not shown). The levels of MBL-A were at the ELISA background in the MBL–/– sera, absent in the C1q–/–/MBL–/– sera, and 40% decreased in the C1q–/– and C3–/– sera. The levels of MBL-C were absent in the MBL–/– and C1q–/–/MBL–/– sera and 60% increased in the Bf–/– sera. An ELISA for determination of factor B levels in sera could not be developed, but by Western blot analysis factor B was absent in the Bf–/– sera and present at equivalent levels in the remaining sera. The major unexpected results were: the heterogeneity in C1q levels in the WT sera, the marked decreases in C1q levels in the C4–/– sera, the decreases in C4 levels in the C1q–/– and C1q–/–/MBL–/– sera, the heterogeneity in C4 levels in the MBL–/– sera, and the increases in MBL-C levels in the Bf–/– sera.


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  		Table III. Levels of complement components in sera from WT and complement-deficient mice

 
C3 activation induced by zymosan

To characterize the properties of sera from mice deficient in specific complement proteins, activation by solid-phase zymosan was examined. Zymosan specifically activates complement through the AP in the absence of Ca2+ required by both the CP and LP. We examined C3 deposition on a plate coated with zymosan particles, mediated by the various sera incubated in the presence of Mg2+ and EGTA and absence of Ca2+. The results showed high levels of C3 activation using 1/100 dilutions of the WT, C4–/–, C1q–/–, MBL–/–, and C1q–/–/MBL–/– sera, indicating the presence of an intact functional AP in these sera (Fig. 2). Little to no C3 activation was observed with 1/10 (data not shown) or 1/100 dilutions of the C3–/– or Bf–/– sera. These results indicate that no mechanism exists to bypass the AP to directly activate C3 when Bf–/– serum is incubated with zymosan.


Figure 2
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  		FIGURE 2. Zymosan-induced C3 activation. 1/100 dilutions of sera from WT mice or complement-deficient mice were incubated on microtiter wells coated with zymosan. C3 activation was determined by ELISA and the data are expressed as OD values, mean ± SEM based on n = 3 for each serum. p < 0.001 for Bf–/ – and C3–/– vs WT.

 
C3 activation in vitro by adCII-IC

To further examine the mechanisms of complement activation in IC diseases, an in vitro system was developed with adCII-IC. The ability of the adCII-IC to activate complement in the presence of various sera lacking one or more complement components was assessed by measurement of the level of C3 deposited on the complexes as determined by ELISA. The amount of C3 fixed to CII alone in the absence of anti-CII Ab was subtracted from the amount of C3 found on the adCII-IC.

Sera from MBL–/– and Bf–/– mice exhibited no differences from WT sera in the levels of adCII-IC-induced C3 activation at dilutions of 1/10 through 1/320 (Fig. 3). However, the levels of C3 activation seen with 11 MBL–/– sera incubated with the adCII-IC were quite heterogeneous with OD values for 1/80 dilutions of sera varying between 0.48 and 1.69 compared with a mean OD for 3 WT sera of 1.05 (data not shown). The relative ability of the MBL–/– sera to activate C3 when stimulated by the adCII-IC was directly correlated with the C4 levels in these sera (Spearman correlation coefficient 0.90, p = 0.002). In contrast, the sera from C4–/–, C1q–/–, and C1q–/–/MBL–/– mice exhibited high levels of C3 activation at dilutions of 1/10 and 1/20 and then decreased rapidly with an absence of C3 activation at dilutions of 1/40 or higher. The sera from C3–/– mice showed minimal levels of C3 activation at all dilutions.


Figure 3
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  		FIGURE 3. C3 activation induced by adCII-IC in vitro. Various dilutions of sera from WT mice or from complement-deficient mice were incubated with CII-IC adherent to microtiter wells. C3 activation was determined by ELISA and was expressed as OD values. The OD values for C3 activation by CII alone were subtracted from the OD values observed with the CII-IC. The data are expressed as mean OD ± SEM, based upon: n = 3 for C4–/–, C3–/–, and C1q–/– sera, n = 5 for C1q–/–/MBL–/– sera; n = 7 for WT and Bf–/– sera; and n = 11 for MBL–/– sera. For C4–/– vs WT: p < 0.001 for dilutions of 1/40 through 1/320. For C1q–/– vs WT: p < 0.02 at 1/20 dilution, and p < 0.001 at dilutions of 1/40 through 1/320. For C1q–/–/MBL–/– vs WT: p < 0.02 at 1/20 dilution, and p < 0.001 at dilutions of 1/40 through 1/320.

 
These results indicate that adCII-IC induce C3 activation in vitro using WT, Bf–/–, or MBL–/– sera with a slow decline observed with serial dilutions of the sera. However, C3 activation mediated by C4–/–, C1q–/–, or C1q–/–/MBL–/– sera is only observed at low dilutions of 1/10 and 1/20, suggesting the involvement of a different pathway of complement activation.

Role of AP in adCII-IC-induced C3 activation in vitro

In an effort to clarify the role of the AP, adCII-IC-induced C3 activation was examined in the presence of a mAb to murine factor B that fully inhibits this complement component in vitro or in vivo (24). This experiment was conducted in VSB with Mg2+ and Ca2+, allowing activation of all three pathways of complement. The anti-factor B mAb completely blocked adCII-IC-induced C3 activation using 1/10 dilutions of sera from C1q–/–, C4–/–, or C1q–/–/MBL–/– mice (Fig. 4). However, sera from WT or Bf–/– mice exhibited only slight decreases in C3 activation using the anti-factor Ab (Fig. 4). The sera from MBL–/– mice exhibited mixed results with two having high values (mean of 1.57) before treatment and decreasing 25% (to 1.18) after treatment with the anti-factor B mAb. The third MBL–/– serum was low before treatment (0.18), decreasing to 0.12 after treatment with the anti-factor B mAb.


Figure 4
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  		FIGURE 4. Role of the AP in C3 activation. 1/10 dilutions of sera from WT, Bf–/–, C1q–/–, C4–/–, MBL–/–, and C1q–/–/MBL–/– mice were incubated on adCII-IC. The data are presented as OD values of C3 activation vs the serum. This experiment was conducted in the usual buffer used for the adCII-IC plate, VBS with 1 mM MgCl2 and 2 mM CaCl2, conditions where all three pathways of complement are active. The sera were also incubated with a mAb specific for murine factor B to neutralize the AP. The data are expressed as mean ± SEM with n = 6 for sera from WT mice and n = 3 for each serum from gene-deficient mice. p < 0.01 for anti-factor B-treated sera from C1q–/–, C4–/–, or C1q–/–/MBL–/– mice vs untreated sera.

 
These results indicate that the AP mediates full adCII-IC-induced C3 activation in vitro in the absence of C1q, C4, or both C1q and MBL. However, using sera from WT mice where all three pathways of complement are intact, the AP is not involved significantly in C3 activation as the CP appears to be primarily responsible. The sera from MBL–/– mice exhibit a partial role for the AP, although the CP mediates over half of the adCII-IC-induced C3 activation.

C3 activation in the absence of Ca2+

To further explore the role of the AP in C3 activation induced by adCII-IC in vitro, experiments were conducted with 1/10 dilutions of sera from WT, Bf–/–, C1q–/–, C4–/–, MBL–/–, and C1q–/–/MBL–/– mice in PBS deficient in Ca2+, containing 5 mM Mg2+ and 10 mM EGTA. This buffer allows C3 activation only by the AP as Ca2+ is absolutely required by both the CP and LP. Under these conditions maximal levels of adCII-IC-induced C3 activation were observed with sera from C1q–/–, C4–/–, and C1q–/–/MBL–/– mice but not with sera from Bf–/– mice (Fig. 5). Importantly, the sera from WT and MBL–/– mice also mediated C3 activation under these conditions. However, the 17 WT sera examined were highly heterogeneous with levels of C3 activation varying from OD units of 0.036 to 2.17. The 3 MBL–/– sera were also heterogeneous with one serum showing a high level of activity and two sera very low levels of activity.


Figure 5
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  		FIGURE 5. C3 activation by the AP alone. C3 activation induced by adCII-IC was determined using PBS deficient in Ca2+ with 5 mM Mg2+ and 10 mM EGTA, conditions where only the AP is active. C3 activation by sera from WT and various gene-deficient mice is shown. The data are expressed as mean ± SEM with n = 17 for sera from WT mice and n = 3 for all remaining sera.

 
These results indicate that under conditions where only the AP is active, i.e., in the absence of Ca2+, WT sera are able to activate C3 induced by adCII-IC, although these sera display a high degree of heterogeneity. The results with the sera lacking MBL also suggest heterogeneity in the ability to use the AP.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results of recently published studies, including those from our laboratory, indicate that inflammation and tissue damage in CAIA are markedly reduced in the absence of factor B but not in the absence of C4, suggesting mediation by the AP alone (22, 23). The role and mechanisms of AP involvement in this adCII-IC disease were further explored in the present studies using mice genetically deficient in C3, C1q, MBL-A/C, or in both C1q and MBL-A/C. These results establish that indeed the AP alone can mediate arthritis in the absence of the CP or LP. C3 activation was also examined in vitro using adCII-IC containing the four mAb used for the in vivo studies on CAIA. The results of these studies confirm that the AP alone can mediate C3 activation. However, when all three pathways of complement are intact, C3 activation in vitro occurs almost entirely by the CP.

Our results clearly show that the AP is capable of initiating complement activation in vivo in the absence of C4 or C1q, or of both C1q and MBL, causing inflammation and tissue damage in an adCII-IC model of arthritis. However, the major role of the AP in IC diseases in vivo appears to be amplification of C3b deposition after initiation by the CP or the LP. The absolute requirement for the AP to mediate disease in vivo may be because initiation of complement activation by the CP or LP does not produce an adequate quantity or quality of C3b deposition on the IgG anti-CII mAb or the nearby cartilage surface. The results of recent experiments further support the key role of the AP as CAIA is absent in mice lacking both MBL and factor D, where only the CP is active, or in mice lacking both C1q and factor D, where only the LP is active (data not shown). In our earlier studies, the level of C3 present in the joints of Bf–/– mice was ~1/3 of the levels found in the WT mice, as determined at day 10 after the administration of the anti-CII mAb, and the Bf–/– mice failed to develop disease (23). This observation suggests that the reduced level of C3 deposited in the joints in the absence of the AP was not sufficient to lead to disease.

We postulate that the low levels of C3 deposition in the joints of Bf–/– mice may have been secondary to a low density or clustered distribution of anti-CII mAb deposited on the articular cartilage. Previous studies have demonstrated that at low densities of Ab against a cell surface Ag, complement activation is mediated almost solely by the AP whereas the CP predominates at high Ab densities (26). Thus, in the presence of a low density of Ab on the target surface, the amplifying activity of the AP is necessary to produce enough surrounding sites of C3b deposition to reach a threshold level of C5 convertase activity. However, at high Ab densities sufficient C3b sites would be available on the IgG molecules not requiring as many adjacent C3b sites on the cell surface. The four mAb to CII used in our studies were all directed to epitopes located on the CB11 region of the collagen, suggesting a clustered distribution. Perhaps using a mixture of anti-CII mAb directed to epitopes more widely dispersed on the collagen molecule would produce a phenotype less dependent on the AP (27). An alternative explanation for the importance of the AP is that C5 convertase activity generated by the AP (C3bBb) may be much more potent and efficient than C5 convertase activity generated by the CP (C4b2a), particularly when C3b is bound to a protective surface such as IgG (28). We did not measure activity of the terminal complement components in these studies.

The mechanism whereby IgG in adCII-IC apparently enhances initiation of C3 activation by the AP in the absence of the CP and the LP remains unclear. The AP is thought to exhibit continuous low-grade activity in vivo through spontaneous hydrolysis of the thioester bond of C3, the "tickover" mechanism (29, 30). In the presence of IgG in IC, particularly in adIC, the short-lived C3b binds to the Fd portion of IgG to become a more stable and potent C3 convertase. Properdin binds to C3b complexed with IgG in a bivalent fashion before binding of factor B, creating a preferred site for C3 convertase generation (31). The binding of C3b to immobilized IgG in the presence of properdin leads to a four to eleven-fold enhancement in C3 cleavage in comparison to C3b alone (32). Thus, the amplification loop of the AP is greatly enhanced by adherent C3b-IgG complexes. The results of recent studies indicate that properdin not only stabilizes the C3 convertase (C3bBb) of the AP but may also directly bind to a target surface via C3b or other ligands to promote the association of C3b with factor B (33). Furthermore, properdin may bind primarily to certain surfaces to initiate the AP, with C3b provided by spontaneous low-grade hydrolysis of C3 in the fluid phase (34).

Mice lacking factor B demonstrated a decreased incidence of disease and a low level of disease activity in CAIA (22, 23). However, in the in vitro studies with adCII-IC described herein, sera deficient in factor B did not lead to lower levels of C3 activation in comparison to WT sera (Fig. 3). If the AP is required for adCII-IC-induced disease in vivo, why did the in vitro studies not demonstrate a similar role? Our results suggest that the amplification loop of the AP may not be fully operative in the in vitro experimental system. A possible explanation is that the IC with bound C3b localized on articular cartilage in vivo may present a more optimal substrate for deposition or activity of factor B than is offered by the IC in vitro. The regulatory protein factor H binds to C3b, accelerates the decay of the alternative pathway C3 convertase (C3bBb), and acts as a cofactor for the factor I-mediated proteolytic inactivation of C3b (35, 36). C3b covalently linked to IgG in soluble IC is highly resistant to the action of regulatory factors H and I because of a lowered affinity of factor H to the bound C3b (37). The binding of factor H to C3b present on adCII-IC in inflamed joints in vivo may be weaker than factor H binding to C3b on the adCII-IC in vitro. Factor H binding to surfaces is greatly enhanced by the presence of sialic acid or other polyanions (38). The binding of anti-CII Ab to CII on the surface of cartilage in vivo may neutralize sialic acid or other polyanions, weakening the interaction of factor H with this surface. Consistent with this hypothesis, IgG bound to a cell converted a nonactivating surface into an activating surface for the AP (15). The lack of a discernable role for amplification by the AP of C3 activation induced by the adCII-IC in vitro may be due to enhanced binding of factor H to the plate engendered by a net negative charge. Experiments are in progress using the inhibitory C-terminal domains of factor H to examine the role of this potent regulatory protein in C3 activation induced by adCII-IC in vitro (39).

Fc receptors may have been responsible for mediating residual inflammation in the complete absence of C3. In CAIA, mice lacking {gamma}-chains exhibited an absence of arthritis whereas mice deficient in Fc{gamma}RIII showed decreased disease activity (40, 41, 42). Furthermore, mice lacking FcRn, the FcR responsible for perinatal IgG transport and for IgG homeostasis in adults, exhibited near complete protection in K/BxN mice, both in the direct and the passive Ab transfer models of arthritis (43). In addition mice transgenic for the activating Fc{gamma}RIIa spontaneously developed arthritis (44), whereas an absence of the inhibitory Fc{gamma}RIIb rendered mice more susceptible to CIA (45, 46, 47).

Measurement of a variety of complement proteins in the sera of mice genetically deficient in specific components revealed some unexpected and interesting findings. C1q levels were markedly decreased in the sera from C4–/– mice and C4 levels were decreased in the sera of C1q–/– and C1q–/–/MBL–/– but not in MBL–/– mice. In addition, MBL-C levels were increased in the sera from Bf–/– mice. WT mice demonstrated a wide variability in the serum levels of C1q. The most striking observation was the heterogeneity in C4 levels in the sera from MBL–/– mice, with a strong direct correlation found between the C4 levels in these sera and the ability to mediate C3 activation in vitro. The reasons for these alterations in complement protein levels are unknown but may reflect compensatory changes in rates of synthesis or turnover in the presence of specific genetic deficiencies. However, it does not appear that the conclusions of our studies were affected by these unexpected variations in levels of complement components.

Important differences may exist in pathophysiologic mechanisms between soluble and adherent IC. In the K/BxN model in C57BL/6 mice, induction of increased vascular permeability in the distal joints by soluble GPI-anti-GPI IC was required to lead to arthritis (48). This localized enhancement in vascular leakage required mast cells, neutrophils and Fc{gamma}RIII, but not complement or cytokines. The AP is also required in the passive serum transfer model of K/BxN arthritis, but it is not known whether the AP can function in this model in the combined absence of the CP and LP as we have now shown in CAIA (49). The role of the AP in soluble IC disease may be through solubilization of precipitated IC by intercalation of C3b into the lattice (50, 51, 52). In contrast, in CAIA in BALB/c mice no localization of the anti-CII Ab occurred without the coinjection of irrelevant soluble IC, enhancing the vascular permeability and allowing the anti-CII Ab to enter the joint (53). However, despite localization of the anti-CII Ab in the joint, no disease occurred without the further administration of LPS. One of the important effects of LPS may be stimulation of the local synthesis of complement components. In addition, LPS has been observed to convert a nonactivating surface for the AP into an activating surface by decreasing the binding of factor H to C3b (54). These in vivo effects of LPS may be relevant to the differences we noted in results between the in vivo studies and the in vitro experiments regarding the role of the AP.

Although the role of anti-CCP Ab in RA is not known, Ab to citrullinated CII epitopes are present in early RA sera (55, 56). It is proposed that anti-CCP Ab are involved in inflammatory arthritis at two levels: firstly, forming soluble IC which facilitate entrance of the anti-CCP Ab into the joint, and secondly binding to citrullinated epitopes on the cartilage to create adIC (57). Soluble CCP-anti-CCP IC have not yet been described in the sera of patients with RA, but their possible existence may provide a mechanism of localized vascular leakage in the joint. Subsequently, Ab reactive with CII epitopes in the articular cartilage, either citrullinated or not, may enter the joint producing adCII-IC with subsequent induction of inflammation and tissue destruction (2).

The AP of complement has been implicated in a variety of human diseases and in many experimental animal models of disease (58). These human diseases include lupus nephritis, rheumatoid arthritis, antiphospholipid Ab syndrome, intestinal and renal ischemia/reperfusion injury, asthma, atypical hemolytic-uremic syndrome, type II membranoproliferative glomerulonephritis, spontaneous fetal loss, and macular degeneration. In many of these diseases the AP may serve as an amplification mechanism for C3b generation initiated by the CP and possibly LP. Thus, one of the potential strategies to inhibit the complement system in human disease would be through blocking the AP (59). This approach might permit minimal interference with the important protective roles that the complement system plays in both innate and adaptive immunity (60, 61).


   Acknowledgment
 
Review of the manuscript by Dr. Joshua Thurman is gratefully acknowledged.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AR51749. Back

2 Address correspondence and reprint requests to Dr. William P. Arend, Division of Rheumatology B115, University of Colorado at Denver and Health Sciences Center, Building M20, Room 3106, 1775 North Ursula St., Aurora, CO 80045. E-mail address: william.arend{at}uchsc.edu Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; adIC, adherent immune complexes; anti-CCP, Abs to cyclic citrullinated peptide; AP, alternative pathway; CII, type II collagen; CAIA, collagen Ab-induced arthritis; CIA, collagen-induced arthritis; CP, classical pathway; GPI, glucose-6-phosphate isomerase; IC, immune complexes; LP, lectin pathway; MBL, mannose-binding lectin; MASP, MBL-associated serine proteases; VSB, veronal saline buffer; WT, wild type. Back

Received for publication May 29, 2007. Accepted for publication July 16, 2007.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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