EXPERIMENTAL CELL RESEARCH
ARTICLE NO.
227, 208–213 (1996)
0269
Complement Proteins Are Present in Developing Endochondral Bone
and May Mediate Cartilage Cell Death and Vascularization
JOSÉ A. ANDRADES,* ,†,1 MARCEL E. NIMNI,* JOSÉ BECERRA,*,† REUBEN EISENSTEIN,‡
MISHEL DAVIS,‡ AND NINO SORGENTE* ,§
*Division of Surgical Research, Children’s Hospital Los Angeles, University of Southern California, 4650 Sunset Boulevard, No. 35,
Los Angeles, California 90027; †Department of Cell Biology and Genetics, Faculty of Sciences, Campus Universitario Teatinos,
University of Málaga, 29071-Málaga, Spain; ‡Department of Pathology, Sinai Samaritan Medical Center, No. 342,
Milwaukee, Wisconsin 53201; and §Vide Pharmaceuticals, Los Angeles, California 90026
Normal endochondral bone formation follows a temporal sequence: immature or resting chondrocytes
move away from the resting zone, proliferate, flatten,
become arranged into columns, and finally become hypertrophic, disintegrate, and are replaced by bone.
The mechanisms that guide this process are incompletely understood, but they include programmed cell
death, a stage important in development and some disease processes. Using immunofluorescence we have
studied the distribution of various complement proteins to examine the hypothesis that this sequence of
events, particularly cell disintegration and matrix dissolution, are complement mediated. The results of
these studies show that complement proteins C3 and
Factor B are distributed uniformly in the resting and
proliferating zones. Properdin is localized in the resting and hypertrophic zone but not in the proliferating
zone. Complement proteins C5 and C9 are localized
exclusively in the hypertrophic zones. This anatomically segregated pattern of distribution suggests that
complement proteins may be important in cartilage –
bone transformation and that the alternate pathway
is involved. q 1996 Academic Press, Inc.
INTRODUCTION
The form for an endochondral bone is specified by
the cartilaginous model that precedes it during fetal
development. Even though chondrocyte hypertrophy
and cartilage resorption precede vascularization [1], a
critical event leading to bone formation is the invasion
of the midregion of the cartilaginous model by blood
vessels. During this process chondrocytes undergo a
series of sequential events that lead to their differentia1
To whom reprint requests should be addressed at Departamento
de Biologı́a Celular y Genética, Facultad de Ciencias, Campas Universitario Teatinos, Universidad de Málaga, 29071-Málaga, Spain.
Fax: 34-5-2132000.
208
0014-4827/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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tion and death and the replacement by bone of the area
where chondrocytes have disintegrated. This transformation is thus another example of programmed cell
death, a process critical to development and to a number of pathologic processes. In endochondral bone formation three distinct zones of cartilage are recognizable: a resting zone composed of rounded, randomly
arranged cells; a proliferating zone composed of proliferating cells that assume a flattened shape and become
arranged into discrete columns; and finally a zone of
hypertrophic cells, composed of large round cells that
ultimately disintegrate, leaving an empty lacuna surrounded by extracellular matrix. Advancing blood vessels penetrate the transverse septa of cartilage, accompanied by mesenchymal cells which can differentiate
into osteoblasts, which synthesize bone matrix which
ultimately replaces the cartilage. Cartilage matrix is
degraded by chondroclasts, cells of the monocyte –macrophage lineage which also enter this area along with
the blood vessels.
Little is known about the signals that regulate chondrocyte differentiation and/or death preceding deposition of bone in the distal hypertrophic zone. Since vascular invasion of the cartilage model is a crucial event,
it can be hypothesized that blood vessels are carriers
of signals that participate in the regulation of the cascade of events in this area. Morphologically, the disintegration of hypertrophic chondrocytes appears to be
an abrupt event, since the time frames of histological
sections only rarely reveal cells intermediate between
the hypertrophic and the shrunken, disintegrating
chondrocytes. The penetration of capillaries into the
growth plate requires dissolution of extracellular matrix. Both because this abrupt disintegration resembles
complement mediated lysis of other cells and because
complement has been shown to be capable of inducing
cartilage matrix degradation [2] we developed the notion that these processes could be complement mediated.
With the exception of the report that C1s is present
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COMPLEMENT IN CARTILAGE GROWTH PLATE
and synthesized in articular cartilage of hamsters [3],
where it may play a role in collagen degradation [4], we
know of no reports of the presence of other complement
proteins in cartilage. In both osteoarthritis and rheumatoid arthritis, complement is thought to play a role
in cartilage destruction. In these conditions, however,
complement is thought to be derived from noncartilaginous sources [5 –7].
To examine our hypothesis that cartilage –bone
transformation is complement mediated we have
mapped the distribution of various complement proteins in the tibias or femurs of 19-day-old fetal rats
using immunofluorescent techniques. In addition, we
used a marker for apoptotic cells to compare their distribution with that of complement.
MATERIALS AND METHODS
Tissues and antibodies. Tibias or femurs from 19-day-old fetal
Fischer rats were cleaned of adhering soft tissue, fixed for 2 h in 4%
phosphate-buffered paraformaldehyde, and stored at 0707C. Frozen
sections, 8 mm in thickness, were cut at 0207C using a Miles TissueTek cryostat. Alternatively, the tibias or femurs were fixed in Bouin’s
solution and embedded in paraffin. Sections 5 mm in thickness were
used for immunofluorescence and histological staining.
Goat antibodies to rat C3 (IgG fraction) and goat antiserum to
human complement components C5 and C9 were purchased from
Cappel (West Chester, PA). Goat antiserum to human properdin and
FITC-labeled rabbit anti-goat IgG were purchased from ICN (Irvine,
CA); goat antiserum to human Factor B was purchased from Calbiochem (La Jolla, CA). Human antibodies were tested for cross-reactivity to rat complement proteins by the suppliers.
Immunofluorescence. Paraffin sections were deparaffinized by
two 10-min changes of xylene and then hydrated in a series of alcohol:water mixtures and finally water. The sections were allowed to
air dry and incubated with 10% normal goat serum diluted with
phosphate-buffered saline (PBS). After 30 min the goat serum was
removed and the appropriate primary antibody added. After 1 h the
slides were washed three times, 5 min each with PBS followed by
the addition of FITC-conjugated secondary antibody. Appropriate
controls were also used. After an additional hour of incubation the
slides were washed with PBS, coverslipped, examined by fluorescence microscopy, and photographed.
After the sections were photographed for fluorescence, the coverslip was removed, and the sections were stained with the hematoxylin– eosin or toluidine blue stains.
Since cartilage matrix is known to bind proteins, including nonspecifically antibodies, particular care was employed to block and wash
the tissue sections carefully. The fluorescence signals are specific;
first, because tissues incubated with control antibody did not fluoresce; second, each antibody had a different distribution of binding
sites in the tissue, which would not occur if the binding were nonspecific. An antibody directed against an envelope protein of herpes
simplex virus, type I, was used as a negative control.
Apoptosis. Rat fetal bone was fixed in 10% neutral Formalin for
2 h, dehydrated in ascending concentrations of ethanol and eliminate
xylene, and embedded in paraffin. Sections from rat fetal bone, and
some from human fetuses obtained from our surgical pathology files,
8 mm thick, were deparaffinized, rehydrated with phosphate-buffered
saline (PBS), and digested with proteinase K (20 mg/ml) for 15 min
at room temperature. After washing in distilled water, the sections
were treated with 2% H 2O 2 in PBS for 5 min to quench endogenous
peroxidase activity and then with reagents from a kit (Apoptag;
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Oncor, Inc., Gathersburg, MD). In this procedure 3* OH ends of DNA
generated by DNA fragmentation during apoptosis are bound to digoxigenin complexed nucleotides using terminal deoxynucleotidase
as the catalyst. A peroxidase-coupled anti-digoxigenin antibody is
then added. After washing, the sections were incubated with 0.02%
H2O2 and 0.05% diaminobenzidine to yield a brown reaction product.
Controls (thymus glands from dexamethasone-treated mice) were
treated identically and gave the expected results.
RESULTS AND DISCUSSION
When a section of a 19-day-old embryonic tibia is
reacted with an antibody to complement protein C3,
the cells of the resting area fluoresce intensely without
localization to any specific area of the cell (Fig. 1a). In
the proliferative zone, the fluorescence persists and, in
those cells adjacent to the hypertrophic area, appears
to be localized preferentially at the cell periphery (Fig.
1b). In the hypertrophic area the fluorescence is markedly diminished and only appears at the cell periphery
(Fig. 1c).
The distribution of Factor B of the alternate complement pathway shows a distribution similar to that
of C3; however, in the resting area cells do not appear
uniformly fluorescent. Some cells are intensely positive, whereas others show only faint fluorescence
(Fig. 2a). In the proliferative and hypertrophic zones
fluorescence is markedly diminished and, in the area
closest to bone, largely limited to the cell periphery
(Fig. 2b).
The distribution of properdin, another protein of the
alternate pathway of complement, shows a pattern of
distribution different from that of C3 or Factor B. Properdin staining appears in the resting, but not the proliferative, area (Fig. 3a). In the resting area properdin
is not present in all cells. It is also present in the hypertrophic area, where it localizes at the cell periphery
(Fig. 3c). Figure 3b shows all the morphological characteristics of a typical growth plate in a section of 19day-old embryonic tibia; adjacent to the joint is the
resting zone (R) with small, uniformly distributed
chondrocytes. Adjacent to the resting zone is the proliferative area (P) of flattened cells arranged in columns
and between this zone and the bone (B) is the zone of
hypertrophy (H).
Complement proteins C5 and C9 are demonstrable
only in the hypertrophic area, where they are localized
at the cell periphery (Fig. 4). They are not uniformly
distributed and cells vary in staining intensity.
Apoptotic nuclei, demonstrated by the visualization
of 3* OH DNA ends were unusual, with rare scattered
stained cells in the distal hypertrophic zone at the cartilage –bone junction.
Complement proteins are synthesized mainly by
macrophages or the liver but other cells also have this
capacity. Fibroblasts, for example, can synthesize C1q,
C1r, and C1s [8]. Hyaline cartilage has also been shown
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ANDRADES ET AL.
FIG. 1. Distribution of complement protein C3 in cartilage. In the resting zone (a) C3 appears evenly distributed in all chondrocytes.
In the proliferative zone (b) C3 appears to be localized to the cell periphery, especially in the areas closer to bone. In the hypertrophic zone
(c) not all cells stain for C3; in positive cells C3 appears as patches on or near the cell surface. 1165 (a), 1250 (b) 1350 (c). *Resting zone
of the adjacent femur.
FIG. 2. Distribution of complement protein Factor B in cartilage. In the resting zone (a) Factor B is not present in all cells and more
abundant in some than in others. In the proliferative and hypertrophic zones (b) not all cells are positive and the fluorescence tends to be
at the cell periphery. 1200 (a, b).
to synthesize C1s [3], and synovial fibroblast-like cells
can synthesize C1r, C1s, C1 inhibitor, C2, C3, Factor
B, and Factor H [9].
It has been shown that cartilage dissolution occurs
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when complement is added to cultured cartilage under
conditions in which it is activated [2]. Complement is
thought to be involved in cartilage destruction in osteoarthritis and rheumatoid arthritis [5, 10, 11], in which
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211
FIG. 3. The distribution of complement protein properdin in cartilage. Properdin appears evenly distributed in many cells of the resting
zone (a, upper portion). In the proliferative zone (a, lower portion; c, upper portion) properdin staining is not seen. In the hypertrophic zone
(c, lower portion) properdin is also present. Autofluorescence is visible in bone and perichondrium. (b) Hematoxylin– eosin-stained section
for orientation: R, resting zone; P, proliferative zone; H, hypertrophic zone; B, bone. 1200 (a, c), 1100 (b).
FIG. 4. Distribution of complement proteins C5 (a) and C9 (b). These antigens are demonstrated at the cell periphery in the hypertrophic,
but not the proliferative, zone. 1400 (a), 1200 (b). *Cartilage– bone junction.
cases it is believed to be of extracartilaginous origin.
Our results show that transforming cartilage contains
proteins of the alternate complement system. The presence of the complement proteins C3, Factor B, and pro-
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perdin, but not C5 or C9, in the resting zone of cartilage
suggest that these components may be synthesized by
cartilage cells. This notion is based on their distribution: in the resting zone there is diffuse staining of the
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ANDRADES ET AL.
FIG. 5. Distribution of the alternative complement pathway proteins and hypothetical reactions in the various zones of the cartilage
growth plate. In the resting zone (R) C3 is activated to C3i, which binds to B to form C3iB. C3iB binds to the cell surface where Factor D
hydrolyzes B to give rise to C3 convertase, and C3iBb is inactivated by Factors H and I, leaving C3i attached to the cell and available for
further degradation. In the resting zone the presence of properdin (P) suggests that C3 convertase could be stabilized; however, even a
stabilized C3 convertase can be inactivated by Factors H and I, when their concentration is appropriate. In the proliferative zone the absence
or decreased concentration of Factors H and I allows C3 convertase to hydrolyze C3 to form the other form of C3 convertase, C3bBb, or C5
convertase C3bBbC3b. The C5 convertase is stabilized in the late proliferative zone by P (C3BbC3bP). Upon meeting C5, diffusing from the
blood, C5 convertase hydrolyzes C5 into C5b and C5a, thus starting the attack phase of the complement cascade resulting in cell lysis. 150.
cells, in the proliferative and hypertrophic zones these
components are more localized at the cell periphery,
and perhaps importantly, the extracellular matrix does
not stain. An alternate source of complement proteins
would be via diffusion from the blood vessels at the
osseous end of the growth plate. If that were the case,
the concentration and hence presumably the staining
intensity of these proteins would be expected to be
higher closer to the bone, but such is not the case. On
the other hand, the presence of properdin in the resting
and hypertrophic zones and our failure to demonstrate
it in the proliferative zone suggest that properdin is
present in the resting zone, but reaches the hypertrophic zone by diffusion from the vessels in the subjacent
bone. Until data directly demonstrating local synthesis
are available, however, this remains conjecture. Whatever their source, the presence of complement proteins
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C3, Factor B, and properdin in resting cartilage suggests that the alternate complement pathway may play
a role in the normal development of this tissue.
Endochondral bone formation and growth involve a
series of tightly regulated events that lead to chondrocyte hypertrophy, disintegration, and replacement of
the cartilage matrix by bone. Even though endochondral bone formation has been extensively studied, the
agents that mediate the process are incompletely understood. Two events are thought to be critical for the
generation of endochondral bone, the disintegration of
chondrocytes and invasion of the cartilaginous model
by blood vessels. The nature of the chemoattractants
for the invading vessels is not known. It is possible that
a fragment of one of the complement proteins (C3a or
Ba) may act as such as agent. C3a is known to be chemotactic for a number of cells and to enhance vascular
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COMPLEMENT IN CARTILAGE GROWTH PLATE
permeability [12 –14]. The presence of C5 and C9,
members of the membrane attack assembly of the complement cascade, only in the hypertrophic area supports the concept that complement localization accompanies and perhaps mediates chondrocyte cell death.
A possible scenario is that as the proliferating cells
migrate toward the bone, spontaneous conversion of
C3 into C3i occurs. C3i complexes with B to form the
inactive C3 convertase, C3iB, which is then activated
by Factor D (C3Bb). Once activated the C3 convertase
hydrolyzes C3 into C3b and C3a, expanding the process
and generating the C5 convertase, R-C3bBbC3b. As the
cell carrying C3bBbC3b migrates further toward the
midshaft, properdin diffusion from the vessels present
in the bone binds and stabilizes the C5 convertase. The
complex hydrolyzes C5 into C5a and C5b. C5b then
binds to cell surface which might initiate the cytolytic
attack phase. Figure 5 summarizes the major steps
in this conjecture and how the various events may be
related to the distribution of the various complement
proteins in prenatal cartilage. We have tried to perturb
this system by injecting complement into mice and zymosan into rats. These experiments yielded inconclusive results with regard to growth plate morphology.
A recent study [15], using organ cultures of chick
embryonic femurs demonstrates apoptosis in the distal
hypertrophic zone. Apoptotic cells, including endothelial cells, can bind and activate components of the alternate pathway of the complement system [16, 17]. The
accumulation of these proteins near the cartilage –bone
junction may thus be a consequence or accompaniment
of cell death rather than its cause. Even though cell
death in this area is programmed, this appears to be
unlikely for two reasons. First, except for the dying
chondrocytes at the very distal end of the growth plate,
chondrocyte nuclei are not shrunken, as they are in
apoptotic cells. Second, DNA OH* ends were demonstrable only rarely in the very restricted area of the
shrunken, dead chondrocytes, while complement proteins were present throughout the cartilage. In addition, the striking, apparently regulated pattern of distribution of the various complement proteins in developing cartilage argues strongly that they play a
significant role, perhaps, as we suspect, by promoting
matrix dissolution. The possibility of an interaction between apoptotic cells and complement, however, remains, since nuclear changes are not the first step in
the apoptotic pathway.
Finally, the integration of local events in preosseous
cartilage is critical to cartilage transformation. Inhibition of mineralization in rickets results not only in retardation of cartilage mineralization, but also widening
of the growth plate and its failure to vascularize.
Whether complement distribution is changed in this
condition remains to be seen. It is, however, intriguing
that mice given toxic doses of vitamin D develop more
extensive vascular calcification if injected with complement and that hypervitaminotic rats show less calcification when injected with zymosan [18, 19].
The authors are grateful for the financial support of National Institutes of Health (NIH No. AGO2577, Osteogenesis: Development,
Modulation and Aging), Vide Pharmaceuticals, and Ministerio de
Educación y Ciencia (DGICYT, Spain), which supported the visit of
Dr. José Becerra and Dr. José A. Andrades to Children’s Hospital
Los Angeles. The authors gratefully acknowledge the contribution of
Natalia Sorgente, who performed the original experiments to detect
complement in cartilage.
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Received January 18, 1996
Revised version received May 30, 1996
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