Acute-phase serum amyloid A (A-SAA) is a major component of the
acute-phase response. A sustained acute-phase response in rheumatoid arthritis
(RA) is associated with increased joint damage. A-SAA mRNA expression was
confirmed in all samples obtained from patients with RA, but not in normal
synovium. A-SAA mRNA expression was also demonstrated in cultured RA
synoviocytes. A-SAA protein was identified in the supernatants of primary
synoviocyte cultures, and its expression colocalized with sites of macrophage
accumulation and with some vascular endothelial cells. It is concluded that
A-SAA is produced by inflamed RA synovial tissue. The known
association between the acute-phase response and progressive joint damage may
be the direct result of synovial A-SAA-induced effects on cartilage
Serum amyloid A (SAA) is the circulating precursor of amyloid A
protein, the fibrillar component of amyloid deposits. In humans, four SAA genes
have been described. Two genes (SAA1 and SAA2) encode A-SAA
and are coordinately induced in response to inflammation. SAA1 and
SAA2 are 95% homologous in both coding and noncoding regions.
SAA3 is a pseudogene. SAA4 encodes constitutive SAA and is
minimally inducible. A-SAA increases dramatically during acute inflammation and
may reach levels that are 1000-fold greater than normal. A-SAA is mainly
synthesized in the liver, but extrahepatic production has been demonstrated in
many species, including humans. A-SAA mRNA is expressed in RA synoviocytes and
in monocyte/macrophage cell lines such as THP-1 cells, in endothelial cells and
in smooth muscle cells of atherosclerotic lesions. A-SAA has also been
localized to a wide range of histologically normal tissues, including breast,
stomach, intestine, pancreas, kidney, lung, tonsil, thyroid, pituitary,
placenta, skin and brain.
To identify the cell types that produce A-SAA mRNA and protein,
and their location in RA synovium.
Materials and methods:
Rheumatoid synovial tissue was obtained from eight patients
undergoing arthroscopic biopsy and at joint replacement surgery. Total RNA was
analyzed by reverse transcription (RT) polymerase chain reaction (PCR) for
A-SAA mRNA. PCR products generated were confirmed by Southern blot analysis
using human A-SAA cDNA. Localization of A-SAA production was examined by
immunohistochemistry using a rabbit antihuman A-SAA polyclonal antibody.
PrimaryRA synoviocytes were cultured to examine endogenous A-SAA mRNA
expression and protein production.
A-SAA mRNA expression was detected using RT-PCR in all eight
synovial tissue samples studied. Figure 1 demonstrates
RT-PCR products generated using synovial tissue from three representative RA
patients. Analysis of RA synovial tissue revealed differences in A-SAA mRNA
levels between individual RA patients.
In order to identify the cells that expressed A-SAA mRNA in RA
synovial tissue, we analyzed primary human synoviocytes (n = 2). RT-PCR
analysis revealed A-SAA mRNA expression in primary RA synoviocytes (n = 2; Fig. 2). The endogenous A-SAA mRNA levels detected in
individual primary RA synoviocytes varied between patients. These findings are
consistent with A-SAA expression in RA synovial tissue (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels
were relatively similar in the RA synoviocytes examined (Fig. 2). A-SAA protein in the supernatants of primary synoviocyte
cultures from four RA patients was measured using ELISA. Mean values of a
control and four RA samples were 77.85, 162.5, 249.8, 321.5 and 339.04 μg/l A-SAA, respectively, confirming the production of A-SAA protein by the
primary RA synoviocytes. Immunohistochemical analysis was performed to localize
sites of A-SAA production in RA synovial tissue. Positive staining was present
in both the lining and sublining layers of all eight RA tissues examined (Fig.
3a). Staining was intense and most prominent in the cells
closest to the surface of the synovial lining layer. Positively stained cells
were evident in the perivascular areas of the sublining layer. In serial
sections stained with anti-CD68 monoclonal antibody, positive staining of
macrophages appeared to colocalize with A-SAA-positive cells (Fig.
3b). Immunohistochemical studies of cultured primary RA
synoviocytes confirmed specific cytoplasmic A-SAA expression in these cells.
The specificity of the staining was confirmed by the absence of staining found
on serial sections and synoviocyte cells treated with IgG (Fig. 3c).
This study demonstrates that A-SAA mRNA is expressed in several
cell populations infiltrating RA synovial tissue. A-SAA mRNA expression was
observed in all eight unseparated RA tissue samples studied. A-SAA mRNA
expression and protein production was demonstrated in primary cultures of
purified RA synoviocytes. Using immunohistochemical techniques, A-SAA protein
appeared to colocalize with both lining layer and sublining layer synoviocytes,
macrophages and some endothelial cells. The detection of A-SAA protein in
culture media supernatants harvested from unstimulated synoviocytes confirms
endogenous A-SAA production, and is consistent with A-SAA mRNA expression and
translation by the same cells. Moreover, the demonstration of A-SAA protein in
RA synovial tissue, RA cultured synoviocytes, macrophages and endothelial cells
is consistent with previous studies that demonstrated A-SAA production by a
variety of human cell populations.
The RA synovial lining layer is composed of activated macrophages
and fibroblast-like synoviocytes. The macrophage is the predominant cell type
and it has been shown to accumulate preferentially in the surface of the lining
layer and in the perivascular areas of the sublining layer. Nevertheless, our
observations strongly suggest that A-SAA is produced not only by synoviocytes,
but also by synovial tissue macrophage populations. Local A-SAA protein
production by vascular endothelial cells was detected in some, but not all, of
the tissues examined. The reason for the variability in vascular A-SAA staining
is unknown, but may be due to differences in endothelial cell activation,
events related to angiogenesis or the intensity of local inflammation.
The value of measuring serum A-SAA levels as a reliable surrogate
marker of inflammation has been demonstrated for several diseases including RA,
juvenile chronic arthritis, psoriatic arthropathy, ankylosing spondylitis,
Behçet's disease, reactive arthritis and Crohn's disease. It
has been suggested that serum A-SAA levels may represent the most sensitive
measurement of the acute-phase reaction. In RA, A-SAA levels provide the
strongest correlations with clinical measurements of disease activity, and
changes in serum levels best reflect the clinical course.
A number of biologic activities have been described for A-SAA,
including several that are relevant to the understanding of inflammatory and
tissue-degrading mechanisms in human arthritis. A-SAA induces migration,
adhesion and tissue infiltration of circulating monocytes and polymorphonuclear
leukocytes. In addition, human A-SAA can induce interleukin-1β, interleukin-1 receptor antagonist and soluble type II tumour necrosis factor
receptor production by a monocyte cell line. Moreover, A-SAA can stimulate the
production of cartilage-degrading proteases by both human and rabbit
synoviocytes. The effects of A-SAA on protease production are interesting,
because in RA a sustained acute-phase reaction has been strongly associated
with progressive joint damage. The known association between the acute-phase
response and progressive joint damage may be the direct result of synovial
A-SAA-induced effects on cartilage degradation.
In contrast to noninflamed synovium, A-SAA mRNA expression was
identified in all RA tissues examined. A-SAA appeared to be produced by
synovial tissue synoviocytes, macrophages and endothelial cells. The
observation of A-SAA mRNA expression in cultured RA synoviocytes and human RA
synovial tissue confirms and extends recently published findings that
demonstrated A-SAA mRNA expression in stimulated RA synoviocytes, but not in
unstimulated RA synoviocytes.