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*Joint Disorders
The Journal of Immunology, 2004, 173: 4684-4691.
Copyright © 2004 by The American Association of Immunologists

Serum Amyloid A-Activating Factor-1 (SAF-1) Transgenic Mice Are Prone to Develop a Severe Form of Inflammation-Induced Arthritis1

Alpana Ray2,*, Deepak Kumar*, Arvind Shakya*, Charles R. Brown*, James L. Cook{dagger} and Bimal K. Ray*

* Department of Veterinary Pathobiology and {dagger} Comparative Orthopedic Laboratory, University of Missouri, Columbia, MO 65211


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transcription factor serum amyloid A-activating factor-1 (SAF-1) has been identified as a regulator of a number of cellular genes. To assess the pleiotropic role of SAF-1 in vivo, we generated SAF-1 transgenic mice, in which CMV immediate-early promoter was used to direct expression of the SAF-1 transgene in multiple organs. Our study shows that overexpression of SAF-1 predisposes animals to arthritis. Although SAF-1 transgenic mice do not spontaneously develop arthritis, they develop a severe form of arthritis when challenged with the Lyme disease agent Borrelia burgdorferi, which is known to promote arthritis development in both humans and mice. CMV-SAF-1 transgenic mice, upon B. burgdorferi infection, showed increased joint swelling and synovial inflammation compared with nontransgenic littermates. Immunohistochemical analysis of joint tissues collected 21 days after B. burgdorferi infection revealed colocalization of matrix metalloproteinase-1, a degradative enzyme that destroys type II collagen, a major architectural component of articular cartilage, and SAF-1 in both SAF-1 transgenic and nontransgenic mice. Further analysis by RNase protection assay and Western immunoblot demonstrated the presence of higher levels of matrix metalloproteinase-1 and SAF-1 in the inflamed joints of SAF-1 transgenic mice compared with their levels in nontransgenic mice. Consistent with these findings, reduced levels of proteoglycans were detected in the inflamed joint cartilage of transgenic mice, indicating damage to the cartilage structure. Together these results suggest a role of SAF-1 in the pathogenesis of inflammation-induced arthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The pain, stiffness, and suffering associated with the pathogenesis of arthritis are primarily due to the destruction of articular cartilage in the joints. The role of articular cartilage is to protect underlying bone against shearing force generated by joint movement and compressive forces generated by joint loading to provide smooth and pain-free articulation. Under normal physiological conditions, cartilage tissue is subjected to a dynamic remodeling process in which low levels of degradative and synthetic activities are balanced, such that the volume of cartilage is maintained. In arthritis, erosion of articular cartilage occurs due to overexpression of matrix-degrading enzymes. Studies suggest that matrix metalloproteinases (MMPs), 3 a group of structurally related endopeptidases, play a critical role in the degradation of extracellular matrix of articular cartilage (1, 2, 3). In fact, increased concentrations of circulating MMPs are also found in rheumatoid arthritis (RA) and osteoarthritis (OA) patients, suggesting that MMP-mediated cartilage degradation holds a key role in the pathogenesis of rheumatoid arthritis (4, 5, 6).

Articular cartilage is primarily composed of type II collagen. It forms a fiber-like structure with three polypeptide chains forming a triple helical structure, called tropocollagen. Collagen fibers are distributed throughout the cartilage as building blocks and provide the functional architectural feature of cartilage (7). This essential component of cartilage is vulnerable to degradation by MMPs. MMP-1 is one of the major enzymes that specifically degrade type II collagen. Due to this unique property, MMP-1 plays an important role in cartilage breakdown. MMP-1 is synthesized as a latent proenzyme and undergoes activation by cleavage of the N-terminal peptide (8, 9). Normally, MMP-1 synthesis is very low in healthy articular cartilage. However, increased levels of MMP-1 are detected in the chondrocytes of OA (10) and RA patients (11), mainly due to an increased rate of transcription of this gene (2). A recent study indicated that the transcription factor serum amyloid A-activating factor-1 (SAF-1), an inflammation-responsive protein, is involved in the induction of MMP-1 during OA (12). Accordingly, increased levels of MMP-1 and SAF-1 were detected, and these were found to be colocalized in the chondrocytes of OA cartilage (12). Although this study identified SAF-1 as a regulatory factor in the increase in MMP-1 gene expression, it provided only indirect evidence of its role in the pathogenesis of the disease. To directly assess the role of SAF-1 in the development of arthritis, we used SAF-1 transgenic mice and examined how these mice respond to the environmental signals that promote arthritis. In this report we show that although SAF-1 transgenic mice do not become arthritic spontaneously, these mice are highly susceptible to arthritis-promoting agents. The SAF-1 transgenic mice developed a severe form of arthritis compared with nontransgenic littermates in response to Borrelia burgdorferi infection, which causes Lyme disease and arthritis in humans and mice. Colocalization of SAF-1 and MMP-1 in the arthritic joints of both SAF-1 transgenic and nontransgenic mice demonstrated the role of SAF-1 in regulating MMP-1 expression. We provide evidence for higher levels of MMP-1 and SAF-1 proteins with concomitant reduction in the level of proteoglycans in the extracellular matrix of the arthritic joints of SAF-1 transgenic mice compared with nontransgenic littermates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of SAF-1 transgenic mice

For generation of CMV-SAF-1 mice, a 1462-bp EcoRI-XbaI cDNA fragment containing full-length coding region of rabbit SAF-1 (13) was inserted into the corresponding sites of pCI-neo mammalian expression vector (Promega, Madison WI). The CMV immediate-early enhancer/promoter region present in the pCI-neo vector allows for strong constitutive expression in a variety of tissues (14). Downstream of the CMV enhancer/promoter region is a chimeric intron composed of 5'-donor and 3'-acceptor site that is found to be necessary for increasing transcriptional efficiency in transgenic mice (15). The pCI-neo vector also contains an SV40 late gene polyadenylation signal element for proper termination and polyadenylation of the expressed mRNA. The identity of the cloned gene was verified by DNA sequence analysis. The assembled gene was liberated from the vector backbone by cleaving with BglII and ClaI enzymes as a 2800-bp transgene. This DNA was microinjected into pronuclei of B6C3F1 zygotes and implanted into ICR foster mice, and resultant CMV-SAF-1 transgenic founder mice were identified by PCR analysis of genomic DNA isolated from tail biopsies. The rabbit SAF-1 transgene was detected using PCR primers against the CMV promoter region (5'-GTAACAACTGCGATCGCCCGCCCCGTTGAC-3') and the SAF-1-coding region (5'-GGGGTTCTGGGCGTGACCCTGAGGTGGCGG-3'), resulting in a 574-bp DNA that spans 451 nt of the vector (CMV promoter and chimeric intron) and 123 nt of the N-terminal sequence of SAF-1. The PCR was performed as follows: 1 min at 94°C, followed by 35 cycles of 15 s at 94°C, 30 s at 60°C, 1 min at 70°C, and 5 min at 70°C for the final extension.

RNase protection analysis (RPA)

Total RNA was prepared from various tissues of transgenic and nontransgenic mice using the guanidinium thiocyanate method (16). Cartilage tissues were frozen in liquid nitrogen and subsequently pulverized to powder form before lysis in guanidinium thiocyanate buffer. RPA was performed as previously described (17) using two different riboprobes, one corresponding to the transgene-specific SAF-1 and the other corresponding to endogenous SAF-1. To monitor transgene-specific SAF-1 expression, a 172-bp region of SV40 late poly(A) region was used. For endogenous SAF-1 expression, a 130-bp region containing the 3'-untranslated region of murine SAF-1 cDNA (18) was used. For MMP-1 expression, a 200-bp region containing the 5'-end of the mRNA was used. Antisense ssRNA probe, labeled with [{alpha}-32P]UTP, was produced by in vitro transcription with T7 RNA polymerase of the linearized pGEM3Z plasmid containing the desired fragments. RPA was performed with the RPA II kit (Ambion, Austin, TX) following the manufacturer’s protocol. Ten micrograms of total RNA was used for each sample. To evaluate the quality and quantity of each RNA sample, {beta}-actin cRNA riboprobe was used as an internal control. Protected RNA fragments were separated in a 7% polyacrylamide-8 M urea gel and visualized by autoradiography.

Western immunoblot assay

Cell extracts (50 µg of protein) were prepared from various tissues of nontransgenic and transgenic mice. Proteins were fractionated using SDS-PAGE and transferred onto a nitrocellulose membrane using an electroblotter. After transfer, relative amounts of proteins in each lane were verified by staining with Ponceau S solution (Sigma-Aldrich, St. Louis, MO). Immunoblotting was performed as described previously (19) with anti-SAF-1 Ab or anti-MMP-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were detected using a chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).

EMSA

Nuclear extracts were prepared from various tissues of mice following the method previously described (20). Protein content was measured by the Bradford method (21). EMSA was performed with equal amounts of proteins using a method described previously (9). Radiolabeled probe containing an SAF-binding element of the SAA promoter was prepared using [{alpha}-32P]dCTP as the substrate to label the double-stranded oligonucleotide probe (13). Two complimentary oligonucleotides, 5'-GGCTTCCTCTCCACCCACAGCCC-3' and 3'-CGAAGGAGAGGTGGGTGTCGGGGG-5', were annealed to prepare the double-stranded SAF-binding element. The DNA-protein complexes were separated in a nondenaturing 6% polyacrylamide gel.

Infection of mice

SAF-1 transgenic and nontransgenic littermate B6C3F1 mice, all 6 wk old, were inoculated in both hind footpads with a single injection of 106 Borrelia burgdorferi organisms in 50 µl of Barbour Stoenner Kelly II medium (Sigma-Aldrich). Details of bacterial preparation and infection have been described previously (22). Control mice received a single injection of medium alone. Swelling of the joint, as a result of bacterial infection-induced arthritis, was assessed by weekly measurement of tibiotarsal joints with a metric caliper (Ralmike’s Tool-A-Rama, South Plainfield, NJ) through the thickest anteroposterior diameter of the ankle. Induction of arthritis was evident from the swelling detected 7 days after the bacterial challenge.

Histology, immunohistochemistry, and proteoglycan staining

Mice were killed 21 days after infection. At the end of experiments, the hind feet of mice from control and treated mice were dissected. The ankles were washed in 70% ethanol, and skin was removed. Samples were fixed in 10% zinc-Formalin and decalcified in a solution containing 10% sodium citrate and 25% formic acid overnight. Formalin-fixed tissues were embedded in paraffin, sectioned, and stained with H&E. Arthritis severity scores were determined in a blinded manner and rated on a scale of 0–3 (23). Grade 0 represents no inflammation, grades 1 and 2 represent mild to moderate inflammation, and grade 3 represents severe inflammation.

Immunohistochemical staining was performed as described previously (12) using affinity-purified anti-SAF-1 rabbit IgG or anti-MMP-1 Ab (Santa Cruz Biotechnology) as the primary Ab; as a control, preimmune rabbit IgG was used. HRP-conjugated goat anti-rabbit IgG was used as the secondary Ab. Tissue sections were cut 5 µm thick, deparaffinized in xylene, and rehydrated in graded ethanol solutions, followed by washing with buffer (50 mM Tris-HCl (pH 7.5) and 0.15 M NaCl). Endogenous peroxidase activity was quenched by immersion in 3% H2O2 in methanol for 20 min, followed by rinses in buffer. The slides were then incubated in 0.1% trypsin solution with 0.1% CaCl2 for 60 min at 37°C to unmask Ags. Nonspecific binding was blocked for 30 min at 37°C with 100% normal goat serum. Slides were incubated overnight at 4°C with either anti-SAF-1 IgG or preimmune IgG as a control at a concentration of 1.0 µg/ml. The slides were rinsed twice in washing buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05% Tween 20), then incubated with secondary Ab. Bound primary Ab was detected using an HRP method with substrate-chromogen solution. Sections were counterstained with Mayer’s hematoxylin solution.

Safranin O-Fast Green staining for proteoglycans in the articular cartilage was performed essentially following the method of Conn and Lillie (24). Briefly, deparaffinized tissue sections in slides were stained with iron hematoxylin for 7 min and washed in distilled water for 10 min. The slides were then stained with Fast Green solution for 3 min, rinsed quickly with 1% acetic acid, and additionally stained in 0.1% safranin O solution for 5 min. The slides were dehydrated in 95% ethanol, absolute ethanol, and xylene using two changes for each solvent, 2 min each. As a control, sections of articular cartilage that were not decalcified were stained as described above. Both decalcified and undecalcified cartilage tissue sections produced very similar staining of safranin O, indicating that decalcification had no adverse effect on the proteoglycan staining. Decalcified, safranin O-stained sections were evaluated by one investigator (J.L.C.) who was blinded to animal group and treatment. Articular cartilage in the ankle joint was subjectively evaluated for intensity and distribution of proteoglycan staining as well as tissue morphology. Articular cartilage in the ankle was then scored for surface defects, cell number and morphology, and proteoglycan staining using the Mankin scoring system (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of SAF-1 transgenic mice

SAF-1 transcription factor is identified as a regulator of several cellular genes. To understand the biological effect of SAF-1 function, we generated SAF-1 transgenic mice by using a promoter that targets the expression of a gene to the broadest possible array of tissue types. The enhancer for the immediate-early genes of the human CMV is shown to direct expression of the transgene in multiple tissues (14). A full-length SAF-1 cDNA was cloned in pCI-neo mammalian expression vector, which carries the human CMV immediate-early enhancer/promoter region. The identity of the clone and proper insertion of the SAF-1-coding region into pCI-neo plasmid was verified by DNA sequence analysis. A linearized fragment of DNA, containing the CMV promoter, an intron, coding region of rabbit SAF-1 protein, and an SV40 polyadenylation signal, was isolated by BglII and ClaI digestion of the pCI-neo-SAF-1 plasmid and purified from agarose gel (Fig. 1A). This DNA was used for injection into fertilized mouse oocytes. A total of 90 offspring was screened by PCR, using primers specific for the transgene spanning the CMV promoter and the SAF-1 gene. A band of 574 bp in size was seen in the transgenic mice (Fig. 1B). Four founder mice, two males and two females, were produced with this transgene. The growth patterns of all these founder mice and the nontransgenic littermates were similar, indicating no severe effect of SAF-1 transgene in the normal growth of the transgenic mice. One male founder failed to reproduce, whereas the other three founders gave rise to individual true-breeding lines that manifested qualitatively similar phenotypes. Both male and female offspring reproduced similar to the nontransgenic littermates and grew normally.



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  		FIGURE 1. Construction of SAF-1 transgene and identification of transgenic mice. A, Map of the CMV-SAF-1 transgene. A full-length rabbit SAF-1 cDNA was inserted at the EcoRI-XbaI sites in pCI-neo mammalian expression vector (Promega). The regions containing CMV promoter, intron, SAF-1-coding sequences, and SV40 poly(A) consensus sequences are outlined. The arrows indicate the transgene-specific primers, which span the CMV promoter and the rabbit SAF-1 gene. B, PCR analysis of transgenic animals. Transgene-specific primers were used to identify the transgenic animals. The primers identified a 574-bp fragment, indicated by an arrow, that is specific to the transgenic construct. The transgenic animals were identified in lanes 2, 5, 7, and 9; nontransgenic animals were in lanes 1, 3, 4, 6, 8, 10, 11, and 12. Molecular size markers are shown in lane 13.

 
Expression of SAF-1 in transgenic mice

Offspring of each founder mouse were examined for transgene expression at the level of RNA and protein (Fig. 2). Because transgenic SAF-1 mRNA contains a 3'-untranslated region of SV40 late region plus the SV40 polyadenylation sequences, this region was used as the marker for assessing the expression level of SAF-1 transgene by RPA. The antisense RNA probe contained sequences present in the 3'-untranslated region of SV40 late region and protects a fragment of 172 nt in length. Analysis of total RNA prepared from different organs indicated expression of SAF-1 transgene in multiple organs, but at a variable level (Fig. 2A). To assess whether SAF-1 transgene expression had any effect on endogenous SAF-1 expression, we used a riboprobe that protects 130 nt of the 3'-untranslated region of mouse SAF-1 mRNA. This region is absent in the rabbit SAF-1 transgene. The results of this RPA indicated no change in the level of expression of endogenous SAF-1 between transgenic and normal nontransgenic mice due to the expression of the SAF-1 transgene (Fig. 2B).



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  		FIGURE 2. Analysis of the expression of SAF-1 transgene. A, A schematic representing the 3' region of SAF-1 transgene used for assessing transgene expression. To examine the transgene expression, 30 µg of total RNA was analyzed by RPA for the transgene-specific SV40 poly(A) RNA. A 172-bp protected fragment indicated expression from the SAF-1 transgene in different tissues. For comparison, RNA from both nontransgenic (N) and transgenic (Tg) mice were used. B, A schematic representing the region used for evaluating expression of endogenous murine SAF-1. The riboprobe used in this assay protects a 130-bp region present in the 3'-untranslated region of the murine SAF-1 gene, which is absent in the SAF-1 transgene. RNA from both nontransgenic (N) and transgenic (Tg) mice were used.

 
The level of SAF-1 protein in various tissues was determined by Western blot analysis using anti-SAF-1 Ab (Fig. 3A). This Ab recognizes both rabbit and mouse SAF-1 proteins. As shown in this figure, SAF-1 protein level was at least 2-fold higher in several organs, including liver, kidney, muscle, and heart, of transgenic mice compared with that of nontransgenic littermates. However, in the brain SAF-1 protein level was increased at least 5-fold. To assess the functional activity of this protein, DNA-binding activity of SAF-1 in various tissues was examined by EMSA using nuclear extracts, which showed moderately higher level of DNA binding activity in transgenic animals (Fig. 3B).



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  		FIGURE 3. Western blot analysis and EMSA. A, Fifty micrograms of protein prepared from indicated tissues of nontransgenic (N) and transgenic (Tg) mice was fractionated by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with anti-SAF-1 Ab. The migration positions of SAF-1 and two m.w. markers are indicated. B, Measurement of SAF-1 DNA binding activity. Nuclear extracts (10 µg of protein) prepared from different tissues, described in A, were incubated with a 32P-labeled probe containing the SAF-1 DNA-binding element. The resultant DNA-protein complexes were resolved in a 6% native polyacrylamide gel.

 
Development of arthritis in SAF-1 transgenic mice

A recent study from our laboratory has shown that SAF-1 is present in abundance in the diseased cartilage tissue of both human and canine OA patients (12). This study has also shown that SAF-1 enhances MMP-1 gene expression in the chondrocyte cells of OA cartilage. MMP-1 is known to cause cartilage breakdown, which is associated with arthritis. Together these data suggested that SAF-1 could play a significant role in the pathogenesis of arthritis. To test this possibility, we addressed the question of whether SAF-1 transgenic mice exhibit an increased propensity to develop arthritis. SAF-1 transgenic mice were examined for up to ~18 mo for detection of spontaneously developed arthritis, which was evaluated by assessing swelling and erythema of ankle joints and paws. Interestingly, these mice showed no signs of arthritis and were indistinguishable from the nontransgenic littermates (data not shown). These results indicated that higher than normal level of SAF-1 protein alone may not be sufficient for inducing any arthritis-related pathogenicity in a complex and multicellular organism such as the mouse. However, the possibility could not be ruled out that an increased SAF-1 protein level may sensitize the animals to other risk factors that promote arthritis.

To determine the propensity of SAF-1 transgenic mice to arthritis, we challenged both nontransgenic and SAF-1 transgenic mice with an arthritis-promoting agent and compared the rate of progression of the disease between these two groups. Infection with the spirochete B. burgdorferi, the causative agent of Lyme disease, creates swelling, ankle thickness, and, if left untreated, ultimately results in arthritis in humans (26). B. burgdorferi infection creates an arthritic condition in mice as well (27). SAF-1 transgenic mice and nontransgenic littermates, eight mice in each group, were inoculated in the hind footpads, and arthritis development was monitored for 21 days. Animals were monitored for changes in ankle thickness vs time postinfection (Fig. 4). On day 14, the ankles of SAF-1 transgenic mice were more swollen than those of nontransgenic littermates, and this trend continued up to day 21. Although a slight remission of severity was noted in the transgenic group on day 21, the overall level of ankle swelling was still higher than that in the nontransgenic group. As a control, two groups of transgenic and nontransgenic mice, four mice in each group, were injected with Barbour Stoenner Kelly medium alone. None of these animals displayed any swelling of the ankles. These results suggested that SAF-1 transgenic mice are more prone to develop severe arthritis when challenged with an arthritis-promoting agent.



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  		FIGURE 4. Evaluation of B. burgdorferi-induced arthritis in SAF-1 transgenic mice. SAF-1 transgenic mice and nontransgenic littermates, eight mice in each group, were inoculated in the hind footpad with B. burgdorferi. Mice were 6 wk old at the time of infection. Details of inoculation are described in Materials and Methods. Hind tibiotarsal joints of mice were measured at different days postinfection. {blacksquare}, SAF-1 transgenic (Tg) mice; {blacktriangleup}, nontransgenic (N) mice.

 
Because swelling of the ankle does not always correlate with arthritis development, the ankles of each of the test animals were histologically examined to detect any potential increase in the accumulation of inflammatory cells, a characteristic feature of inflammatory arthritis. A representative result is shown in Fig. 5. Microscopic analysis of the H&E-stained tissues indicates that the joints of both nontransgenic and SAF-1 transgenic mice have similar morphological patterns. However, after induction of arthritis by B. burgdorferi infection, SAF-1 transgenic mice exhibited much higher levels of accumulated inflammatory cells compared with the infected nontransgenic littermates. This pattern of staining was observed in all other mice in each group. For a semiquantitative evaluation of inflammation, based on infiltrate accumulation, all histological samples were scored in a blinded manner (Fig. 6). The data show that transgenic mice are more susceptible to the inflammatory response and exhibit severe arthritis compared with nontransgenic mice.



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  		FIGURE 5. Histopathology of tibiotarsal joints of SAF-1 transgenic mice and nontransgenic littermates. The hind tibiotarsal joints of four different groups of mice, eight in each group, were examined. Joint tissue samples were prepared as described in Materials and Methods, stained with H&E, viewed under a microscope, and photographed at x400 magnification. A representative sample from each group of mice is shown. a and c, Untreated wild-type and transgenic mice, respectively. b and d, B. burgdorferi-treated wild-type and transgenic mice, respectively. Increased accumulation of inflammatory cells, including neutrophil infiltrates in d, is indicated by arrows.

 


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  		FIGURE 6. Comparison of the clinical scores between arthritic SAF-1 transgenic and nontransgenic mice. Arthritis severity scores were determined by assessing relative levels of infiltrated inflammatory cells as observed in each group of mice after H&E staining. Severity scores were rated on a scale of 0 (no inflammation) to 3 (severe inflammation). The mean clinical scores (n = 8) were plotted. The error bar represents ±SD. *, p < 0.005 compared with the transgenic untreated group of mice.

 
Colocalization of SAF-1 and MMP-1 in the joints of transgenic arthritic mice

Increased accumulation of inflammatory cells, as shown in Fig. 5, most likely activates the expression of numerous genes in cells surrounding the inflamed joint. One of the consequences of this event is the onset of cartilage degradation due to the increased expression and activity of MMPs. Rationale for examining MMP-1 in our studies is its unique property of degrading type II collagen, which is the major constituent of cartilage. Cartilage architecture is built predominantly with collagen fibers; therefore, the structure disintegrates when type II collagen is depleted. We investigated whether the severity of arthritis in SAF-1 transgenic mice correlates with the expression levels of SAF-1 and its target, MMP-1. Immunohistochemical analysis was performed with the ankle joint sections of each mouse in both transgenic and nontransgenic groups under arthritic and untreated conditions. The presence and localization of SAF-1 and MMP-1 proteins from one representative member from each group are shown in Fig. 7. Similar results were observed with all animals in each group of mice. Serial sections of joint tissue from different animals were probed with anti-SAF-1 and anti-MMP-1 Abs. The immunohistochemical patterns of the SAF-1 and MMP-1 Abs indicated that the same group of inflammatory cells coexpresses both proteins.



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  		FIGURE 7. Colocalization of SAF-1 and MMP-1 in arthritic joints. Serial sections of tibiotarsal joints were analyzed by H&E staining and by immunohistochemical staining with anti-MMP-1 or anti-SAF-1 Ab, as indicated. Joint tissue sections were obtained from untreated nontransgenic mice (a–c), untreated SAF-1 transgenic mice (d–f), B. burgdorferi-treated arthritic nontransgenic mice (g–i) and B. burgdorferi-treated arthritic SAF-1 transgenic mice (j–l). b, e, h, and k, Stained with anti-MMP1 Ab; c, f, i, and l, stained with anti-SAF-1 Ab. b and c, e and f, and k and l were counterstained in weak hematoxylin. Magnifications, x200.

 
Increased SAF-1 and MMP-1 expression in joint tissues of arthritic mice

Immunohistochemical analysis, described in Fig. 7, also indicated that both SAF-1 and MMP-1 were expressed at higher levels in the arthritic mice. However, the relative abundance of these proteins among the transgenic and nontransgenic mice was not yet known. To evaluate the relative abundance of SAF-1 and MMP-1 in the joints of arthritic mice and to assess whether relative abundance correlated with the severity of arthritis, we performed Western blot analysis (Fig. 8A). This assay indicated higher levels of SAF-1 and MMP-1 proteins in the joints of arthritic SAF-1 transgenic mice compared with arthritic nontransgenic littermates. To assess whether the increased level of these proteins was due to increased expression, a quantitative RPA was performed, which revealed an increase in mRNA levels as a result of bacterial infection-induced inflammation (Fig. 8, B and C). The effect was more pronounced in the transgenic animals compared with the nontransgenic mice, indicating that SAF-1 transgenic mice are capable of producing more of the matrix-degrading enzymes.



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  		FIGURE 8. MMP-1 and SAF-1 protein and mRNA levels are higher in the joints of arthritic SAF-1 transgenic mice. A, Fifty micrograms of protein from ankle joints (pooled from four mice) of nontransgenic (N) and SAF-1 transgenic (Tg) mice were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-SAF-1 or anti-MMP-1 Ab, as indicated. Lane 1 contains protein from the joints of untreated nontransgenic mice, lane 2 contains protein from the joints of untreated SAF-1 transgenic mice, lane 3 contains protein from the joints of B. burgdorferi-treated nontransgenic mice, and lane 4 contains protein from the joints of B. burgdorferi-treated SAF-1 transgenic mice. The migration positions of MMP-1 and SAF-1 are indicated. B, Total RNA (50 µg) from the joint tissues of nontransgenic (N) and transgenic (Tg) mice, untreated or treated with B. burgdorferi, were analyzed by RNase protection assay as described in Materials and Methods. Radiolabeled probes specific for MMP-1, SAF-1, and {beta}-actin were used, and protected RNAs were detected by autoradiography. C, Radioactivity in each protected RNA sample in B were measured, normalized against {beta}-actin, and plotted as relative expression compared with untreated normal mice RNA.

 
Decreased proteoglycan content in arthritic joint of transgenic cartilage

High levels of the matrix-degrading enzyme, MMP-1, in the inflamed joint tissue of SAF-1 transgenic mice suggested the possibility of cartilage damage under this condition. To test this possibility, the level of proteoglycans, a major component of healthy cartilage, was assessed by staining with safranin O (Fig. 9). This dye produces an intense red color due to its interaction with glycosaminoglycans, which are covalently linked to proteoglycans. Staining intensity correlates well with the proteoglycan content, and loss of dye staining is an indication of loss of proteoglycans that are normally embedded within the fibrillar network of type II collagen in articular cartilage. As shown in Fig. 9, transgenic mice consistently showed lower levels of staining of safranin O, indicating a lower level of proteoglycan in these mice compared with the nontransgenic littermates. Additional reduction of the safranin O stain was observed in the arthritic joints of SAF-1 transgenic mice (Fig. 9d). Cartilage pathology was assessed using the histological grading system of Mankin et al. (25) as shown in Fig. 9B. Although severely degenerated cartilage was not observed in any of the transgenic and infected mice, loss of proteoglycans was most evident in these animals. In addition, the lamina splendens (superficial tangential collagen layer) in infected animals subjectively appeared less distinct and less organized (blue lining in Fig. 9, b and d, compared with that in a and c). These subjective findings corresponded well with Mankin scores and together indicate that B. burgdorferi-induced arthritis had detrimental effects on articular cartilage, which was most severe in the transgenic animals.



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  		FIGURE 9. Histological analysis of cartilage tissue of normal and arthritic tibiotarsal joints of SAF-1 transgenic mice and nontransgenic littermates. Hind tibiotarsal joints of four different groups of mice, four in each group, were examined. A, Joint tissue samples were prepared as described in Materials and Methods, stained with safranin O, viewed under a light microscope, and photographed at x400 magnification. A representative sample from each group of mice is shown. a, Nontransgenic; b, transgenic; c, nontransgenic treated with B. burgdorferi; d, transgenic treated with B. burgdorferi. B, Assessment of cartilage based on staining intensity, cellularity, and integrity of the articular surface. Specimens were scored for cartilage pathology using the histological-histochemical grading system described by Mankin et al. (25 ) with a grading scale of 0–4, where 0 represents normal, and 4 represents lack of safranin O staining, hypocellularity, and deformity of cartilage surface structure.

 
Together these data indicate that inflammation of the arthritic joint in transgenic mice overexpressing SAF-1 leads to increased production of matrix-degrading enzyme, resulting in the loss of proteoglycans, an indication of cartilage degradation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In considering an in vivo model in which to test the biologic effects of SAF-1 function, a mouse model in which a transgene is driven by a strong CMV early enhancer/promoter was developed. Our results indicate that an inappropriate level of SAF-1 predisposes animals to the pathogenesis of arthritis, in which subjects develop arthritis more frequently and with higher severity than subjects expressing this protein at a normal level. This conclusion is supported by evidence of increased clinical scores, synovial inflammation, increased levels of infiltrating inflammatory cells, and biochemical analysis demonstrating higher levels of MMP-1 activity in arthritic joints of SAF-1 transgenic mice compared with the nontransgenic littermates.

Increased MMP-1 synthesis by activated synovial fibroblasts and infiltrating macrophage cells is partly responsible for cartilage degradation in arthritic joint tissues. Normally the activity of MMP-1 is tightly regulated at the transcriptional level by controlling its synthesis, but a post-translational event involving its interaction with specific inhibitors, including the tissue inhibitors of metalloproteinases, is also an important regulatory process (3, 28). During arthritis, an increased rate of MMP-1 gene transcription causes an imbalance in the cellular level of this protein, which is primarily due to the activation of transcription factors regulating MMP-1 expression. Despite the great body of information on the role of MMP-1 in collagenolysis and cartilage degradation, a direct analysis of the effect of MMP-1 in the pathogenesis of arthritis is not yet available. Two transgenic mouse models are reported showing that targeted overexpression of MMP-1 in the skin of transgenic mouse causes hyperkeratosis and promotes tumorigenesis (29, 30), and in the lung causes progressive adult-onset emphysema (31, 32). SAF-1 was recently identified as one of the regulators of MMP-1 (12). The studies reported in this paper reveal that SAF-1 transgenic mice synthesize increased levels of MMP-1 in joint tissues and exhibit early signs of cartilage degradation due to reduction of articular cartilage proteoglycan levels in response to B. burgdorferi-induced inflammatory arthritic conditions (Figs. 8 and 9). Together these findings provide the first in vivo demonstration of an association between MMP-1 induction and onset of cartilage degradation in an inflammatory arthritis.

SAF-1 transgenic mice exhibited higher levels of SAF-1 protein in various organs, which correlated with increased SAF-1 DNA binding activity. Although the phenotype and growth pattern of SAF-1 transgenic mice were indistinguishable from those of nontransgenic littermates, these mice were highly susceptible to arthritis-promoting signals. Infection of SAF-1 transgenic mice with B. burgdorferi microorganisms promoted the development of arthritis at a higher rate and more severely than in control nontransgenic littermates. B. burgdorferi infection is known to cause Lyme disease in humans and arthritis at later stages of infection. Notably, the development of arthritis in humans as a consequence of Lyme disease normally takes a long period of time (33). This indicates that a chronic inflammatory condition is most likely present that eventually leads to the arthritic condition. This may explain why we did not observe any severe cartilage degradation in mice exposed to B. burgdorferi for only 21 days (Fig. 9B). Our studies, however, have shown significant signs of inflammation that appeared during this period (Figs. 4–6). Compared with nontransgenic mice, at 21 days of infection the joints of SAF-1 transgenic mice were more swollen, contained higher levels of inflammatory cells, and had reduced cartilage space. Consistent with these findings, higher levels of MMP-1 (Figs. 7 and 8) and reduced levels of proteoglycans (Fig. 9) in the inflamed joint cartilage suggested potentially abnormal cartilage health.

Bacterial challenge of B. burgdorferi in mice evokes a host response by which several inflammatory mediators, including cytokines and chemokines, are expressed, and these molecules play a determining role in the pathogenesis of Lyme arthritis (22). Due to the increased level of neutrophil recruitment in transgenic mice (Fig. 5) after bacterial infection, it is likely that robust chemokine activity is associated with transgenic mice. Additional studies are needed to assess what role inflammatory mediators and neutrophils play in the onset of arthritis in the SAF-1 transgenic model.

An interesting feature of this study was that transgenic mice did not develop arthritis spontaneously despite constitutively higher levels of SAF-1 expression. These mice also did not produce excessive MMP-1 unless they received the inflammatory challenge. This was surprising, because in cultured cells SAF-1 alone could induce transcription from the MMP-1 promoter at a detectable level. It is possible that in transgenic mice SAF-1 expression was much less than that achieved in transfected cells. When expressed at a suboptimal level, SAF-1 requires other transcription factors binding to the MMP-1 promoter for enhanced transcription. This assumption is supported by an earlier study that used c-fos transgenic mice (34). In c-fos transgenic mice, an increase in c-Fos transcription factor protein levels caused an increase in the level of MMP-1, but not in MMP-3 and MMP-10 levels (34). MMP-1, MMP-3, and MMP-10 all contain c-Fos/AP-1 binding elements in their promoters, and in transfected cells the overexpression of AP-1 increased the transcription of all three gene promoters.


   Acknowledgments
 
We thank Brad Wagner (University of Cincinnati, Transgenic Mouse Core Facility) for help in the development of transgenic founder mice. We also thank Howard Wilson for assistance with preparing the figures.


   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 U.S. Public Health Service Grant DK49205. Back

2 Address correspondence and reprint requests to Dr. Alpana Ray, Department of Veterinary Pathobiology, University of Missouri, 126A Connaway Hall, Columbia, MO 65211. E-mail address: rayal{at}missouri.edu Back

3 Abbreviations used in this paper: MMP, matrix metalloproteinase; OA, osteoarthritis; RA, rheumatoid arthritis; RPA, RNase protection analysis; SAF, serum amyloid A-activating factor. Back

Received for publication March 26, 2004. Accepted for publication July 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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