Inflammatory arthritis is characterized by massive infiltration of mononuclear and polymorphonuclear cells into the joints, resulting in synovitis, destruction of articular cartilage, and bone loss (). Such bone loss includes 1) bone erosions affecting the subchondral bone and bone at the joint margins, 2) periarticular bone loss adjacent to the inflamed joints, and 3) generalized osteoporosis ([2, 3]). Periarticular bone loss is a characteristic feature of early arthritis but has not been studied as extensively as bone erosions and generalized osteoporosis.
The mechanisms involved in inflammation-mediated bone loss in arthritis are only partly understood, and several different cell types are implicated. Osteoclasts, derived from hematopoietic stem cells, are responsible for bone resorption. The presence of macrophage colony-stimulating factor (M-CSF) leads to increased proliferation and survival of osteoclast precursor cells, as well as up-regulated expression of RANK in these cells (). RANKL, which is expressed by synovial fibroblasts (), osteoblasts (), osteocytes (), and activated Th cells (), stimulates RANK, which leads to osteoclast formation (). The interaction between RANK and RANKL is inhibited by the decoy receptor osteoprotegerin (OPG), which is ubiquitously expressed. The presence of M-CSF and RANKL is essential for osteoclastogenesis to occur; however, osteoclastic bone resorption is enhanced by inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), IL-1, and IL-17 ([10-13]). Synovial macrophages are also important in inflammation-mediated bone loss due to their prominent number in inflamed synovial tissue and are, together with neutrophils, key cells producing inflammatory cytokines and chemokines that attract and activate T cells ([14, 15]). Activated Th cells comprise a large proportion of the invading cells in inflamed synovial tissue and may therefore be of importance in pathogenesis ([16, 17]). Th17 cells, a subgroup of the Th cells implicated in the pathogenesis of arthritis (), produce the inflammatory cytokines IL-17 and RANKL, both of which are important in osteoclastogenesis.
The roles of NADPH oxidase (NOX) and reactive oxygen species (ROS) in autoimmune diseases such as rheumatoid arthritis (RA) have been intensively studied (). Neutrophils and macrophages phagocytize antigens and, as a direct response, produce ROS, which are essential for the activation of proteolytic enzymes and the prevention of antigen diffusion (). ROS are well-known immune promoters (), and it has been shown, experimentally, that excessive production of ROS may lead to accelerated joint destruction and osteoclast activation ([22-24]). However, it has also been shown that limited ROS production in Ncf1-mutated mice, caused by impaired NOX-2 function, results in enhanced disease severity in several different animal models of arthritis ([25, 26]). Thus, the exact role of ROS in arthritis is not completely understood.
In the present study, we used the antigen-induced arthritis (AIA) model to examine the influence of inflammatory arthritis on periarticular bone loss. This model is characterized by leukocyte infiltration and synovitis, together with bone and cartilage destruction, and shows several clinical and histopathologic features similar to those in human RA (). The aim of this study was to investigate periarticular bone loss in AIA and to study the associated effects on local cellular distribution in bone marrow and synovial tissue. Finally, we used the AIA model to determine the importance of NOX-2–derived ROS in periarticular bone loss.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Bone loss in arthritis is a complex physiologic process and is dependent on several different cell types and cytokines. The inflammation in arthritic diseases may result in different types of bone loss and can cause both generalized bone loss, i.e., osteoporosis, as well as local bone loss, including bone erosions and periarticular osteopenia (). The exact mechanism behind inflammation-mediated bone loss is not clearly understood and needs to be further studied in order to facilitate the development of new arthritis-preventing drugs. Several different experimental models are available to study the pathogenesis of arthritis. The murine model of AIA results in local monarthritis, with no generalized bone loss, and has mainly been used to study the pathophysiology underlying synovitis and joint destruction, including bone erosions. In contrast, periarticular bone loss in AIA has been less extensively investigated.
In the current study, we used computed tomography to investigate the effect of AIA on periarticular bone loss. We also evaluated the impact of inflammation on 1) various cell types in synovial tissue, 2) local effects on inflammatory cells in bone marrow, 3) local effects on ROS production in bone marrow, and 4) gene expression in different joint-associated compartments. Furthermore, we used the AIA model to determine the importance of NOX-2–derived ROS for periarticular bone loss.
AIA has many histologic features of RA, including infiltration of inflammatory cells into the joint, synovitis, and joint destruction (), and has been shown to be dependent on Th cells as well as synovial macrophages and neutrophils but independent of cytotoxic T cells and B cells ([31-33]). In accordance with this, we observed clear synovitis and joint destruction after the initiation of AIA and increased frequencies of monocyte/macrophages, neutrophils, and T cells in synovial tissue from arthritic knees compared with nonarthritic knees. Furthermore, expression of the cytokines IL-17A, TNFα, IL-6, and IL-1β was induced in arthritic synovial tissue but not nonarthritic synovial tissue. Induction of these cytokines in synovial fluid from patients with RA has been associated with disease activity ([34-36]). We also observed a significant decrease in periarticular trabecular BMD in both the distal femur and proximal tibia on the arthritic side versus the nonarthritic side, using peripheral QCT.
Periarticular bone loss in the rat model of AIA has been observed using histomorphometry (). Similar to the findings of that study, we detected changes only in the trabecular and not in the cortical bone compartment in the metaphyseal regions of the distal femur and proximal tibia; no effects were seen in cortical BMD in the diaphyseal part of the femur or tibia. Trabecular bone has a more porous structure and remodels more actively than cortical bone and therefore is more susceptible to changes such as physiologic status, diet, or drug treatment. It has also been shown that biologic differences between trabecular and cortical bone can be characterized using quantitative PCR ([38-40]). In the current study, we observed significant up-regulation of the osteoclastogenesis-related genes RANKL, RANK, and M-CSF in trabecular but not cortical bone in the arthritic side compared with the nonarthritic side.
The inflammation-mediated bone loss was associated with an increased frequency of osteoclast precursors in bone marrow and an increased number of osteoclasts in the epiphyseal regions of the distal femur and proximal tibia. It has been suggested that mediators originating from inflammatory tissue affect osteoclasts by paracrine mechanisms, because the number of osteoclasts dramatically decreases with growing distance from the affected joint (). In accordance with this, we detected effects on the frequency of preosteoclasts only in bone marrow from the distal part of the femur (closest to the inflamed joint) and not in bone marrow from the proximal part of the femur.
We next studied effects of arthritis on inflammatory cells in the bone marrow and observed that these cells were affected by inflammation in a local manner. The frequency of inflammatory cells in bone marrow from the distal but not the proximal part of the femur was increased in the arthritic side compared with the nonarthritic side, with increased frequencies of macrophages, neutrophils, and preosteoclasts in the area closest to the inflamed joint. Thus, inflammation has very local effects on bone marrow cells, suggesting that the factors mediating the effects signal in a paracrine manner. It is well known that there are variations in the transverse direction of bone marrow cell populations within a femur (), but such differences in the longitudinal direction have not been previously recognized.
ROS are widely considered to be involved in several destructive conditions; however, ROS may have a dual role. High ROS production in pathologic conditions such as RA increases osteoclast activity, but ROS can also provide hyperoxidative stress and thereby induce apoptosis of osteoclasts (). ROS have a primarily proinflammatory role in RA and mediate tissue damage, but low levels of ROS have been shown to have a regulatory function in arthritis ([21, 25]). Circulating neutrophils and neutrophils in synovial fluid from patients with RA have increased ROS production due to higher NOX-2 activity ([44, 45]). In accordance with this, we observed an increased capability of neutrophils and monocytes to produce intracellular ROS in bone marrow from the distal part of the femur, closest to the inflammation, but not from the proximal part. These data indicate that this effect of arthritis induction on ROS production is strictly local.
To determine the importance of ROS production in periarticular bone loss, we used Ncf1-mutated mice, which are incapable of producing NOX-2–derived ROS. The Ncf1*/* mice displayed no difference compared with their controls regarding arthritis development, including synovitis and joint destruction or periarticular bone loss. Thus, we conclude that ROS production via NOX-2 is not mandatory for the development of arthritis or inflammation-mediated periarticular bone loss in AIA. Mutated Ncf1 has previously been shown to both enhance () and reduce () arthritis severity in different murine models of arthritis compared with wild-type mice. Hence, ROS production can have different roles depending on the model used. NOX-2 is responsible for the majority of the ROS production, and the Ncf1 mutation results in loss of detectable respiratory burst activity ([21, 26, 47-49]). However, we cannot exclude the possibility that minor ROS production by other oxidases may be involved in the inflammation-mediated bone loss observed in this model.
RA is a complex disease with high heterogeneity among patients (), and currently there is no animal model of arthritis that totally reflects the pathogenesis. It is therefore important to choose the appropriate animal model in order to address the question being posed. We show that use of the AIA model is suitable to investigate local inflammation–mediated periarticular bone loss and propose that this model may be useful to increase knowledge regarding local bone loss, not only in RA, but also in other monarthritic diseases, including reactive arthritis and gout. This animal model makes it possible to compare the arthritic and nonarthritic joints in the same animal and is therefore ideal for investigating different antiarthritis therapies and their abilities to protect against local bone loss. Furthermore, the AIA model has no strain restrictions (), and by using different genetically modified animals, which may be on different genetic backgrounds, the mechanisms behind local bone loss could be further elucidated.
In conclusion, AIA results in periarticular bone loss associated with local effects on inflammatory cells and osteoclasts. Furthermore, using this AIA model, we determined that NOX-2–derived ROS production is not essential for inflammation-mediated periarticular bone loss. Thus, AIA can be used as a model to investigate the pathogenesis of local inflammation–mediated bone loss, and increased knowledge of the pathophysiology behind such bone loss in arthritis may help in identifying beneficial therapeutic strategies to protect bone in inflammatory diseases.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Engdahl had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Engdahl, Lindholm, Stubelius, Ohlsson, Carlsten, Lagerquist.
Acquisition of data. Engdahl, Stubelius, Lagerquist.
Analysis and interpretation of data. Engdahl, Lindholm, Stubelius, Carlsten, Lagerquist.