Previously, we reported that an extracellular matrix protein, osteopontin (OPN), is involved in various autoimmune diseases using a neutralizing polyclonal antibody against OPN generated in rabbits. However, the antibody cannot be used for long-term mouse models of chronic inflammatory disease because of the induction of antibodies against anti-OPN rabbit IgG. In this study, we generated a new antibody, anti-mouse OPN mouse IgG (35B6). 35B6 inhibited the cell adhesion of mouse and human OPN to Chinese Hamster Ovary (CHO) cells or CHO cells expressing α4 or α9 integrin. It was reported that OPN is highly expressed and has an important role in a chronic liver disease, non-alcoholic steatohepatitis (NASH). 35B6 injection twice a week for 8 weeks attenuated liver inflammation and fibrosis in a NASH mouse model, suggesting 35B6 is beneficial for the treatment of NASH. 35B6 was preferable to the rabbit anti-OPN antibody for investigating the in vivo function of OPN in mouse models of long-term disease.

Osteopontin (OPN) is a multifunctional protein that has been linked to various intractable inflammatory diseases. One way by which OPN induces inflammation is the production of various functional fragments by enzyme cleavage. It has been well appreciated that OPN is cleaved by thrombin, and/or matrix metalloproteinase-3 and -7 (MMP-3/7). Although the function of thrombin-cleaved OPN is well characterized, little is known about the function of MMP-3/7-cleaved OPN. In this study, we found a novel motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved mouse OPN binds to α9β1 integrin. Importantly, this novel motif is involved in the development of anti-type II collagen antibody-induced arthritis (CAIA). This study provides the first in vitro and in vivo evidence that OPN cleavage by MMP-3/7 is an important regulatory mechanism for CAIA.

The adaptive immune system generates memory cells, which induce a rapid and robust immune response following secondary Ag encounter. Memory CD8(+) T cells are a critical component of protective immunity against infections and cancers. Therefore, understanding the mechanism whereby memory CD8(+) T cells are generated and maintained is important for inducing effective memory CD8(+) T cell response. Recent studies have demonstrated that the inflammatory cytokine IL-12 favors the generation of terminal effector CD8(+) T cells rather than memory precursor effector CD8(+) T cells by regulating the expression of the transcription factor T-bet. In this study, we report that the inflammatory cytokine osteopontin (Opn) modulates memory CD8(+) T cell generation during influenza virus infection. Although Opn wild-type and Opn knockout (KO) mice had similar numbers of virus-specific effector CD8(+) T cells, virus-specific effector CD8(+) T cells generated in Opn KO mice showed low levels of T-bet expression and an increased memory precursor cell population compared with cells generated in Opn wild-type mice. This resulted in the persistently increased number of memory CD8(+) T cells in Opn KO mice. Studies with bone marrow-derived dendritic cells demonstrated that Opn deficiency in bone marrow-derived dendritic cells results in low levels of IL-12 production in response to the stimulation with influenza virus. Thus, we hypothesize that Opn modulates the generation of memory precursor effector CD8(+) T cells by regulating cytokine milieu during the acute phase of virus infection. This finding may provide new insight into the role of Opn in adaptive immune response.

The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macrophages (MΦs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MΦs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MΦ (RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80hiMac-1low MΦs residing in the red pulp showed different expression patterns of surface markers compared with F4/80+Mac-1hi monocytes/MΦs. Studies with purified cell populations demonstrated that F4/80hiMac-1low MΦs regulated CD4+ T cell responses by producing soluble suppressive factors, including TGF-β and IL-10. Moreover, F4/80hiMac-1low MΦs induced the differentiation of naive CD4+ T cells into functional Foxp3+ regulatory T cells. Additionally, we found that the differentiation of F4/80hiMac-1low MΦs was critically regulated by CSF-1, and in vitro-generated bone marrow-derived MΦs induced by CSF-1 suppressed CD4+ T cell responses and induced the generation of Foxp3+ regulatory T cells in vivo. These results suggested that splenic CSF-1–dependent F4/80hiMac-1low MΦs are a subpopulation of RPMs and regulate peripheral immune homeostasis.

Osteopontin is critically involved in rheumatoid arthritis; however, the molecular cross-talk between osteopontin and joint cell components that leads to the inflammatory joint destruction is largely unknown. We found that not only osteopontin but also tenascin-C and their common receptor, alpha(9) integrin, are expressed at arthritic joints. The local production of osteopontin and tenascin-C is mainly due to synovial fibroblasts and, to a lesser extent, synovial macrophages. Synovial fibroblasts and macrophages express alpha(9) integrin, and autocrine and paracrine interactions of alpha(9) integrin on synovial fibroblasts and macrophages and its ligands contribute differently to the production of proinflammatory cytokines and chemokines. alpha(9) integrin is also involved in the recruitment and accumulation of inflammatory cells. Inhibition of alpha(9) integrin function with an anti-alpha(9) integrin Ab significantly reduces the production of arthrogenic cytokines and chemokines and ameliorates ongoing arthritis. Thus, we identified alpha(9) integrin as a critical intrinsic regulator that controls the development of autoimmune arthritis.

Osteopontin (OPN) contains at least two major integrin recognition domains, Arg159-Gly-Asp161 (RGD) and Ser162-Val-Val-Tyr-Gly-Leu-Arg168 (SVVYGLR), recognized by αvβ3 and α5β1 and α4 and α9 integrins, respectively. OPN is specifically cleaved by thrombin and matrix metalloproteinase (MMP)-3 or MMP-7 at a position of Arg168/Ser169 (R/S) and Gly166/Leu167 (G/L), respectively. We in this study examined the requirement of residues within SVVYGLR for the α4 and α9 integrin recognition and how MMP-cleavage influences the integrin recognition. The residues, Val164, Tyr165, and Leu167 are critical for α4 and α9 integrin recognition in both cell adhesion and cell migration. The residue Arg168 is additionally required for α9 integrin recognition in cell adhesion and this explains why α9 integrin binds to only thrombin cleaved form of OPN. α4 integrin is able to bind to SVVYG (MMP-cleaved form of RAA OPN-N half), while α9 integrin is not, supporting the above notion that Arg168 is additionally required for α9 integrin-mediated cell adhesion. The residue Val163 is important for α4, but not for α9 integrin recognition in cell migration. Importantly, we found that the replacement of Arg168 by Ala (R168A mutant) induces the augmentation of cell migration via α4 and α9 integrins.

Osteopontin (OPN) is a T helper type 1 immunoregulatory cytokine that plays a critical role in various inflammatory disorders. OPN exerts proinflammatory reactions through interaction with integrin receptors. OPN function can be modulated by protease digestion. However, the molecular mechanisms that regulate OPN function in vivo have not been elucidated. There are two putative heparin-binding domains (HBDs) within the OPN molecule, which may bind both heparin and heparin-like glycosaminoglycans such as syndecan. We show that expression of OPN and syndecan-4 is significantly up-regulated after concanavalin-A (ConA) injection. Syndecan-4 binds to one of the HBDs of OPN, which overlaps with the thrombin cleavage site of OPN. When OPN is associated with syndecan-4, syndecan-4 masks both the thrombin cleavage and the integrin binding sites within OPN. Importantly, syndecan-4–deficient (Syn4KO) mice are more susceptible to hepatic injury, and the thrombin-cleaved form of OPN is significantly elevated in Syn4KO mice as compared with wild-type mice after ConA injection. Finally, we demonstrate that administration of purified syndecan-4 protects mice from ConA-induced hepatic injury. Thus, syndecan-4 is a critical intrinsic regulator of inflammatory reactions via its effects on OPN function and is a potential novel therapeutic tool for treating inflammatory diseases.

Osteopontin (OPN) has been implicated in various helper T cell type 1 immunity-mediated diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), Crohn's disease, and fulminant hepatitis. Increased expression of OPN has been detected in pathological foci of these diseases. RA and fulminant hepatitis have been successfully treated by administration of neutralizing anti-OPN antibody in mice. Antibody treatment may elicit side effects including allergic reactions against heterologous antibody proteins, thus necessitating humanization of antibody. To provide alternative means to neutralize OPN function, in this study we explored the possibility of using OPN small interfering RNA (siRNA) to silence OPN gene expression. In vitro, OPN siRNA efficiently silenced the expression of both exogenous and endogenous OPN gene. After hydrodynamic intravenous injection of OPN siRNA, OPN siRNA was efficiently delivered to the liver, which resulted in the efficient silencing of OPN gene expression in liver. In a murine model of concanavalin A (ConA)-induced fulminant hepatitis, OPN expression was elevated in liver and severe hepatic necrosis was induced. Importantly, after OPN siRNA treatment, the OPN expression level in liver was significantly reduced and liver tissue injury was ameliorated, as reflected by the significant reduction of serum alanine aminotransferase levels and almost normal liver histology. Thus, this study indicates that OPN siRNA delivery has therapeutic potential in various inflammatory diseases in which OPN play a critical role by silencing OPN gene expression in vivo.

It has been shown that musculoskeletal tissues undergo dynamic tissue remodeling by a process that is quite sensitive to the mechanical environment. However, the detailed molecular mechanism underlying this process remains unclear. We demonstrate here that after denervation-induced mechanical stress deprivation, tendons undergo dynamic tissue remodeling as evidenced by a significant reduction of the collagen fibril diameter. Importantly, the transient up-regulation of osteopontin (OPN) expression was characteristic during the early phase of tendon tissue remodeling. Following this dynamic change of OPN expression, matrix metalloproteinase (MMP)-13 expression was induced, which presumably accounts for the morphological changes of tendon by degrading tendon collagen fibrils. The modulation of MMP-13 expression by OPN was specific, since the expression of MMP-2, which is also known to be involved in tissue remodeling, did not alter in the tendons under the absence or presence of OPN. We also demonstrate that the modulation of MMP-13 expression by OPN is due to the signaling through cell surface receptors for OPN. Thus, we conclude that OPN plays a crucial role in conveying the effect of denervation-induced mechanical stress deprivation to the tendon fibroblasts to degrade the extracellular matrices by regulating MMP-13 expression in tendon fibroblasts.

Objective—Osteopontin (OPN), a noncollagenous adhesive protein, is implicated in atherosclerosis, in which macrophages within atherosclerotic plaques express OPN. However, it is not known whether the elevated OPN expression is a cause or result of atherosclerosis. Methods and Results—We generated mice that lacked OPN and crossed them with apolipoprotein (apo) E–deficient mice and analyzed these mice with a mixed C57BL/6 129 background after 36 weeks on a normal chow diet. In female mice, OP / E / and OP / E / mice had significantly smaller atherosclerotic and inflammatory lesions compared with OP / E / mice, and that was reflected by smaller area of MOMA-2–positive staining. In male mice, however, there was no significant difference in the atherosclerosis lesion areas among 3 genotypes. In both OP / E / and OP / E / mice, typical atherosclerotic lesions were detected, which include necrotic core, foamy cell collections, and cholesterol clefts. However, we found that vascular mineral-deposited areas in 60-week-old male OP / E / mice were significantly increased compared with those in OP / E / male mice. Conclusions—These results suggest that OPN plays a promoting effect in atherosclerosis and inhibitory effect in vascular calcification. The suppression of OPN expression in females should be considered a therapeutic possibility in atherosclerosis. (Arterioscler Thromb Vasc Biol. 2003;23:1029-1034.)

Osteopontin (OPN), a noncollagenous adhesive protein, may possibly be implicated in atherosclerosis, in which macrophages and activated T lymphocytes could have higher OPN levels within the atherosclerotic plaques. However, it is not known whether a higher OPN level is a cause or a result of atherosclerosis or whether it has a promoting or inhibitory effect on atherosclerosis. To clarify the role of OPN in atherosclerosis, we developed a transgenic mouse (OPN-TG) in which the exogenous OPN gene was designed to be expressed by hematopoietic cells, expressing OPN, which carried the immunoglobulin enhancer (Eμ)/SV40 promoter. In OPN-TG, the expression of exogenously transfected OPN RNA was found in lymphoid organs, such as the thymus and spleen, and the kidney. In the present study, OPN-TG mice were assigned into two groups, an atherogenic diet group (15% fat, 1.25% cholesterol) for 3 months or a standard diet group (4% fat), and both groups were compared with wild-type C57BL/6 mice to investigate the relationship between osteopontin and the atherosclerotic lesion. In wild-type mice, OPN mRNA was detected in kidney, but not in lymphoid tissues. In both OPN-TG and wild-type mice fed with control diets, atherosclerotic lesions were not found in the aortic sinus or the thoracic and abdominal aorta. In both OPN-TG and wild-type mice fed with atherogenic diets, a high incidence of atherosclerotic lesions was noted in the aortic sinus. The atherosclerotic lesions were significantly larger in OPN-TG as compared with those in control littermate mice (size: 33.8% ± 23.4% vs 10.9% ± 20.4%, respectively, P < 0.05). Activated foamy macrophages within atherosclerotic plaque in OPN-TG expressed a considerably larger amount of OPN compared with such macrophages in control mice. The OPN protein detected in the atherosclerotic lesions was not due to the deposition of serum OPN, but mainly due to in situ production by the infiltrating macrophages. Thus, these results suggest that OPN is atherogenic and that macrophages expressing OPN can be easily activated and thus promote atheromatous lesions if a high fat diet is consumed.

Five cases of calcium deposits in the menisci of the knee out of 5440 cases complaining of the pain of the knee joint in our clinic were characterized roentgenographically and symptomatically. One of these five cases was noted ossification of meniscus roentgenologically and histologically. Another one case revealed multiple calcification occurring in fibrocartilage disc and hyaline cartilage surfaces (elbow, hand, symphysis, hip and hand).The clinical evidence of these calcification was often very acute and resemble a septic arthritis, and called as “pseudogout” or “chondrocalcinosis articularis”.Otherwise, five menisci of 136 menisci removing from the knee joints of 34 cadavera revealed the finding of the spotted or linear calcification.